WO2011148135A1

WO2011148135A1 - Translocation across eukaryotic cell membranes based on oomycete protein sequence motifs

Info

Publication number: WO2011148135A1
Application number: PCT/GB2011/000801
Authority: WO
Inventors: Stephan Wawra; Pieter Van West; Severine Grouffaud; Paul Birch; Stephen Whisson; Christopher John Secombes; Andrew Justin Radcliffe Poter
Original assignee: The University Court Of The University Of Aberdeen
Priority date: 2010-05-25
Filing date: 2011-05-25
Publication date: 2011-12-01
Also published as: GB201008720D0

Abstract

This invention relates to proteins comprising sequence elements found in the AVR3a protein from the potato late blight pathogen Phytophthora infestans. The present invention also related to derivatives of these proteins and methods of modulating their function and activity. The invention describes a number of uses of these proteins, including the use to direct protein translocation into eukaryotic cells.

Description

TRANSLOCATION ACROSS EUKARYOTIC CELL MEMBRANE BASED ON OOMYCETE PROTEIN SEQUENCE MOTIFS

Technical field

The present invention relates generally to proteins comprising sequence elements found in the AVR3 protein from the potato late blight pathogen

Phytophthora infestans and SpHtpl from Saprolegnia parasitica. Also described are variants of these proteins, along with uses of the proteins and methods of modulating their function and activity.

Background art

Several Prokaryotic and Eukaryotic microbial pathogens have evolved intriguing mechanisms to translocate effector proteins into their host cells. Many eukaryotic pathogens have evolved mechanisms that translocate effector proteins into the host cells. These effectors modulate molecular processes in the hosts in order to establish an infection and/or suppress their hosts' immune responses (Birch et al., 2009; Hein et al., 2009; Mattoo et al., 2007; Stergiopoulos and de Wit, 2009). For example, pathogenic bacteria have developed a number of secretion systems, which allow a direct translocation of effector proteins into the cells under attack (Buttner and He, 2009; He et al., 2004; Salmond and Reeves, 1993). Whilst the bacterial translocation machineries are well described, little is known about how effectors from Eukaryotic pathogens are delivered into their host cells.

One phylum of eukaryotes containing many pathogenic species that utilizes such methods are the oomycetes. The group of oomycetes comprise devastating pathogens of plants and animals. They have evolved a host targeting system, thought to be similar to the Plasmodium PEXEL system, whereby effector proteins are translocated into their host cells. Recently, a tetrameric amino acid sequence motif, RxLR (Arg, any amino acid, Leu and Arg), was discovered that is common to all characterised oomycete effector proteins and crucial for the translocation of the effector proteins from oomycete plant pathogens.

The RxLR-motif, often followed by an EER (Glu, Glu and Arg) motif, is usually located within the 40 amino acids C-terminal to the cleavage site of a canonical signal peptide sequence (Birch et al., 2006; Dou et al., 2008a; Grouffaud et al., 2008; Rehmany et al., 2005; Whisson et al., 2007). Secretion of the RxLR (±EER) effectors takes place via haustoria, which are structures formed by the pathogen that are in intimate contact with the extra-haustorial membrane formed by the host during an infection (Whisson et al., 2007). It is generally assumed that the translocation process takes place after the effector has entered the extra-haustorial space between the haustorium and the extra-haustorial membrane (Birch et al., 2009).

It has been well documented that the RxLR motif plays an important role in the infection process of pathogenic oomycetes (Allen et al., 2004; Armstrong et al., 2005; Shan et al., 2004; van Poppel et al., 2008; Whisson et al., 2007), however, details about the translocation mechanism that these RxLR proteins use and how the effectors cross the extra-haustorial membrane are scarce.

The RxLR-motifs in the effectors from oomycetes are similar in sequence and relative location as the Plasmodium export element (PEXEL), which is also a host targeting signal found in effector proteins from malaria parasites (Hiller et al., 2004; Marti et al.,

2004) . Like the RxLR-motif, the pentameric PEXEL motif, RxLxE/D/Q (Arg, any amino acid, Leu, any amino acid, Glu or Asp or Gin), has been shown to direct effector proteins from Plasmodium spp. into red blood cells (RBC). The similarities of the RxLR- and PEXEL- systems has led to the idea that both groups of Eukaryotic pathogens might utilise a similar mechanism of delivering effectors inside their host cells. Indeed, a transgenic Plasmodium falciparum strain expressing a construct of GFP fused to the RxLR leader sequence of AVR3a from the oomycete potato pathogen Phytophthora infestans was able to translocate the chimeric GFP-protein inside the RBC (Bhattacharjee et al., 2006; Haldar et al., 2006). Reciprocally, a transgenic and virulent P. infestans strain expressing a construct where the PEXEL motif of the histidine rich protein HRPII from P. falciparum was fused to the avirulent form of the effector domain of AVR3a, was able to trigger a hypersensitive response (HR) in leaves of potato strains able to recognise this AVR3a effector domain (Grouffaud et al., 2008). Only three avirulence genes encoding RxLR proteins from P. infestans have been studied in detail so far, namely AVR3a (Armstrong et al.,

2005) , AVR4 (van Poppel et al., 2008) and AVR-blb1 (formally known as ipiO) (Vleeshouwers et al., 2008), but hundreds of other genes encoding potential RxLR effectors have been found in the genome of P. infestans highlighting the important role that the RxLR motif potentially has in the host pathogen interaction (Haas et al., 2009). The important role of the oomycete RxLR motif in establishing a successful infection has been well documented (Allen et al., 2004; Armstrong et al., 2005; Dou et al., 2008b; Shan et al., 2004; van Poppel et al., 2008; Whisson et al., 2007). However, information about the actual mechanism by which the RxLR effectors are

translocated into the host cells is missing. Whisson et al. (2007) showed that a chimeric protein with the RxLR-EER motif from AVR3a fused to (..-glucuronidase (GUS) is successfully translocated into potato leaf cells by transformants of P.

infestans expressing the reporter gene. Whisson ef al. (2007) also showed that virulent P. infestans stains transformed with avirulent AVR3a variants in which the (R)RxLR-EER motif was replaced with (A)AAAA-EER, RxLR-AAA, (A)AAAA-AAA, or ( )KMIK-DDK had a diminished ability to translocate RxLR effector proteins into host cells during pathogen-host interaction and failed to trigger an HR in susceptible potato leaves (Whisson et al., 2007). These experiments suggested that the RxLR and EER motifs may be required for the translocation process.

Dou et al. (2008) published an experiment whereby the RxLR-EER leader sequence of an effector protein from Phytophthora sojae, AVR1b, fused to GFP is taken up into onion root cells once these are soaked for 12 h in a concentrated solution of the recombinant protein. Based on these observations the authors argued that the entry of RxLR effectors into host cells does not require a pathogen encoded machinery and that translocation is solely based on the presence of the RxLR motif (Dou et al., 2008b). Furthermore, van West er al. (submitted) demonstrated that the RxLR- protein SpHtpl from S. parasitica can also translocate into fish cells in the presence and absence of the pathogen.

The Plasmodium RxLxE/D/Q-amino acid sequence has been shown to be a protease cleavage site for the ER resident aspartic protease plasmepsin V with leucine in the P1 position whereas the amino acid in P1 ' position becomes subsequently N- acetylated (Boddey et al., 2010; Chang et al., 2008; Russo et al., 2010). It is thought that the PEXEL cleavage is required for protein sorting and trafficking to specific regions of the parasitovorous vacuole (PV) membrane. In addition, Gehde et al. (2009) showed that PEXEL proteins may need to be unfolded in the PV in order to be translocated (Gehde et al., 2009). These observations seem to fall into place with the recent discovery of a protein translocon containing host and parasite derived components. This protein transport channel is specific for the PEXEL mediated translocation (de Koning-Ward et al., 2009). Whether a similar transport system is Disclosure of the invention

Based on the available literature, the accepted working model and hypothesis was that the translocation process of oomycete RxLR effector proteins is mediated by the RxLR-motif. In contrast to this, the results presented herein demonstrate that the translocation of the RxLR effector AVR3a from P. infestans, is not directly mediated by the RxLR-motif, but rather by the AVR3a effector domain in a pathogen- independent manner.

The present inventors have discovered that the RxLR effector proteins SpHtpl and AVR3a bind to tyrosine-O-sulfate, which is a common post-translational modification of solvent-exposed and extracellular proteins. The results described herein suggest that the sulfated aryl-moiety that is recognised by the tested RxLR-effectors is present in plants, fish and humans. This conclusion is supported by the findings that, (i) sulfatase treatment of cells to specifically remove the sulfate groups from the aryl- O-sulfate moieties of surface molecules strongly decreases the observed uptake of RxLR effector proteins into cells, indicating that the interaction with aryl-O-sulfate is required for the transport of the RxLR-effector proteins across the cell membrane, and (ii) both AVR3a and SpHtpl physically interact with tyrosine-O-sulfate which seems to cause structural rearrangements within the proteins. In case of AVR3a^22' ^{1 7}(His)₆ this structural rearrangement subsequently leads to aggregation of the protein.

For AVR3a the present inventors have shown that the sulfate binding and

translocation activity is solely dependent on the effector domain of this protein (AVR3a^{60 147}), although the presence of the RxLR leader peptide increases the efficiency of both activities. However, for the RxLR protein SpHtpl , it appears the sulfate binding and translocation activities require the presence of the RxLR leader sequence (i.e. the entire SpHtpl^24"198 fragment).

This difference in behaviour was investigated using the 'K IK' and Ά5' RxLR motif variants of Whisson et al. (2007) (see above). Using these variants the present inventors have determined that an interaction between the leader peptide and effector domain of RxLR proteins promotes the adoption of a specific conformation by the proteins. It is believed that this new conformation is a 'transition state' in a host cell. The existence of such a partially-unfolded transition state is consistent with the observation that the presence of the E. coli HSP70 chaperone DnaK effectively inhibits the uptake of RxLR proteins into cells.

In the case of SpHtpl , it appears that the 'wild-type' conformation of the effector domain is significantly different from the 'transition' state, meaning that interaction with the RxLR leader sequence is required for translocation. In contrast, it appears that the 'wild-type' conformation of the effector domain from AVR3a 'wild-type' is more easily converted into the 'transition' state, meaning that the protein is able to translocate in the absence of a RxLR leader sequence, albeit with reduced efficiency.

No evidence was found that the RxLR-leader sequence of either AVR3a or SPHtpl possesses any ability for self-translocation. However, the present inventors have established that a dimerisation site is present within the RxLR leader sequence of AVR3a, which is capable of interacting with another RxLR protein (for example, SpHtpl or a chimeric protein consisting of an RxLR leader peptide fused to a reporter such as mRFP) to form a heteromeric complex. Thus RxLR proteins that are capable of translocation (such as AVR3a^60"147 and SpHtpl^24"198) may be able to shuttle into cells other RxLR-leader containing proteins that lack the ability to translocate.

In short, the work of the present inventors indicates that the RxLR-translocation system is a far more dynamic, flexible and complex system than has hitherto been suggested. This finding has implications for new uses of RxLR-effector proteins and variants thereof, as well as other aspects that exploit the newly discovered interaction between RxLR-effector proteins and aryl-O-sulfate moieties. These aspects are described below.

Methods and compositions for enhancing protein translocation

Accordingly, in a first aspect the invention provides for the use of a recombinant polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to the amino acid sequence AVR3a^60"147, AVR3a^{22 147} or SpHtpl ^24-198 shown in Figure 14 to enhance translocation of a payload across the plasma membrane of a eukaryotic cell, wherein said membrane comprises a surface aryl-O-sulfate moiety, whereby said motif interacts with said aryl-O-sulfate moiety causing translocation of said polypeptide and payload. The "surface aryl-O-sulfate moiety" may be a tyrosine-O-sulfate residue comprised within a surface protein. In other embodiments, "the surface aryl-O-sulfate moiety" may be comprised within a surface glycan. In some embodiments the polypeptide may comprise one or more RxLR leader sequences which, in embodiments comprising an amino acid sequence motif having identity to AVR3a^60"147, may optionally be directly N-terminal to that motif.

As referred to herein, the terms "AVR3a", "AVR3a^1"147", "AVR3a^22"147", "AVR3a^60'147" and "AVR3a^22'59" encompass both the 'ΚΙ' (C¹⁹, K⁸⁰ and ⁰³) and ΈΜ' (S¹⁹, E⁸⁰ and 0³) isoforms of A R3a (see Figure 14). For example, a reference to "AVR3a^60"147" should be interpreted as a reference to "AVR3a(KI)^60'147 or AVR3a(EM)^60"147". The Kl isoform, but not the EM isoform is able to activate the innate immunity triggered by potato resistance gene R3a [Bos et al. 2009]. The reason for this difference is not known. However, it is possible that the Kl isoform may have a higher affinity for sulfate than the EM isoform.

In some embodiments of the present invention, the amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to the amino acid sequence of AVR3a^1'147, AVR3a^22'147, A R3a^60'147 or AVR3a^22'59 shown in Figure 14 comprises on or more of the following amino acids; C or S at position 19; K or E at position 80; I or M at position 103. For example, the amino acid sequence motif may comprise the amino acids C¹⁹, K⁸⁰ and P⁰³ or S ⁹, E⁸⁰ and M¹⁰³.

As used herein the term "surface aryl-O-sulfate moiety" is used to mean an aryl-O- sulfate moiety that is exposed to the extracellular environment of the cell when in its native sub-cellular location. The surface aryl-O-sulfate moiety may be comprised within a surface molecule such as a protein or a glycan.

The term "surface molecule" as used herein means a molecule which has at least a portion exposed to the extracellular environment of the cell when the molecule is in its native sub-cellular location. The surface molecule may be a protein. Surface proteins are typically synthesised and, if appropriate, post-translationally modified within the eukaryotic cell prior to their arrival at the cell surface. For example, the addition of sulfate groups to tyrosine residues of surface proteins is catalysed by tyrosylprotein sulfotransferase enzymes that reside in the Golgi apparatus of eukaryotic cells. Sulfation takes place as the surface proteins travel through the the cell surface. As used herein, if a protein is destined to become a "surface protein" it is referred to as such throughout the process of synthesis and export, even though at these stages the surface protein may not yet be exposed to the

extracellular environment.

Typically, the amino acid sequence motif interacts with said aryl-O-sulfate moiety by specifically binding the aryl-O-sulfate moiety. In some embodiments the binding interaction between the amino acid sequence motif and the aryl-O-sulfate moiety has a disassociation constant (Kd) of no greater than 50μΜ, 100μΜ, 150μΜ, 160μΜ, 200μΜ, 250μΜ or 300μΜ. The binding interaction between the amino acid sequence motif and the aryl-O-sulfate moiety is an important step in a process which results in the translocation of the polypeptide and payload across the plasma membrane of a eukaryotic cell i.e. causes the movement of the polypeptide and payload from the extracellular space, or a topologically equivalent compartment, into the cytosol of the eukaryotic cell.

Where the aryl-O-sulfate moiety is comprised within a molecule that is synthesised within the cell prior to its exposure to the extracellular environment, the addition or removal of a sulfate group from the aryl-O-sulfate moiety (e.g. a tyrosine residue of a surface protein) may occur whilst the molecule is in situ on the cell surface or when the molecule is within the cell (e.g. during the synthesis of the molecule). Both of these possibilities are contemplated in the present invention. Therefore, if an agent is described as a "desulfating agent" it may remove sulfate groups from molecules in situ on the cell surface (e.g. treatment of a cell with an aryl-sulfatase) or may remove / inhibit the addition of sulfate groups to aryl moieties when the molecules are within the cell (for example, during the synthesis of surface molecules). In some embodiments the desulfating agent may cause a decrease in the activity of tyrosylprotein sulfotransferases).

The invention may be used to selectively translocate a payload into members of a specific cell population in an organism, wherein members of the specific cell population have a higher concentration or number of aryl-O-sulfate moieties on their surface molecules (e.g. tyrosine-O-sulfate residues on their surface proteins) than members of otherwise comparable cell populations. The selectivity of the

translocation can be determined assessing the presence or amount of the payload in the ER or cytoplasm of a representative member of targeted specific cell population and comparing it to the presence or amount of the payload in the ER or cytoplasm of a representative member of a comparable non-targeted specific cell population. If the payload has been selectively targeted, the payload will be found at higher levels in the ER or cytoplasm of the member of the specifically targeted cell population. A comparable cell population may, for example, be cells of the same or similar type from another location in the organism, or cells of a different type found in the same location of the organism.

In some embodiments, the concentration or number of aryl-O-sulfate moieties on the surface molecules of a specific cell population may be increased by exposing the cell population to a sulfating agent as defined herein. In one embodiment the cell population is (i) exposed to a sulphating agent in order to increase the concentration or number of aryl-O-sulfate moieties on the cells' surface molecules, and then (ii) exposed to a molecule having a translocation sequence comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to the amino acid sequence AVR3a^60'147, AVR3a^22"147 or SpHtp ^24-198 shown in Figure 14. Said translocation sequence may be associated with a payload as set out below. In this way, the initial exposure of the cell population to the sulfating agents may be used to increase the efficiency with which the translocation sequence is translocated into the members of the specific cell population. Thus, cell populations not normally characterized by their expression of surface molecules having aryl-O-sulfate moieties may be targeted by the translocation sequences described herein.

In another aspect the invention provides a composition comprising, (i) a translocation sequence comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to the amino acid sequence AVR3a^60"147, AVR3a^{22' 47} or SpHtpl^24"198 shown in Figure 14, wherein the polypeptide sequence directs translocation of the polypeptide into a eukaryotic cell; and (ii) a payload coupled to the translocation sequence.

The payload may be coupled to the translocation sequence through any one of a number of different bonds. The payload and translocation sequence may be covalently bonded together and/or may associate through electrostatic bonds, hydrogen bonds, hydrophobic association or van derWaals interactions. In some embodiments the payload and translocation sequence each comprise a dimerisation described below), and associate with each other via this dimerisation domain or sequence.

The translocation sequence may be derived from an AVR protein (avirulence protein), as exemplified by AVR3a of Phytophthora infestans or SpHtpl of

Saprolegnia parasitica. The translocation sequence may further comprise at least one RxLR or RxLxE/D/Q leader sequence such as to form a multimer with the payload, which is translocated. For example, in some embodiments both the translocation sequence and the payload each include at least one RxLR leader sequence such as to form a translocation sequence/RxLR leader . RxLR

leader/payload multimer, which is translocated.

In some embodiments, the translocation sequence does not comprise an RxLR leader sequence within the N-terminal most 10, 20, 30, 40, 50 or 60 amino acids. Similarly, in some embodiments, where the payload comprises a polypeptide, the payload does not comprise an RxLR leader sequence within the N-terminal most 10, 20, 30, 40, 50 or 60 amino acids. In some embodiments, where the payload comprises a polypeptide, the payload does not comprise an RxLR leader sequence within the N-terminal most 10, 20, 30, 40, 50 or 60 amino acids.

The payload may be any type of molecule, for example a polypeptide, nucleic acid or small organic molecule. The payload may be a molecule with which the translocation sequence is not associated with in its native setting. For example, the payload may be an "exogenous protein" i.e. a protein that is not natively expressed in the eukaryotic cell into which it is being translocated. Exogenous proteins include, for example, fusions of native proteins, or fragments of native proteins, with non-native polypeptides or other molecules.

In some embodiments the payload is a therapeutic agent, a marker (e.g. GFP) or a protective agent (e.g. an agent that protects the cell from the effects of a cytotoxin to which the cell is subsequently). In some embodiments the payload is a cytotoxic molecule (i.e. a molecule, which when bound to or taken up by a target cell stimulates the death and lysis of the cell) or an RNA molecule such as a siRNA.

Cytotoxic molecules include members of the following groups or families: nitrogen - mustard types (e.g. melphalan), anthracyclines (e.g. adriamycin, doxorubicin, and (e.g. methotrexate). Also encompassed by the term "cytotoxic molecules" as used herein are enzymes intended to catalyse the conversion of a non-toxic prodrug into a cytotoxic drug (for example a HSV-Thymidine Kinase / Ganciclovir system). The prodrug may be systemically administered.

In a further aspect, the invention provides a method of producing the compositions defined above, the method comprising the steps of: (i) providing a translocation sequence comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to the amino acid sequence AVR3a^{60 47}, AVR3a^22"147 or SpHtpl^24"198 shown in Figure 14, wherein the

translocation sequence directs translocation of the polypeptide into a eukaryotic cell, and at least one RxLR leader sequence; (ii) providing a payload comprising at least one RxLR or RxLxE/D/Q leader sequence; and (iii) combining the translocation sequences and the payload in vitro so as to form multimers comprising at least one copy of the translocation sequence and at least one payload.

The present invention also provides a multimer having: (i) a first subunit comprising a translocation sequence comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 00% identity to the amino acid sequence of AVR3a^60"147, AVR3a^22"147 or SpHtpl ^24-198 shown in Figure 14, wherein the translocation sequence directs translocation of the polypeptide into a eukaryotic cell, and at least one RxLR leader sequence; and (ii) a second subunit comprising a payload and at least one RxLR leader sequence. The multimer may further comprise at least one additional subunit, wherein the additional subunit has at least one RxLR leader sequence and either (i) comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl^24"198 shown in Figure 14, wherein the translocation sequence directs translocation of the polypeptide into a eukaryotic cell, or (ii) a payload. The multimer may comprise two or more non-identical payload subunits.

In one aspect the invention provides a composition or multimer as described above for use in a method of treatment of the human or animal body. The composition or multimer may be used to selectively deliver a therapeutic agent, a marker, a protective agent, a cytotoxin, or siRNA into a target eukaryotic cell. The invention also provides for the use of a composition or multimer as described above in the treating a disorder comprising administering an effective amount of a composition or multimer as described above to a patient in need thereof.

The present inventors have determined that, unexpectedly, a dimerisation site is present within the RxLR leader sequence of AVR3a, which is able to interact with another RxLR protein (SpHtpl ) to form a heteromeric complex. Accordingly, in another aspect the invention provides a method of producing a multimer, the method comprising the steps of: (i) providing a first polypeptide comprising at least one RxLR leader sequence; (ii) providing a second polypeptide comprising at least one RxLR leader sequence; and (iii) combining the first and second polypeptide sequences so as to form multimers. The first polypeptide and/or the second polypeptide may be recombinant. In come embodiments the first and second polypeptide sequences are combined in vitro.

Also provided by the present invention is the use of a first polypeptide sequence comprising at least one RxLR leader sequence and a second polypeptide sequence comprising at least one RxLR leader sequence to form a multimer.

As used herein the term "RxLR leader sequence" is used to mean a polypeptide that has at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to either the amino acid sequence AVR3a^22"59 or SpHtpl^24"68 shown in Figure 14 and is able to form a dirtier with a second RxLR leader sequence. The RxLR leader sequence may comprise one or more "RxLR motifs" or "RxLxE/D/Q' motifs". In some embodiments the RxLR leader sequence interacts with AVR3a^60'147 or SpHtpl^69"198. This interaction may be such that a 1 : 1 mixture of solutions of the RxLR leader sequence and AVR3a^60"147 results in aggregation of the peptides (for instance, see Example 6). The interaction and/or aggregation may be suppressed by the interaction of AVR3a^60"147 with phosphate or sulfate ions (see Example 6).

As used herein, the term 'RxLR motif is used to mean an amino acid sequence having the sequence "RXLR" wherein X = any amino acid and R/L have the normal meaning in the one-letter amino-acid code. The term 'RxLxE/D/Q' motif is used to mean an amino acid sequence having the five-residue sequence "RXLX(D/E/Q)" wherein X = any amino acid, R/L have the normal meaning in the one-letter amino- acid code, and (D/E/Q) indicates the final position may be any one of D, E or Q. As used herein, any reference to a "RxLR motif should be understood as including a reference to an "RxLR" or a "RxLR-EER" motif unless explicitly stated otherwise. The term "RxLR-EER motif is used to mean a polypeptide comprising an RxLR motif as defined above, followed in a N to C terminal direction by the amino acid sequence "EER". In some embodiments the "EER" an aspartate residue ("D") is upstream of the EER motif to form a longer "DEER" motif. The "EER" motif may be directly after the RxLR motif or, alternatively, the RxLR and EER motifs may be separated by not more than 5, 10, 15, 20, 25, 30, 40, 50 or 60 amino acids.

Methods and compounds for blocking polypeptide translocation

In one aspect the present invention provides for the use of a desulfating agent or a chaperone agent to inhibit or block translocation of a polypeptide across of the plasma membrane of a eukaryotic cell, wherein the polypeptide comprises an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl^24"198 shown in Figure 14. The protein whose translocation is inhibited or blocked may be an exogenous pathogenic protein or effector protein. In some embodiments the desulfating agent or chaperone agent inhibits the binding of the amino acid sequence motif to a surface molecule including an aryl-O-sulfate moiety (e.g. a surface protein including a tyrosine-O-sulfate residue).

The desulfating agent may specifically reduce the sulfation of a surface molecule including an aryl-O-sulfate moiety (e.g. a surface protein including a tyrosine-O- sulfate residue), whereby the amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22'147 or SpHtpl^24'198 shown in Figure 14 interacts with said aryl-O-sulfate moiety of said surface molecule to cause translocation of said polypeptide and payload. The desulfating agent may be an aryl-sulfatase enzyme.

As used herein, "desulfating agent" refers to any agent that decreases the number, concentration and/or density of aryl-O-sulfate moieties on surface molecules (e.g. tyrosine-O-sulfate residues on surface proteins) of the eukaryotic cell. The agent may be of any type, for example, it may be a protein, a nucleic acid such as an RNA, or a small molecule. The decrease in aryl-O-sulfate moieties may be specific, that is the levels of other post-translational modifications of surface proteins (e.g.

phosphorylation, glycosylation) may be unaffected. Correspondingly, the term number, concentration and/or density of aryl-O-sulfate moieties on surface molecules (e.g. tyrosine-O-sulfate residues in surface proteins) of the eukaryotic cell. The desulfating or sulfating agents may be produced by the target cell, by another cell of the organism comprising the target cell, or may be from an exogenous source.

Desulfating agents include any agent that reduces the activity or expression of enzymes that add sulfate groups to molecules exposed on the extracellular surface of the target cell, or increase the activity of enzymes that remove sulfate groups from molecules exposed on the extracellular surface of the target cell. Example desulfating agents include sulfatase enzymes, such as aryl sulfatases (e.g. sulfatase type VI from Aerobacter aerogenes) or agents that increase the activity or expression of these enzymes. Also included as desulfating agents are inhibitors of the activity or expression of sulfate transferase enzymes, such as small molecule inhibitors of enzyme activity or RNAi encoding fragments of the sulfate transferase enzymes. Sulfate transferaes enzymes which may be targeted for inhibition include the tyrosylprotein sulfotransferase enzymes such as TPST1 (Uniprot identifier 060507), TPST2 (Uniprot identifier 060704) or homologues thereof.

Sulfating agents include any agent that increases the activity or expression of enzymes that add sulfate groups to molecules exposed on the extracellular surface of the target cell, or decreases the activity of enzymes that remove sulfate groups from molecules exposed on the extracellular surface of the target cell. Example sulfating agents include tyrosylprotein sulfotransferase enzymes such as TPST1 (Uniprot identifier 060507), TPST2 (Uniprot identifier O60704) or homologues thereof. Also included as sulfating agents are inhibitors of the activity or expression of sulfatase enzymes (e.g. sulfatase type VI from Aerobacter aerogenes) or inhibitors of the activity or expression of sulfatase enzymes, such as small molecule inhibitors or RNAi encoding fragments of the sulfatase enzymes.

An "exogenous pathogenic protein" as used herein is an exogenous protein as defined herein whose translocation into the target cell is associated with

pathogenesis of the target cell, or the organism of which it forms a part. Examples of exogenous pathogenic proteins include effector proteins, such as AVR3a or SpHtpl

Examples of "exogenous pathogenic proteins" include the Plasmodium falciparum effector GBP130, the Phytophthora infestans effector AVR3a, the Saprolegnia Phytophthora species, e.g. Phytophthora infestans, Phytophthora sojae,

Phytophthora ramorum, Phytophthora parasitica, Phytophthora capsici, Phytophthora nicotianae, Phytophthora cinnamomi, Phytophthora cryptogea, Phytophthora drechsleri, Phytophthora cactorum, Phytophthora cambivora, Phytophthora citrophthora, Phytophthora citricola, Phytophthora megasperma, Phytophthora palmivora, Phytophthora megakarya, Phytophthora boehmeriae, Phytophthora kernoviae, Phytophthora erythroseptica, Phytophthora fragariae, Phytophthora heveae, Phytophthora lateralis, Phytophthora syringae; any Pythium species, e.g. Pythium ultimum, Pythium aphanidermatum, Pythium irregulare, Pythium

graminicola, Pythium arrhenomanes, Pythium insidiosum; any downy mildew species; any Peronospora species, e.g. Peronospora tabacina, Peronospora destructor, Peronospora sparsa, Peronospora viciae; any Bremia species, e.g.

Bremia lactucae; any Plasmopora species, e.g. Plasmopora viticola, Plasmopara halstedii; any Pseudoperonospora species, e.g. Pseudoperonospora cubensis, Pseudoperonospora humuli; any Sclerospora species e.g. Sclerospora graminicola; any Peronosclerospora species, e.g. Peronosclerospora philippinesis,

Peronosclerospora sorghi, Peronosclerospora sacchari; any Sclerophthora species, e.g. Sclerophthora rayssiae, Sclerophthora macrospora; any Albugo species, e.g. Albugo Candida; any Aphano yces species, e.g. Aphanomyces cochlioides, Aphanomyces euteiches, Aphanomyces invadans, Aphanomyces astaci; any Saprolegnia species, e.g. Saprolegnia diclina, Saprolegnia salmonis, Saprolegnia ferax; any Achlya species; any rust fungi; any smut fungi; any bunt fungi; any powdery mildew fungi; any Puccinia species, Puccinia striijormis, Puccinia graminis, Puccinia triticina (syn. Puccinia recondita), Puccinia sorghi, Puccinia schedonnardii, Puccinia cacabata; any Phakopsora species, e.g. Phakopsora pachyrhizi,

Phakopsora gossypii; any Phoma species, e.g. Phoma glycinicola; any Ascochyta species, e.g. Ascochyta gossypii; any Cryphonectria species, e.g. Cryphonectria parasitica; any Magnaporthe species, e.g. Magnaporthe oryzae; any

Gaeumannomyces species, e.g. Gaeumannomyces graminis; any Synchytrium species, e.g. Synchytrium endobioticum; any Ustilago species, e.g. Ustilago maydis, Ustilago trifici, Ustilaginoidea virens; any Tilletia species, e.g. Tilletia indica, Tilletia caries, Tilletia foetida, Tilletia barclayana; any Erysiphe species, e.g. Erysiphe necator (formerly Uncinula necator); any Blumeria species, e.g. Blumeria graminis; Podosphaera oxyacanthae; any Alternaria species, e.g. Alternaria alternata; any Botrytis species, e.g. Botrytis cinerea; any Diaporthe species, e.g. Diaporthe phaseolorum; any Fusarium species, e.g. Fusarium graminearum, Fusarium Leptosphaeria maculans, Leptosphaeria maydis; any Macrophomina species, e.g. Macrophomina phaseolina; any Monilinia species, e.g. Monilinia fructicola; any Mycosphaerella species, e.g. Mycosphaerella graminicola, Mycosphaerella fijiensis, Mycosphaerella tassiana, Mycosphaerella zeae-maydis; any Phialophora species, e.g. Phialophora gregata; any Phymatotrichopsis species, e.g. Phymatotrichopsis omnivora; any Taphrina species, e.g. Taphrina deformans; any Aspergillus species, e.g. Aspergillus flavus, Aspergillus parasiticus, Aspergillus fumigatus; any Verticillium species, e.g. Verticillium dahliae, Verticillium albo-atrum, Rhizoctonia solani, Ophiostoma ulmi (syn. Ceratocystis ulmi), Ophiostoma novo-ulmi; any Septoria species, e.g. Septoria avenae; any Pyrenophora species, e.g. Pyrenophora tritici- repentis; any Colletotrichum species, e.g. Colletotrichum graminicola; any Sclerotinia species, e.g. Sclerotinia sclerotiorum; any Sclerotium species, e g Sclerotium rolfsii; any Thielaviopsis species, e.g Thielaviopsis basicola; any Coccidioides species, e.g. Coccidioides immitus; any Paracoccidioides species, e.g. Paracoccidioides braziliensis; any Pneumocystis species, e.g. Pneumocystis carinii; any Histoplasma species, e.g. Histoplasma capsulatum; any Cryptococcus species, e.g. Cryptococcus neoformans; any Candida species, e.g. Candida albicans; any apicompiexan parasite species such as: any Plasmodium species, e.g. Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae; any Babesia species, e.g. Babesia bovis, Babesia bigemina; any Cryptosporidium species, e.g. Cryptosporidium parvum; any Toxoplasma species, e.g. Toxoplasma gondii; any Trypanosomatid species such as: any Trypanosoma species, e.g. Trypanosoma brucei, Trypanosoma cruzi, Trypanosoma congolense, Trypanosoma vivax; any Leishmania species, e.g. Leismania donovani. Any amebozoan parasites; any Entamoeba species, e.g.

Entamoeba histolytica; any Mastigamoeba species; any Schistosoma species; any Onchocerca species; any Giardia species; any microsporidial species; any

Enterocytozoon species; any Encephalitozoon species, e.g. Encephalitozoon cuniculi, etc.

As used herein, a "chaperone agent" is an agent that binds, sequesters or otherwise interacts with unfolded or partially unfolded polypeptides. For example, the chaperone agent may be a chaperone protein, such as a member of the Hsp70 protein family (e.g. E.coli DnaK - Uniprot accession number P0A6Y8, or a polypeptide having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity thereto) or a member of the Hsc70 protein family (e.g. A.thaliana Hsc70-1 - Uniprot accession number P22953, or a polypeptide having at least 20, The chaperone protein may be in its ADP-bound form. The agent may be extracellular or intracellular. For example, chaperone agent may be added to the media surrounding the eukaryotic cell, or may be synthesised within the eukaryotic cell.

In some embodiments, the chaperone agent may be a regulator of Hsp70 family chaperones, such as a J-domain protein or a member of the Hsp90 protein family (e.g. H. sapiens Hsp-90-alpha - Uniprot accession number P07900, or a polypeptide having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity thereto) or the Hsp 00 protein family.

Processes for increasing pathogen resistance

In a further aspect the invention provides a process for increasing the pathogen resistance of a eukaryotic cell, wherein said process comprises introducing a genetic modification into the cell such as to inhibit or block the translocation of an effector peptide of the pathogen across the cell plasma membrane into the cell cytoplasm, the process comprising: (i) providing a genetically modified cell; (ii) providing a construct comprising a translocation sequence, which sequence comprises an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60'147, AVR3a^22"147 or SpHtpl ^24'198 shown in Figure 14; (iii) exposing the genetically modified cell to said construct; (iv) determining the presence or amount of the construct in the ER or cytoplasm of the genetically modified cell; and (v) correlating a reduction in the presence or amount of the construct in the cytoplasm of the genetically modified cell compared with a corresponding determination in the wild-type cell with the ability of the genetic modification to inhibit or block the translocation of an effector peptide of the pathogen across of the cell plasma membrane. The genetically modified cell may be a plant cell.

The genetic modification of the cell may be such that it reduces the number or concentration of aryl-O-sulfate moities (e.g. tyrosine-O-sulfate residues) on the modified cell's surface relative to a wild-type cell. For example, the genetic modification of the cell may cause, relative to the wild-type cell, (i) an increase in the activity or expression of one or more aryl sulfatase enzymes, or (ii) a decrease in the activity or expression of one or more tyrosylprotein sulfotransferase. For example, one or more genes encoding for a tyrosylprotein sulfotransferase may have been some embodiments the target cell(s) may have been transformed with one or more additional copies of an aryl sulfatase such that the level of enzyme activity and/or expression is increased.

In some embodiments, the genetic modification causes, relative to the wild-type cell, an increase in the activity or expression of an agent that either (i) competes with the amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 00% sequence identity to the amino acid sequence AVR3a^60"147,

AVR3a^22"147 or SpHtpl^24'198 shown in Figure 14 for binding tyrosine-O-sulfate residues on the cell surface or (ii) mimics an aryl-O-sulfate moiety of a cell surface molecule (e.g. a tyrosine-O-sulfate residue of a cell surface protein). For example, the agent may comprise an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl^24"198 shown in Figure 14.

In some embodiments, the genetic modification causes, relative to the wild-type cell, an increase in the activity or expression of a chaperone agent as defined herein.

In some embodiments, the increase in the activity or expression of the agent is upregulated in response to the onset of infection, for example the infection of the organism comprising the genetically modified cell by an organism expressing amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60'147, AVR3a^22"147 or SpHtpl ^4"198 shown in Figure 14.

The up-regulation may be stimulated by a change in the levels of signalling molecules in the environment of the target cell. For example, in a number or organisms, signalling molecules of the cytokine family are released in response to the onset of infection by pathogenic organisms (e.g. interleukins or interferons). Accordingly, in some embodiments the change in enzyme activity and/or expression is triggered by the presence of a signalling protein, such as a cytokine (e.g. an interleukin or interferon) or other elicitor or effector protein (e.g. AVR proteins, pathogen-associated molecular patterns (PAMPS) or pattern recognition molecules of the host that trigger defense responses in the host. Methods for making the activity and/or expression of sulfate moiety modifying enzymes responsive to the signalling molecules released at the onset of infection are well-known in the art. For example, will result in a sulfatase whose expression is triggered in response to the presence of the relevant cytokine.

The construct comprising the translocation sequence may further comprise at least one RxLR leader sequence. In some embodiments the construct includes an effector peptide of the pathogen, optionally having its native signal peptide and\or

translocation sequence. In some embodiments the construct is an artificial fusion construct comprising: (i) a payload; (ii) a translocation sequence having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60-147, AVR3a^22-147 or SpHtp ^24-198 shown in Figure 14; and, optionally, (iii) an N-terminal signal sequence. The construct may also comprise a detectable group which is optionally a fluorescent protein, GUS or other suitable reporter group or molecule.

The present invention also provides for the use of an agent or genetic modification as described herein for enhancing the pathogen resistance of a eukaryotic cell by inhibiting or blocking the translocation of an effector peptide of the pathogen across the cell plasma membrane into the cell cytoplasm, wherein the effector peptide comprises an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60'147, AVR3a^22"147 or SpHtpl ^24-198 shown in Figure 14. The eukaryotic cell may be a plant cell.

Treatment with desulfating and chaperone agents

In another aspect the invention provides a desulfating agent or a chaperone agent for use in a method of treatment of the human or animal body, wherein the desulfating agent or chaperone agent inhibits or blocks translocation of a polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 00% sequence identity to the amino acid sequence AVR3a^60-147, AVR3a^22-147 or SpHtpl^24-198 shown in Figure 14 across the plasma membrane of a eukaryotic cell.

Also provided is a desulfating agent or a chaperone agent for use in a method of treatment of a disorder associated with pathogenic cell invasion, wherein the desulfating agent or chaperone agent inhibits or blocks translocation of a polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, AVR3a^60'147, AVR3a^22-147 or SpHtpl ^24-198 shown in Figure 1 across the plasma membrane of a eukaryotic cell. As used herein, a "disorder associated with pathogenic cell invasion" is a disorder that results from, or is characterized by, the translocation of an "exogenous pathogenic protein" into the host (target) cell.

Examples of disorders associated with pathogenic cell invasion include malaria, oomycete infections on plants and animals (e.g potato blight, saprolegniosis), fungal infections on plants and animals, protist infections, and other disorders caused by organisms that express an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60'147, AVR3a^22-147 or SpHtpl ^4-198 shown in Figure 14.

In another embodiment, the invention provides a desuifating agent or a chaperone agent for use in the manufacture of a medicament for the treatment of a disorder associated with pathogenic cell invasion, wherein the desuifating agent or chaperone agent inhibits or blocks translocation of a polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60-147, AVR3a^22-147 or SpHtpl ^{2 -198} shown in Figure 14 across the plasma membrane of a eukaryotic cell. The invention also provides a method of treating a disorder associated with pathogenic cell invasion comprising administering to a patient in need thereof an effective amount of a desuifating agent or a chaperone agent that inhibits or blocks translocation of a polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60-147, AVR3a^22-147 or SpHtpl ^{2 -198} shown in Figure 14 across the plasma membrane of a eukaryotic cell.

Inhibition of Tyrosine-Q-sulfate binding

In a yet further aspect the invention provides for the use of an agent which inhibits binding of an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60-147, AVR3a^22-147 or SpHtpl ^{2 -198} shown in Figure 14 to a surface molecule including an aryl-O-sulfate moiety (e.g. a surface protein including a tyrosine-O- sulfate residue) to inhibit or block translocation of a polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60-147, AVR3a^22-147 or SpHtpl ^24-193 shown in Figure 14 across the plasma membrane of a eukaryotic cell. In membrane of polypeptides comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl ^{2 - 98} shown in Figure 14. In some embodiments the protein whose translocation is inhibited or blocked is an exogenous pathogenic protein.

Examples of suitable agents are agents that are: (i) intended to compete with the polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 00% sequence identity to the amino acid sequence AVR3a^50'147, AVR3a^22"147 or SpHtpl^24'198 shown in Figure 14 for binding aryl-O-sulfate moieties (e.g. tyrosine-O-sulfate residues) on the cell surface; or (ii) an aryl-O-sulfate moiety mimetic (such as a tyrosine-O-sulfate residue mimetic) intended to compete with the aryl-O-sulfate moieties on the cell surface for binding the polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl ^{2 198} shown in Figure 14. The agent may comprise an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^{22" 47} or SpHtpl^24"198 shown in Figure 14, of or a variant thereof.

Processes for increasing pathogen resistance

In another aspect the invention provides a process for providing a pathogen resistance composition for use in a eukaryotic cell, wherein the composition comprises an agent which inhibits or blocks the translocation of an effector peptide of the pathogen across the plasma membrane of the cell into the cell cytoplasm, the process comprising: (i) providing a test agent and a eukaryotic cell;

(ii) providing a construct comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60'147, AVR3a^22"147 or SpHtpl^24"198 shown in Figure 14; (iii) exposing the cell to said construct in the presence and absence of the agent; (iv) determining the presence of amount of the construct in the ER or cytoplasm of the cell; (v) correlating a reduction in the presence or amount of the construct in the cytoplasm of the cell with the ability of the agent to inhibit or block the translocation of an effector peptide of the pathogen across the membrane of the cell.

Examples of suitable agents are agents that are either: (i) intended to compete with residues) on the cell surface; (ii) an aryl-O-sulfate moiety mimetic intended to compete with the aryl-O-sulfate moieties on the cell surface for binding the construct; (iii) intended to specifically remove the sulfate groups from aryl-O-sulfate moities on cell surface molecules (e.g. the tyrosine-O-sulfate residues from cell surface proteins); or (iv) specifically prevent the addition of sulfate groups to the aryl moieties of cell surface molecules (e.g. tyrosine residues of cell surface proteins). The agent may be a peptide or protein provided by expression from nucleic acid introduced into the plant cell.

Treatment with desulfating agents

In one aspect the invention provides an agent which inhibits binding of an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22'147 or SpHtpl ^{2 198} shown in Figure 14 to a surface molecule having an aryl-O-sulfate moiety (e.g. a protein having a tyrosine-O-sulfate residue) for use in a method of treatment of the human or animal body. The method of treatment may be of a disorder associated with pathogenic cell invasion.

Also provided is the use of an agent which inhibits binding of amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl^24" ¹⁹⁸ shown in Figure 14 to a surface molecule having an aryl-O-sulfate moiety (e.g. a protein having a tyrosine-O-sulfate residue) for use in the manufacture of a medicament for the treatment of a disorder associated with pathogenic cell invasion. The present invention also encompasses a method of treating a disorder associated with pathogenic cell invasion comprising administering to a patient in need thereof an effective amount of an agent which inhibits binding of an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl^24"198 shown in Figure 14 to a surface molecule having an aryl-O-sulfate moiety (e.g. a protein having a tyrosine-O-sulfate residue).

Production of transgenic plants

As would be apparent to the skilled person, a transgenic plant may be produced by creating a construct bearing a nucleic acid capable of directing the expression of the required agent, or bearing the required genetic modification. The host plant cell may established within the cell. The heterologous nucleic acids may recombine with the host cell genome.

Nucleic acid can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-01 16718, NAR 12(22) 871 1 - 87215 1984), particle or microprojectile bombardment (US 5100792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green ef al. (1987) Plant Tissue and Cell Culture, Academic Press), electroporation (EP 290395, WO 8706614 Gelvin Debeyser) other forms of direct DNA uptake (DE 4005152, WO 9012096, US 46846 ), liposome mediated DNA uptake (e.g. Freeman er a/. Plant Cell Physiol. 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U.S.A. 87: 1228 (1990d) Physical methods for the transformation of plant cells are reviewed in Oard, 1991 , Biotech. Adv. 9: 1-1 1.

Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has also been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (see e.g. Hiei ef al. (1994) The Plant Journal 6, 271-282)). Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium alone is inefficient or ineffective. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the

transformation process, eg bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co- cultivation with Agrobacterium (EP-A-486233).

It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.

Thus a further aspect of the present invention provides a method of transforming a plant cell involving introduction of a nucleic acid as described herein into a plant cell and causing or allowing recombination between the nucleic acid and the plant cell genome to introduce the nucleic acid into the genome.

The invention further comprises a plant host cell, optionally present in a plant, having amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147,

AVR3a^22"147 or SpHtpl ^24-198 shown in Figure 14, wherein said plant host cell comprises (i) a exogenous nucleic acid capable of directing the expression of a desulfating agent or an agent that inhibits the binding of aryl-O-sulfate as described herein; and/or (ii) a genetic modification as described above. The desulfating agent or the agent that inhibits the binding of aryl-O-sulfate may either (i) bind to or comprises an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence

AVR3a^60"147, AVR3a^22'147 or SpHtpl^24"198 shown in Figure 14, or (ii) reduce the number or concentration of sulfate groups on aryl moieties of cell surface molecules (e.g. tyrosine residues on cell surface proteins). In some embodiments the desulfating agent or an agent that inhibits the binding of aryl-O-sulfate is secreted to the extracellular compartment of the cell.

Detailed description of the invention

Overview

As described above, the previously accepted working model and hypothesis was that translocation of proteins such AVR3a from P. infestans was directly mediated by the RxLR-motif. Accordingly, the initial aim of the current study was to use the RxLR- effector AVR3a from the potato late blight pathogen Phytophthora infestans was to unravel some unknown aspects of the host targeting process. However, the results of the current study indicate that the previous model and hypothesesis are incorrect.

The current study demonstrates that AVR3a binds to the surface of plant, fish and human cells. Uptake of AVR3a is autonomously and does not require any pathogen- encoded machinery. Although AVR3a and other RxLR-effectors bind to PIP3 and PIP4, interaction with these phospho-lipids does not appear to be essential for the translocation process.

Instead all tested RxLR effector proteins were found to bind tyrosine-O-sulfate, which is a common post-translational modification of solvent-exposed and extracellular proteins. Binding and uptake of AVR3a and other RxLR-effectors into cells was significantly reduced after removing the sulfate groups with aryl-sulfatase.

Furthermore, binding to the sulfated tyrosines and the uptake of AVR3a is leader peptide, although the presence of a RxLR leader sequence comprising the RxLR motif increases the efficiency of protein translocation.

No bona fide evidence was obtained to substantiate previously made claims that the RxLR-motif possesses the ability for self-translocation. In fact, recombinant proteins whereby the RxLR-leader sequence is fused to mRFP or a His-tag are unable to translocate autonomously. Interestingly, it was discovered that the RxLR-leader sequence functions as a dimerisation site. The peptide region that includes the RxLR motif (the RxLR leader sequence) is able to form a heteromeric complex with another RxLR-protein (SpHtpl) and RxLR-containing constructs, enabling translocation of the latter. For example, the RxLR-mRFP fusion protein can only translocate into cells when AVR3a containing the effector domain is added together with the fusion protein, suggesting that AVR3a can act as a shuttle molecule for other RxLR proteins via heteromerisations through the RxLR leader peptide.

Each of these results is discussed in more detail below and in the "Examples" section.

The invention

It is herein demonstrated that the initial step in the translocation process that directs the P. infestans RxLR-effector AVR3a across the cell membrane is achieved by the binding of the effector domain to a presumably conserved cell surface protein carrying a tyrosine-O-sulfation site. The uptake does not require any pathogen encoded machinery. Furthermore, the observation that the amino acids in the location of the RxLR-EER sequence encode for a dimerisation motif, which is able to interact with a randomly chosen RxLR protein from a fish pathogenic oomycete raises the question about a potential interplay of RxLR effectors via the RxLR-leader sequence.

Despite the different origin of the RxLR effectors SpHtpl , AVR3a and the PEXEL effector PfGBP130, the present inventors observed in immuno-localisation experiments that AVR3a and the PfGBP130 possess an affinity to Eukaryotic cell surfaces (Figure 1/2/8) comparable to the previously reported SpHtpl [van West ef al. submitted]. This phenomenon was investigated in more detail using different constructs of the P. infestans AVR3a elicitor. Interestingly, the binding and cellular uptake of AVR3a is not mediated by the RxLR leader sequence but is encoded within sequence (Figure 1/8). Furthermore, this protein is able to interact with cells derived from onion, trout and human and, if mRFP fusion constructs are utilised, is clearly visible in fish and human cells after a 30 minute incubation time using relatively low amounts of the recombinant proteins.

Dou et al. (2008) described that the RxLR-EER motif of the P. sojae homolog of AVR3a, AVR1 , fused to GFP, is able to direct this recombinant protein into soybean root- and onion cells (Dou et al., 2008b). However, these results were obtained with solutions containing protein concentrations 63 times higher (8 mg/ml) than those used in this study and up to 24x longer incubation times (12 h). Therefore, the present inventors believe that these results are likely attributable to a nonspecific protein uptake of cells exposed to a highly saturated protein solution.

In general, the first step of the AVR3a uptake into cells must be the ability of this protein to bind to a molecule on the outer surface. Since different cell types can be targeted by AVR3a it either has to interact with a common lipid, a conserved surface protein or a common glycoside/glycoprotein. In the process of deciphering how the RxLR proteins attach to eukaryotic cell membranes, the present inventors identified a subset of phospholipids that interact with AVR3a, SpHtpl and PfGBP130

(Figure 11a). However, the identified phospholipids are in general present on the inner membranes under normal growth conditions (Ardail et al., 1990; Balla et al., 2008; Clague et al., 2009; Gillooly et al., 2000; Gozani et al., 2003; Lemmon, 2008; Munnik and Testerink, 2009; Pettitt et al., 2006; Sorice et al., 2009; Vermeer et al., 2009) and in the light of the controls described here, lipid binding of RxLR effectors is likely to be irrelevant for their translocation into the cell. This conclusion is based on our results showing that: i) treatment of cells with aryl-sulfatase, which does not dephosphorylate phospholipids (Davolio et al., 986), significantly reduces the intake of the tested RxLR protein constructs; ii) urea denatured AVR3a^{22 174}(His)₆ shows the same phospholipid binding pattern as the native protein on lipid spot membranes (Figure 11a2M 1b) whilst interaction is tost when the charged denaturant Gnd-HCI is used (Fig. 11c); iii) sub-stoichometric amounts of ADP bound DnaK are sufficient to suppress the lipid binding of native AVR3a^{22' 7} (His)₆ (Figure 11e). This suggests that the phospholipid binding properties of the tested RxLR effectors is accomplished by a (sub)-fraction of partially unfolded protein molecules; and (iv) no interaction between AVR3a^22"17 (His)₆ and inositol-1 ,4-bisphosphate head group could be observed in isothermal titration experiments (Fig. 12A). The above results indicate that the interaction with the phospholipids is most likely based on charge-charge (electrostatic) interactions of the RxLR-proteins to a phosphorylated inositol head group and seems to require a specific spacing of presumably charged residues within the amino acid sequence (Figure 11 d,c).

Furthermore, all lipids identified are usually located on the inside of eukaryotic cells. Cardiolipin is found in the inner and outer membrane of the mitochondria (Ardail et al., 1990; Sorice et al., 2009) whereas Ptdlns(3)P is located in early endosomes (Clague et al., 2009; Gillooly et al., 2000). Pidlns(4)P appears in mammalian cells predominantly in the Golgi (Balla et al., 2008; Lemmon, 2008) and cytoplasmatic expression of Ptdlns(4)P binding proteins in planta labelled the plasma membrane as well suggesting its presence on the inner surface of the plasma membrane (Munnik and Testerink, 2009; Vermeer et al., 2009). For Ptdlns(5)P it is still unclear but is probably nuclear localised (Gozani et al., 2003; Pettitt et al., 2006). Certainly, these lipids become exposed to the outer surface of cells if these are aged (Connor et al., 1994) or apoptotic (Fadok et al., 2000; Fadok et al., 1992). Once inside the cell the ability of RxLR proteins to bind this specific set of phospholipids could perhaps become important for intracellular trafficking or function.

Even though the phospholipid binding of RxLR proteins could be artificial, the fact that all tested effectors bind to the same lipid subset demonstrates that they must share a common property. A first glimpse of the origin of this property came from the observation that AVR3a^{22" 7}(His)₆ is stabilised by phosphate ions (Figure 4A,C). Interestingly, the present inventors discovered that the chemically and structurally similar double-charged sulfate ion is an even more efficient stabilising agent

(Figure 4B,C). Thus, the present inventors hypothesised that protein tyrosine sulfation, a common post-translational modification (PTM) of cell surface proteins (Chang et al., 2009), might play a role in the cell surface interaction of AVR3a.

Consistent with a role for a tyrosine-O-sulfate interaction, AVR3a^{2 "147}(His)₆ is able to interact with Fmoc-Tyr(S0₃)-OH and H-Tyr(S0₃)-OH in a way that results in aggregation of this protein (Figure 4D/12F). In contrast the A R3a^{22 147}-mRFP fusion protein did not show any precipitation. However, isothermal titration experiments utilising this construct and Fmoc-Tyr(S0₃)-OH as a titrant resulted in complex thermogrammes suggesting that the interaction is accompanied by large structural changes within the protein (Figure 12D). Thus, it seems that the fused mRFP is only tipping the balance away from the aggregation reaction by permitting a higher but, in contrast to AVR3a^22'147(His)_6l the structural changes upon the interaction are less pronounced and allowed a thermodynamic characterisation of this interaction (Figure 12C).

At present, data on the biological role of tyrosine sulfation in proteins are scarce. However, this modification is thought to be important for cell surface ligand-receptor interactions especially in pathogen host interactions (Choe et al., 2005; Farzan et al., 1999). To our knowledge there are no thermodynamic data available describing the interaction of tyrosine-O-sulfate to proteins recognising this PTM. Therefore, the parameters measured for the SpHtp1^{24 1 8}(His)_s - Fmoc-Tyr(S0₃)-OH interaction represent the first thermodynamic data for this type of interaction. In addition, the specificity and strength of the interaction with Fmoc-Tyr(S0₃)-OH only represents one site of a larger protein-protein binding interface. Nevertheless, this PTM is one important feature that seems to influence the translocation of the RxLR effectors AVR3a and SpHtpl , since the aryl-sulfatase treatment of the cell surface protein continuum strongly reduced their binding and uptake (Figure 5/6/13). In addition, this effect was reversed if the cells were allowed to recover after the aryl-sulfatase treatment (Figure 13H).

Whisson et al. (2007) provided strong evidence that the translocation of the AVR3a elicitor from the P. infestans parasite into potato leaf cells is dependent on the presence of the RxLR-EER motif when they showed that translocation of AVR3a relied on an intact RxLR-motif in order to trigger a hypersensitive response (HR). They also showed that P. infestans transformants carrying alanine replacements within the conserved amino acids of the AVR3a RxLR-EER sequence failed to deliver the protein and were not able to trigger an HR in plants expressing the R3A resistance gene. Moreover, the fusion of the AVR3a RxLR leader sequence (amino acids 22-59) to the non-RxLR protein GUS enabled transformants to deliver this fusion constructs into potato cells (Whisson et al., 2007). Later on Grouffaud ef al. (2009) used P. infestans transformants to show that the RxLR-motifs from different oomycetes were functionally interchangeable with the Plasmodium PEXEL motif of the HRPII when they were N-terminally fused to the effector domain of AVR3a (Grouffaud et al., 2008). However, the present inventors conclude from the present study that only the AVR3a effector domain is necessary for the translocation of the protein and not the RxLR leader sequence. These seemingly contradictory findings are brought into agreement by the novel discovery that the sequence in the area of the RxLR-motif harbours a dimerisation site. The A R3a^22'147(His)₆ construct is clearly a dimeric protein whereas the construct lacking the RxLR peptide is monomeric (Figure 9A,B). When the RxLR leader sequence (amino acids 22-59) of AVR3a, which includes the RxLR motif, is fused to a His-tag this construct is able to form a tetrameric complex, however, this was not observed with AVR3a^{22 147}(His)₆ (Figure 9A,C). This suggests that the oligomerisation ability of the RxLR fragment is restricted in the full-length protein, and therefore, the folding of this amino acid stretch is dependent on the effector domain of AVR3a.

Compared to the experimental system presented here, Whisson et al. and Grouffaud et al. both relied on transgenic P. infestans strains, which contain an endogenous AVR3a protein. This wild type AVR3a protein contains the same RxLR leader sequence as was used in the RxLR-GUS fusion construct. Here the present inventors have demonstrated that the RxLR leader sequence of AVR3a fused to mRFP is not taken up by HEK293 and RTG2 cells and does not bind to onion cells (Figure 1/3/8/10). However, this construct is translocated in the presence of a His- tagged AVR3a^{22 47} carrying both the RxLR-motif and the effector domain

(Figure 3D, G, J; 10B, E). Moreover, SpHtp1^{2 '198}(His)₆ is also capable to interact with AVR3a^{22 59}-mRFP(His)₆ and is also able to permit an affinity towards onion, fish and human cells and deliver this protein into RTG2 and HEK293 cells

(Figure 3E,H,K; 10C.F). Consequently, the present inventors conclude that the observations of Whisson et al. are attributable to the production of a full length endogenous AVR3a by their P. infestans strain. The latter probably forms mixed dimers with the RxLR-GUS fusion, enabling this construct to be translocated into the cells. Theoretically this is possible since the present inventors show that AVR3a^22"59- mRFP(His)₆ forms heteromers with either SpHtp1^{24 198}(His)₆ or AVR3a²²-^{1 7}(His)₆ (Figure 3 J,K).

Mutations within the RxLR motif

The ability of the RxLR leader sequence to form dimers explains the earlier observations that RxLR-GUS fusions are able to translocate (when expressed in a background containing a translocation-competent RxLR protein such as AVR3a or SpHtpl). However, this explanation does not fully account for the observations of Whisson et al. that an intact RxLR motif is required for AVR3a to trigger a To investigate this further, the present inventors generated His-tagged protein versions of the AVR3a amino acids 22-59 containing the 'KMIK' and Ά5' mutations and compared those peptides to the WT polypeptide. Both RxLR mutants

AVR3a^{22 59}(His)₆ KMIK and A5 had very similar CD-spectra but compared to the WT polypeptide showed a larger random structural content (Figure 15B). No significant difference could be observed when the CD-spectrum of the homolog mutated peptide AVR3a^{22 59}(His)₆ KMIK was compared to the on carrying an alanine replacement at the same position.

Interestingly, these mutations did not abolish the property of this amino acid stretch to form dimers but had a lower propensity to form the tetrameric state (Figure 15C,D,E). This indicates that the mutants possess different dynamic properties then the wild type polypeptide.

To further analyse these mutants the present inventors performed reconstitution experiments with the AVR3a construct containing only the effector domain

AVR3a^60"147(His)₆. These experiments made use of the observation that the full length construct AVR3a^{22 1 7}(His)₆ strongly aggregates in the absence of phosphate ions, whereas the stability and tendency to aggregate of the effector domain alone (AVR3a^{eo' 47}(His)₆) is not effected by phosphate. Mixing of the WT AVR3a^22'59(His)₈ peptide with AVR3a^60'147(His)₆ resulted in an increased aggregation reaction which could be partially suppressed by high concentrations of phosphate (Figure 16A). The conclusion of these observations is that both protein fragments must be able to interact with one another.

The fact that the aggregation process is not completely suppressed by phosphate indicates that the non-covalent complex formed by AVR3a^{22 59}(His)₆ and

AVR3a^60"147(His)₆ does not have the same properties as the covalent complex of the same peptides (AVR3a^22"147(His)₅). Normalised CD-spectra of AVR3a^22"147(His)₆, AVR3a⁶⁰-¹⁴⁷(His)₆ and a mix of AVR3a⁶°-¹⁴⁷(His)₆ with AVR3a^22'59(His)₆ indicate that the interaction between AVR3a⁶°-¹⁴⁷(His)₆ with AVR3a^22'59(His)₆ does not involve large secondary structural rearrangements and is therefore most likely attributable to tertiary interactions (Figure 16E). These observations are in agreement with the finding that the stabilisation effect of phosphate on AVR3a²²~¹⁴⁷(His)₆ is due to a stabilisation on a secondary structural level but does not affect the dimerisation of the However, the aggregation process taking place in a mix of AVR3a^{60 147}(His)₆ and AVR3a^22'59(His)₆ prevents an exact secondary structural determination since the spectra can only be recorded immediately after mixing and thus slow conformational rearrangements are not observable.

In contrast to the WT RxLR peptide, addition to AVR3a^60'147(His)₆ of either of the KMIK or A5 variants of AVR3a^{22 59}(His)₆ is unable to even partially restore the phosphate dependency property seen in AVR3a^{22 147}(His)₆ (Figure 16B). Thus, it appears the KMIK and A5 variants are not able to interact with the effector domain of AVR3a.

The above result is consistent with the results of ITC titration using the AVR3a fragments. In these assays the addition of the WT AVR3a^22'59(His)₆ to

AVR3a^{60"1 7}(His)₆ resulted in an endothermic reaction; a control titration of this peptide into dialysis buffer gave an exothermic reaction, most likely caused by the dilution heat of the peptide (Figure 16C). In contrast, the addition of

AVR3a^{22 59}(His)₆ KMIK to AVR3a⁶°-¹⁴⁷(His)₆ did not give the endothermic profile seen with the WT RxLR sequence, instead giving an exothermic profile matching that seen in the control titration (Figure 16D). This result indicates that the mutations within the tetrameric RxLR motif of AVR3a described by Whisson et al. (2007) diminish the interaction of the AVR3a RxLR leader sequence (amino acids 22-59) to the effector domain of this protein.

An interaction between the RxLR leader sequence and the effector domain in SpHtpl could explain why the deletion of the RxLR leader sequence in SpHtpl abolishes translocation if it is assumed the RxLR/effector interaction is of higher structural relevance in SpHtpl than in AVR3a. This theory is supported by the observation that the recombinant SpHtpl protein construct that lacks the RxLR-leader has a significantly altered secondary structure compared to that of the construct containing this amino acid stretch [van West ei al. submitted]. In case of AVR3a, the deletion of the RxLR-EER motif leads to only relatively minor structural differences (e.g. seen in Figure 16E), possibly explaining why this protein retains a translocation ability without the RxLR leader sequence, albeit diminished relative to the 'full-length' AVR3a AVR3a^22"147.

Returning to the observations of Whisson et al. (2007) that an intact RxLR motif is explained by the above. Whilst, as the present inventors have shown, mutation within the RXLR-EER does not prevent AVR3a from entering cells, it does reduce the efficiency of the uptake (Figure 19A 4). In 2006 Bos et al. (2006) it is shown that a certain threshold concentration of AVR3a protein is required to trigger an HR in susceptible plants. Therefore, a possible explanation of the results observed by Whisson et al. is that whilst the KKMIK-DDK AVR3a variant was able to self- translocate, it was not able to accumulate beyond the critical threshold required to trigger a hypersensitive reaction (HR). Thus no HR was observed under the conditions employed by Whisson et al. (2007) using transformant assays.

A partially unfolded transition state may be involved in AVR3a translocation

The present inventors have shown that AVR3a binds to a cell surface protein carrying a tyrosine-O-sulfate, a specific extracellular PTM. In vitro] AVR3a^{22"1 7}(His)₆ interacts with H-Tyr(S0₃)-OH in a way that results in the aggregation of the protein. Isothermal titration measurements using Fmoc-Tyr(S0₃)-OH and AVR3a^{22 1 7}(His)₆ indicates that the binding involves larger structural rearrangements of the protein .

Also described herein is how AVR3a^{22 1 7}(His)₆ is capable of adopting at least three different stable structures in vitro. At a protein concentration of ~ 10 μΜ an a-helical conformation with a high melting point over 0°C is present (Figure 18A.B). If the protein concentration in solution is ~250 μΜ a second a-helical state is observed characterised by an unfolding transition temperature of ~32°C (Figure 18B,C,D). At present it is not clear if the observed change in the temperature stability of

AVR3a^{22"1 7}(His)₆ is caused by a shift in the monomer-dimer equilibrium with the dimer as the predominant form, but the exponential decay of the melting temperature with increasing protein concentration could suggest such a model. A third soluble state was observed at protein concentrations of -1.2 mM that shows characteristics of a native unfolded conformation (Figure 18D red cross peaks). According to the law of mass action these states have to be always in equilibrium; nonetheless, the probability of adopting one of these conformations is dependent on the environmental conditions (e.g. protein concentration).

The present inventors investigated how the biological function of AVR3a^{2 ' 7}(His)₆ may be linked to the structural state of the protein. It was reasoned that if binding of AVR3a^{22 147}(His)₆ to the cell surface was linked to large structural changes within the protein there may be a partially unfolded 'transition state'. If this was the case, the unfolded proteins such as ADP-bound DnaK (the high affinity state); these agents should be able to influence the uptake and therefore the translocation of the protein. This effect was studied via life cell imaging using the previously described mRFP tagged versions of AVR3a, AVR3a^{22 147}-mRFP(His)₆ and AVR3a⁶°-¹⁴⁷-mRFP(His)₆ . In both cases, substoichometric amounts of DnaK strongly reduced the uptake of the mRFP fluorescence which supports the hypothesis that AVR3a partially unfolds upon binding to its target on the cell surface (Figure 19A, 21).

In order to understand in more detail how binding and translocation of AVR3a is linked to the conformational equilibrium of the protein, a way was needed to alter the protein structure without changing the protein concentration so as to obtain comparable results. In theory, the half life of protein structures is dependent on the environment the polypeptide chain (Pauling, 1939 (first edition)). However, it is not yet technically feasible to change the environment in a way the significantly changes the structural equilibrium of AVR3a but does not deleteriously affect the cells being monitored. Accordingly, the present inventors decided to change the structural equilibrium of AVR3a^{22"1 7}(His)₆ by unfolding the protein in 6 M urea and then initiating the refolding of the protein in the presence of the cells. Using this approach the assayed cells would be exposed to an amount of urea that did not alter the translocation of non-denatured protein and more importantly was acceptable for the survival of the cells themselves.

TEM immuno-gold stained sections of either HEK293 or RTG2 cells that were exposed to refolding AVR3a^{2 47}(His)₆ showed gold particles mainly in the nuclei of these cells (Figure 19B1.2). There, the gold particles formed conglomerates, which could occasionally be observed in the cytosol of these cells and implies a vesicular transport of the protein under these conditions. However, due to the lack of ultra- structural preservation resulting from the preparation method, cellular membranes are not clearly visible and thus the theory remains unproven. Since these results are distinct from the observation made using "native" AVR3a^{22"1 7}(His)₆, these

experiments indicate that at least one translocated conformation of AVR3a is close to the unfolding branch of the AVR3a folding pathway. The different localisation also means that all of the different uptake mechanisms should be studied when comparing "native" folded AVR3a with AVR3a in the process of refolding.

In contrast to AVR3a^22"147(His)₆, the presence of the mRFP enables optical imaging of the molecule to unfold (Broering and Bommarius, 2008). Interestingly, the results obtained using the mRFP construct AVR3a^22'147-mRFP(His)₆ differed to those described above for the (His)₆ construct. On exposing RTG2 cells to 6 urea treated AVR3a²²-^{1 7}-mRFP(His)₆ (1 M Urea final concentration on the cells), no red

fluorescence in the nuclei of the cells could be observed (Figure 19A3, 21 -3). The fluorescence localisation pattern appeared similar to non-urea treated AVR3a^22"147- mRFP(His)₆ but had a clearly weaker intensity. Hence, it appears that the presence of the folded mRFP either seems to block the nuclear uptake or vastly accelerates the refolding kinetics of AVR3a^22'147 domain. Nevertheless, it can be concluded that if a nuclear localisation of AVR3a is of biological importance, a protein unfolding mechanism for the translocation of the protein into the nucleus must be involved.

Definitions

Compositions and formulations

The agents, compositions and multimers of the invention may be provided in substantially isolated form, e.g. free or substantially free of material with which they are associated with in a host cell used for their production.

The agents, compositions and multimers of the invention may be in the form of a salt, particularly a pharmaceutically acceptable salt. These include basic salts, such as an alkali or alkaline earth metal salt, e.g. a sodium, potassium, calcium or magnesium salt. The salt may also be an acid addition salt such as those formed with

hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. A potassium salt is preferred.

The agents, compositions and multimers of the invention may be prepared in the form of a pharmaceutical composition. The composition may be in the form of a liquid, gel or solid. Administration of the agents, compositions and multimers of the invention the invention will depend on the nature of the molecule and the host.

If the host organism is a plant, application is generally in the form of a foliar spray or molecule in a concentration sufficient to block effector molecules of pathogens, which are likely to attack the plant. The compositions may include one or more than one blocking molecule. For example, a preparation for application to plants may include molecules that block the effector proteins of one or of several different types of pathogen. In addition, the blocking molecules may be administered to plants in conjunction with other beneficial substances, such as fertilizers, various pesticides, growth factors, etc. Administration methods that are particularly suitable for animal subjects are described below.

Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. Oral, nasal and topical administration may include administration by way of aerosols.

Topical formulations may also be present in the form of creams, ointments or gels, depending upon the site of intended use. Topical compositions of the invention may be in any pharmaceutical form normally used for topical application, in particular in the form of an aqueous, aqueous-alcoholic or oily solution, an oil-in-water or water-in- oil or multiple emulsion, an aqueous or oily gel, a liquid, pasty or solid anhydrous product. The composition may also contain the usual adjuvants in the cosmetics and dermatological fields, such as one or more of a hydrophilic or lipophilic gelling agent, hydrophilic or lipophilic active agent, preserving agent and antioxidant. When the composition of the invention is an emulsion, the proportion of the fatty phase can range from 5 to 80% by weight, and preferably from 5 to 50% by weight, relative to the total weight of the composition. The oils, the emulsifiers and the co-emulsifiers used in the composition in emulsion form are chosen from those used conventionally in the field considered. The emulsifier and the coemulsifier are present in the composition in a proportion ranging from 0.3 to 30% by weight, and preferably from 0.5 to 20% by weight, relative to the total weight of the composition.

Oils which can be used include mineral oils (liquid petroleum jelly), oils of plant origin (avocado oil, soybean oil), oils of animal origin (lanolin), synthetic oils

(perhydrosqualene), silicone oils (cyclomethicone) and fluoro oils

(perfluoropolyethers). Fatty alcohols (cetyl alcohol) fatty acids and waxes (carnauba wax, ozokerite) can also be used as fatty substances. Emulsifiers and co-emulsifiers which can be used include, for example, of fatty acid esters of polyethylene glycol, such as PEG 20 stearate, and fatty acid esters of glycerol, such as glyceryl stearate.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

For solid compositions, conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium carbonate, and the like may be used. The active compound as defined above may be formulated as suppositories using, for example, polyalkylene glycols, acetylated triglycerides and the like, as the carrier. Liquid pharmaceutically administrate compositions can, for example, be prepared by dissolving, dispersing, etc, an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see "Remington: The Science and Practice of Pharmacy", 20th Edition, 2000, pub.

Lippincott, Williams & Wilkins. The composition or formulation to be administered will, in any event, contain a quantity of the active compound(s) in an amount effective to alleviate the symptoms of the subject being treated.

For oral administration, a pharmaceutically acceptable non-toxic composition is formed by the incorporation of any of the normally employed excipients, such as, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, sodium crosscarmellose, starch, magnesium stearate, sodium saccharin, talcum, form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations and the like.

Parenteral administration is generally characterized by injection, either

subcutaneously, intramuscularly or intravenously. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, triethanolamine sodium acetate, etc.

Another approach for parenteral administration employs the implantation of a slow- release or sustained-release system, such that a constant level of dosage is maintained. See, e.g., US Patent No. 3,710,795.

Dosage forms or compositions containing active ingredient in the range of 0.1 to 95% with the balance made up from non-toxic carrier may be prepared. Preferably, percentages of active ingredient of 0.1 % to 50% in solution are employable.

Compositions of the agents, compositions and multimers of the invention may also comprise a second active agent, including a different polypeptide of the invention including those described herein, a different antibacterial agent, or another agent intended to treat a second symptom or cause of a condition to be treated.

Eukaryotic cell

The Eukaryotic cells defined herein may be plant cells or animal cells. The animal cells may be human, or may be non-human. For example, the cells may be from humans, cattle, sheep, pigs, goats, horses, cats, dogs, chickens, turkeys, bees, salmon, trout, bass, catfish, shellfish, crayfish, lobsters, shrimp or crabs.

Example plant cells include cell from wheat, maize, rice, sorghum, barley, oats, millet, soybean, common bean (e.g. Phaseolus species), green pea (Pisum species), cowpea, chickpea, alfalfa, clover, tomato, potato, tobacco, pepper, egg plant, grape, strawberry, raspberry, cranberry, blueberry, blackberry, hops, walnut, apple, peach, passionfruit, coconut, date and oil palm, citrus, safflower, carrot, sesame, common bean, banana, citrus (e.g. orange, lemon, grapefruit), papaya, macadamia, guava, pomegranate, pecan, Brassica species (canola, cabbage, cauliflower, mustard etc), cucurbits (pumpkin, cantaloupe, squash, zucchini, melons etc), cotton, sugar cane, sugar beets, sunflower, lettuce, onion, garlic, ornamental cut flowers, grasses used in lawns, athletic fields, golf courses and pastures (e.g. Festuca, Lolium, Zoysia, Agrostis, Cynodon, Dactylis, Phleum, Phalaris, Poa, Bromua and Agropyron species), etc.

The nature of the Eukaryotic cell that is targeted for, for example, desulfation or the translocation of a protein comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl ^24-198 shown in Figure 14 depends on the aspect of the invention. The cell may be healthy or diseased. For example, in aspects where the amino acid sequence motif is coupled to a cytotoxin the targeted Eukaryotic cell may be a diseased or pathogenic cell e.g. a cancer cell. In contrast, when the amino acid sequence motif is coupled to a protective agent the targeted Eukaryotic cell may be a healthy cell.

Percentage Identity

As used herein, the term "percentage sequence identity" refers to identity as measure over the entire length of the SEQ ID in question.

For example, a polypeptide comprising a sequence motif having 70% sequence identity to SEQ ID NO: 1 would contain a contiguous polypeptide where:

(Number of amino acids identical to SEQ ID NO 1) / Total number of amino acids in SEQ ID NO 1 = 0.7

The percent identity of two amino acid or two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or more preferably, the comparison is done by comparing sequence information using a computer program. An exemplary, preferred computer program is the Genetics Computer Group (GCG; Madison, Wis.) Wisconsin package version 10.0 program, 'GAP' (Devereux et al., 1984, Nucl. Acids Res. 12: 387). The preferred default parameters for the 'GAP' program includes: (1) The GCG implementation of a unary comparison and the weighted amino acid comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745,1986, as described by Schwartz and Dayhoff, eds., Atlas of Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; or other comparable comparison matrices; (2) a penalty of 30 for each gap and an additional penalty of 1 for each symbol in each gap for amino acid sequences, or penalty of 50 for each gap and an additional penalty of 3 for each symbol in each gap for nucleotide sequences; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps.

While the invention has been described in conjunction with the exemplary

embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

Some particular embodiments of the invention will now be discussed.

Figures Figure 1.

Localisation of the AVR3a-mRFP fusion constructs AVR3a^{22'1 7}-mRFP(His)₆, AVR3a^{60 7}-mRFP(His)₆ and AVR3a" ⁵⁹-mRFP(His)₆ on living onion-, RTG2 and HEK293 cells using confocal microscopy.

The mRFP fusions of AVR3a containing the C-terminal effector domain (AA60-147) are able to bind to onion cells (A,B), to the cells of the fish cell line RTG2 (D,E) and to the human HEK293 cells (G,H). Both protein constructs show identical localisation patterns on the respective cell types and an uptake into the cells is clearly visible with RTG2 and HEK293 cells. Compared to AVR3a²²-^{1 7}-mRFP(His)₆, AVR3a^60'147- mRFP(His)₆ shows a weaker binding/uptake signal. The mRFP fusion AVR3a^22"59- mRFP(His)₆ carrying only the RxLR-EER motif and mRFP does not show any affinity to any of the chosen cell types (C,F,I). J (and K) show magnified images of RTG2 (HEK293) cells incubated for 30 min with AVR3a^22'147-mRFP(His)₆. These images clearly show that in addition to the outer surface binding an accumulation of red fluorescence dots appear around the nucleus in the cells. The RTG2 monolayers were incubated for 30 min at RT in L15-media (+10 % FCS), the HEK293

monolayers for 30 min at 37°C in DMEM-media (+ 10 % FCS) and the onion cells for 1 h at RT in PBS with either 3 μΜ AVR3a ^{2 M7}-mRFP(His)_6l 3 μΜ AVR3a^60"147- mRFP(His)₆ or 15 μ AVR3a^22'59-mRFP(His)₆. Red Chanel: mRFP fluorescence; white channel: DIC.

Figure 2.

A) Immuno-gold staining of high pressure frozen HEK293 cells incubated with 20 μΜ of either AVR3a^{22 147}(His)₆ or AVR3a^{22 147}(His)₆.

Immuno-staining was carried out as described in material and methods. Clusters of gold particles (010 nm) visible on the AVR3a ^{2 147}(His)₅ and AVR3a^{2 '147}(His)₆ treated cells are indicated with arrows. The samples were prepared without uranyl-acetate in the substitution solution and often showed gold labelling close to the mitochondrial membrane (A,B). Very few gold particles were detected in the BSA treated controls with this method (C). Cy indicates the cytosol, t are mitochondria, E is the extracellular space and the asterisks (*) indicate the plasma membrane.

B) Co-localisation of AVR3a^{22 U7}-mRFP(His)₆ and AVR3a^{6(M 7}-mRFP(His)₆ with the mitochondrial marker MitoTracker green FM (500 nM). The mRFP fluorescence partially overlaps (yellow areas) with the fluorescence of the mitochondrial specific dye. Green channel: MitoTracker green FM fluorescence; red channel: mRFP fluorescence; white channel: DIC.

Figure 3.

A: Coomassie blue stained native PAGE showing:

AVR3a^{22 59}-mRFP(His)₆ (lane 1); SpHtpl ²⁴-¹⁹⁸(His)₆ (lane 2); a mix of both protein constructs (lane 3). In the lane showing the mixed sample one species of

SpHtpl ^24'193(His)₆ has disappeared (a) and a new species appeared (b), thus showing a direct protein-protein interaction between both constructs.

B: unstained native PAGE loaded with:

AVR3a²²-⁵⁹-mRFP(His)₆ (lane 3); AVR3a^{22 147}(His)₆ (lane 2); SpHtp1²⁴- ⁹⁸(His)₆ (lane 4); mixture of AVR3a²²-⁵⁹-mRFP(His)₆/AVR3a²²-¹⁴⁷(His)₆ (lane 1); mixture of

AVR3a^22'59-mRFP(His)₆/SpHtp1^{2 198}(His)₆ (lane 5).

The two red fluorescent bands of the samples containing AVR3a^{22 59}-mRFP(His)₆ are indicated with c,d. The letter e in lane 3 highlights the adhesion of the loading dye bromphenol blue to AVR3a^{22 59}-mRFP(His)₆ which is not present in the lanes (1 ,5) where this protein is mixed with the non-mRFP constructs.

The mixed samples contained 42.8 μΜ of either SpHtpl ^24'198(His)₆ or

AVR3a^22'147(His)₆ and 80 μΜ of AVR3a ^{2 59}-mRFP(His)₆ and were incubated for 30 min on ice prior to loading the gel. The native PAGE was loaded using following protein amounts: 18.2 pg SpHtp1^{24 8}(His)₆, 14.4 pg AVR3a^22"147(His)₆, 36.4 pg AVR3a^{2 '59}-mRFP(His)₆, 54.6 pg AVR3a ^2"59-mRFP(His)₆/SpHtp1²⁴-¹⁹⁸(His)₆ and 50.8 pg AVR3a^22'59-mRFP(His)₆/ AVR3a^{22'1 7}(His)₆.

C-K: Mixed heteromerisation of AVR3a^{22 59}-mRFP(His)₆ with AVR3a^{22 l47}(His)₆ and SpHtpl ^24'198(His)₆ enables the mRFP construct to bind onion cells and bind/enter RTG2- and HEK293 cells.

The cells were incubated for 1 h with 15 μΜ of AVR3a^22"59-mRFP(His)₆ alone or in combination with 50 μΜ of the non-mRFP proteins AVR3a^{22 "147}(His)₆ and

SpHtpl ^{24 198}(His)₆. Onion cells were incubated in PBS at RT, RTG2 cells in U S- media containing 10 % FCS at RT and HEK293 cells were incubated in DMEM- media containing 10 % FCS at 37°C. Subsequently the cells were washed 5x 5 min AVR3a^22"59-mRFP(His)₆ was applied to the onion cells is shown in panel C for HEK293 in panel F and for RTG2 cells it is shown in panel I. No mRFP fluorescence was observed on either of the cell types under these conditions. However, when mixed with AVR3a^{22 147}(His)₆ (D,G,J) or SpHtp1^{24 198}(His)₆ (Ε,Η,Κ) mRFP

fluorescence was detectable.

Figure 4.

A/B: Phosphate and sulfate dependency of AVR3a^{22 47}(His)₆.

AVR3a ^{2 1 7}(His)₆ shows a strong tendency towards aggregation in 10 mM Tris/HCI buffer pH7.2 at 22°C. The protein can be efficiently stabilised by the addition of HP04^2"- and S04^2' ions. Aggregation curves were monitored at 310 nm on a UV-Vis spectrometer for 10 μΜ protein in the presence of different phosphate- (A) and sulfate- (B) ion concentrations. The black circles (·) in the inlets show the integral of the aggregation curves which is plotted against the phosphate/sulfate-ion

concentration. Assuming a constant aggregate size these data have been fitted with a single exponential function (y=y₀ ⁺ae^"bx) to obtain EC₅₀ values. The EC₅₀ for the phosphate ion stabilisation under this experimental conditions was 5.5 mM, the EC₅₀ for the sulfate ion stabilisation was calculated to be 2.7 mM.

C: Unfolding transition temperature of AVR3a^{22 47}(His)₆ in different buffers.

The unfolding was followed using the change in fluorescence of CyrPro orange when released from unfolding AVR3a^{2 47}(His)₆ ( 0 μΜ). Each transition temperature is the average of 8 individual experiments. Compared to phosphate, approximately half the concentration of sulfate is needed to accomplish the same thermal stabilisation effect. The pH of all used buffers was 7.2.

D: Effect of Fmoc-Tyr(S0₃)-OH on AVR3a^{22 W}(His)₆.

The graph shows that the addition of Fmoc-Tyr(S0₃)-OH to 10 μΜ AVR3a^{22"1 7}(His)₆ in 50 mM sodium phosphate buffer pH 7.2 results in the aggregation of the protein (monitored at 310 nm, 22°C). The black circles (·) in the inlet show the integral of the aggregation curves plotted against the Fmoc-Tyr(S0₃)-OH concentration. The data points can be described by a four parameter logistic function:

y=(max-min)/(1+(x/EC₅₀)^'Hi"^s'^ope. This function describes a saturated situation.

Therefore, it is highly likely that the amount of protein available is the limiting factor at high Fmoc-Tyr(S0₃)-OH concentrations and suggests an effective interaction between the protein and the tyrosine sulfation. The EC₅₀ value of this interaction is 1.3 mM Fmoc-Tyr(S0₃)-OH.

Figure 5.

Effect of aryl-sulfatase treatment of onion-, RTG2- and HEK293 cells on the binding and uptake of AVR3a²²- ⁴⁷-mRFP(His)₆.

The cells were incubated with the respective media with or without 1.1 U of sulfatase type VI from Aerobacter aerogenes. After 3 hours of sulfatase treatment 3 μ of AVR3a ^{2'1 7}-mRFP(His)₆ was added and the cells were further incubated for 1 h. Non-sulfatase treated onion cells incubated with A R3a^{22 1 7}-mRFP(His)₆ (A) showed mRFP fluorescence on the outline of the cells. In contrast, the sulfatase treated onion cells under identical conditions showed strongly reduced mRFP fluorescence (B). The same observations were made when using RTG2 and HEK293 cells. There, untreated cells showed a strong binding and uptake of the mRFP protein (C,E) whereas sulfatase VI treatment nearly abolishes the binding of the protein (D,F). Viability controls are given in Figure 13.

Figure 6.

Aryl-sulfatase treatment of RTG2 cells reduces the binding affinity of

SpHtp1²⁴-¹⁹⁸(His)₆.

The washed monolayers were incubated for 3h with L15-media containing 10% FCS in presence or absence of 1.1 U of sulfatase type VI from Aerobacter aerogenes prior the addition of SpHtp1^24'198(His)₆ (20 μΜ final concentration). Subsequent wash steps, fixation and immuno-staining are described in the material and method's section. Green channel: anti His FITC488; red channel: FM4-64 FX; blue channel: nuclear stain TO-PRO-3 iodine.

Figure 7.

Panel A-C: Normalised CD-spectra, calculated secondary structure contents and temperature dependent CD-spectra of the recombinant produced protein fragments of AVR3a, AVR3a^{22 147}(His)₆ (A), AVR3a^60'147(His)₆ (B) and

AVR3a^{22 59}(His)₆ (C).

AVR3a^{22"1 7}(His)₆ has a melting temperature (T_M) of 42°C, the T_M of AVR3a⁶°-¹⁴⁷(His)₆ is 48°C, whereas AVR3a^{2 "59}(His)₆ does not show a significant change in secondary structure within the investigated temperature range. All spectra were recorded in 50 m sodium phosphate buffer pH 7.2 containing either 5 μΜ AVR3a^{22" 7}(His)₆, Jasco 710 CD-spectrophotometer and a 1 mm cuvette. The secondary structure content was calculated using K2D (http://www.embl.de/~andrade/k2d/).

Panel D-H: Coomassie SDS-PAGES of the indicated purified recombinant proteins.

The theoretical retention according to the respective size of the protein constructs are indicated with black arrows. The SDS-PAGES of AVR3a ^{2 147}-mRFP(His)₆ and AVR3a^22"S9-mRFP(His)₆ show several bands. ALDI-TOF masses revealed that the two additional bands of AVR3a^{22 147}-mRFP(His)₆ positioned close to the 25 kDa marker band on the SDS-PAGEs are fragments of the full length protein, which seems to possess a cleavage site within the mRFP domain. AVR3a^{22 59}-mRFP(His)₆ seems to possess the same cleavage site. It is not entirely clear if the fragments contribute to the overall fluorescence of the mRFP proteins since the mRFP domain either lacks the first 35AA or 64AA. The latter case would most certainly prevent the formation of the chromophore and so would not contribute to fluorescence

(Yarbrough et al., 2001 ). The absence of the first 35AA means that two anti-parallel β-sheets are missing that shield the central chromophore forming a-helix from the solvent (pdb code: 2vad). Therefore, one would expect a shifted UVA is absorption for mRFP, which was not observed. Thus, all data regarding the protein

concentrations of the mRFP constructs are approximate concentrations. The present invnetors assumed that all fragments show a typical mRFP absorption and the values were corrected by the amount of full length protein deduced from densitometric analysises of the SDS-PAGE bands of the respective samples.

Figure 8

Panel A-H: Immuno-localisation on HEK293 cells of the recombinant protein constructs.

AVR3a²²-¹⁴⁷-mRFP(His)e (A), AVR3a⁶°-¹⁴⁷-mRFP(His)₆ (B), AVR3a^22'59-mRFP(His)₆ (C), AVR3a^{22 l47}(His)₆ (D), AVR3a⁶°-¹⁴⁷(His)₆ (E), AVR3a^{22 59}(His)₆ (F) and the Plasmodium falciparum GBP130^{65 196}(His)i₈ (G) on HEK293 cells. Panel H shows untreated immuno-stained HEK293 cells.

I-L: Immuno-localisation on RTG2 cells of the recombinant protein constructs.

AVR3a^22"147(His)₆ (I), AVR3a⁶°-^{1 7}(His)₆ (J) and AVR3a²²-⁵⁹(His)₆ (K) on RTG2 cells. Panel L shows untreated immuno-stained RTG2 cells. The washed HEK293 (RTG2) monolayers were incubated in DME - (L15-) media containing 10 % FCS, 20 μΜ of the respective protein and 2 μΜ of FM4-64 Fx for 30 min. Subsequent wash steps, fixation and immuno-staining are described in the material and method's section. The localisation pattern of AVR3a^{22 1 7}(His)₆ and AVR3a^60"147(His)₆ are comparable for both cell types. Green channel: anti His FITC488; red channel: FM4-64 FX; blue channel: nuclear stain TO-PRO-3 iodine.

M: Immuno-gold staining of high pressure frozen HEK293 cells incubated with 20 μΜ of either AVR3a^{22 147}(His)₆ or AVR3a^{22"l 7}(His)₆.

The samples were prepared with uranyl-acetate in the substitution solution. Immuno- staining was carried out as described in material and methods. Clusters of gold particles (010 nm) visible on the cells are indicated with arrows. Gold particles were mainly detected close to the extracellular cell membranes (A,B). These samples only occasionally showed small clusters of gold particles inside the cells. Very few gold particles were detected in the BSA treated controls with this method (C).

Figure 9.

Size exclusion profiles of the respective protein constructs recorded at 280 nm.

The column calibration run is shown in J and the corresponding calibration curve in K. Size exclusion profiles were quantified using gausian peak integration.

According to the calibration the AVR3a construct AVR3a^{22 1 7}(His)₆ (A) runs as a dimer whereas the fragment lacking the RxLR-EER motif AVR3a^{6tM 7}(His)₆ (B) shows the retention expected for the monomeric protein.

Interestingly, the RxLR peptide of AVR3a, AVR3a^22'59(His)₆ (C), shows an equilibrium between a tetramer- and dimer with the dimeric form as the dominant species. It is not clear if the peak corresponding to the monomeric mass is part of the equilibrium or solely caused by the degradation product of this peptide.

The fusion construct of amino acids 22-147 of AVR3a to mRFP (D) does not run as a dimer but shows a retention that corresponds to a molecular mass of + 20 kDa. In addition, ca. 30% of the protein sample is eluted from the column within the exclusion volume. The AVR3a mRFP fusion lacking the RxLR sequence, AVR3a^{60 147}-mRFP(His)₆ (E), shows an apparent mass of +4.7 kDa. Around 14% of the protein is eluted within the exclusion volume.

AVR3a^22'59-mRFP(His)₆ (F), has a retention corresponding to a molecular mass of +10.5 kDa.

The majority of SpHtp1^{2 "198}(His)₆ (G) elutes from the column within the exclusion volume. However, it is not clear if this is due to aggregation of the protein or if it forms higher oligomeric structures. Two peaks appear within the resulution of the column corresponding to a tetrameric and monomeric state of the protein.

H shows the running profile for a mix of AVR3a^22"59(His)₆ with

AVR3a ^2"147-mRFP(His)₆. All peaks found for the individual proteins are found in this run as well but are broader. In addition the amount of AVR3a^{2 147}-mRFP(His)₆ found in the exlusion volume is reduced by ca. 5x compared to the main peak for this construct.

The chromatogram for a protein mix of SpHtp ^{24 198}(His)₆ with AVR3a^{22 59}(His)₆ is shown in I.

Figure 10.

Mixed heteromerisation enables AVR3a^22'59-mRFP(His)₆ to enter RTG2- and HEK293 cells.

RTG2 cells were incubated with 15 μ of AVR3a ⁵⁹-mRFP(His)₆ ± the indicated non-mRFP protein constructs in L15-media containing 10% FCS for 1 h at RT. For the HEK293 cells DMEM media was used and the cells were incubated at 37°C.

Subsequently the cells were washed 3x in medium before imaging. The signal obtained if only AVR3a^{22 59}-mRFP(His)₆ was applied is shown in panel A for RTG2 and in panel D for HE 293 cells no mRFP fluorescence was observed under these conditions. However, when mixed with AVR3a^22"147(His)₆ (B,E) or SpHtp1²⁴-¹⁹⁸(His)₆ (C,F) mRFP fluorescence is detectable inside cells of both cell lines.

Figure 11.

11a. Lipid binding profiles of:

SpHtp1²⁴-¹⁹⁸(His)₆ (1 ), AVR3a²²-^{1 7}(His)₆ (2), PfGBP^65"196(His)₁₈ (3), SpHtp1⁶⁹-¹⁹⁸(His)₆ R84A/L86A E88A (7). The lipid membranes purchased from TebuBioscience S-6000 (A), P-6001 (B) and P-6002 (C) were incubated with 20 μΜ protein in PBS containing 5 % milk powder and 0.1 % Tween 20 for 20 min. The SpHtpt^{24 " 98}(His)₆ sample contained 0.5 % pluronic F68. All protein variant's that showed signals after antibody detection seem to have a common binding pattern. The lipid recognised on membrane A is sulfatide, those on membrane B are phosphatidylinositol(3)-, phosphatidylinositol(4)- and phosphatidylinositol(5)-phosphate and those on membrane C are phosphatidylglycerol (PG), cardiolipin and phosph.atidylinositol(4)- phosphate. The binding of the proteins to the lipids is independent of the RxLR-motif of AVR3a and the RxLxE/D/Q motif of GBP130.

11 b. Urea-denatured AVR3a ^{2 147}(His)₆ binds to lipids.

The urea transition of AVR3a^{22 1 7}(His)₆ was measured using circular dichroism spectroscopy (CD). 5 μΜ protein was incubated for 30 min in a 50 m sodium phosphate buffered solution containing the indicated urea concentration. The CD spectra were recorded in a 1 mm sample cell. The urea transition point of AVR3a^22" ¹⁴⁷(His)₆ is 4.68 . In order to analyse the lipid binding ability of urea-denatured AVR3a^{2 1 7}(His)₆, the membranes where equilibrated for 10 min using a phosphate buffered urea solution (50 mM sodium phosphate, 8 M urea, 0.1 % Tween 20 (v/v), 5 % milk powder (w/v) pH 7.2). AVR3a^{2 " 47}(His)₆ was denatured for 30 min in the same solution before it was added to the membranes. After 20 min incubation the membranes were washed 7x 5 min with 50 mM sodium phosphate containing 8 M urea, 0.1 % Tween 20 (v/v) and 5 % milk powder (w/v) (pH 7.2). The antibody incubation was carried out after the membranes were re-equilibrated with PBS. The present inventors did not observe any difference between the lipid-binding properties of native and urea-denatured AVR3a^{22"1 7}(His)₆.

11c. Lipid binding of guanidinium hydrochloride (GdnHCI) denatured

AVR3a^{22 147}(His)₆.

The same procedure described in 4b was used only that the 8 M urea was substituted with 6 M guanidinium hydrochloride (GdnHCI). No lipid binding of GdnHCI denatured AVR3a^{22"1 7}(His)₆ was detected.

11 d. Lipid binding profile on membrane B (P-6001 ) of urea denatured

AVR3a^{22 59}(His)₆ (left) and SpHtpl ^{69 196}(His)₆ (right) according to the protocol outlined in 4b. No lipid binding could be observed for these constructs. 11e. Lipid binding of AVR3a^{22 147}(His)₆ and PfGBP130^65"196(His)₁₈ in presence of substoichometric amounts of DnaK.

20 μ AVR3a^{22 147}(His)₆ and GBP130^{65 196}(His)₁₈ were incubated with the lipid spot membranes in the presence of 1 μΜ E.coli DnaK (Uniprot accession number P0A6Y8). In the case of GBP130^{65 196}(His)i ₈ DnaK is able to suppress the lipid binding completely whereas the signals for AVR3a^{22 1 7}(His)₆ were strongly reduced and only visible after long exposure times (>10 h).

11 f. Lipid binding profile of AVR3a"^"147(His)₆ on pre-treated membrane B.

The membranes were pre-treated for 3 h at RT with either 1 U of alkaline

phosphatise (left) or 1 U of aryl-sulfatase in L15 medium containing 10 % FCS. The membranes were than washed with PBS and subsequently incubated for with 20 μ AVR3a^{22 147}(His)₆ in PBS containing 5 % milk powder and 0.1 % tween 20.

All protein incubations were carried out for 20 min at RT. All blots were probed with a HRP coupled penta-His antibody using a titre of 1 :25,000. All films were developed for 3 min if not indicated otherwise.

Figure 12.

Isothermal calorimetric titration (ITC) and aggregation control measurements.

(A) Isothermal titrations of inositol (blue line— ) and inositol 1 ,4 bisphosphate (red line— ) to AVR3a^{22 147}(His)₆.

A protein concentration of 172 μΜ (200 μΙ) was used as bait in 50 mM sodium phosphate pH 7.2. The inositol concentration was 123 mM and the concentration for inositol 1 ,4 bisphosphate was 24 mM. After an initial delay of 300 s the first titration step used 0.4 μΙ and was followed by 19 steps with a 2 μΙ volume. The delay interval was set to 150 s. No physical interactions between the small molecular compounds and the AVR3a^{2 147}(His)₆ protein could be observed. For better visualisation of the graphs showing the inositol titrations an offset of 0.2 peal was used. The control titration of inositol 1 ,4 bisphosphate (green line— ) and inositol (black line— ) into the dialysis buffer showed that the dilution heat is minimal for this compound under identical conditions.

(B) Titration of Fmoc-Tyr(S0₃)-OH vs. AVR3a^{22 1 7}(His)₆ (black line— ) compared to 8 M urea denatured AVR3a^{2 147}(His)₆ vs. inositol 1,4 bisphosphate (red line — ) (25°C, in 50 mM sodium phosphate pH 7.2). 20 μΙ of 15 mM Fmoc-Tyr(S0₃)-OH was titrated into 200 μΙ of 201 μΜ

AVR3a^{22" 7}(His)₆. The initial delay was 300 s, the first titration step used 0.4 μΙ and was followed by 18 steps using 2 μΙ and 4 steps with 0.4 μί. The delay interval was set to 150 s. A strong heat generation could be observed for each titration step. The control titration of Fmoc-Tyr(S0₃)-OH into the dialysis buffer showed that the dilution heat is minimal for this compound under identical conditions (blue line— ).

Since the present inventors observed that 8 M urea denatured AVR3a^{2 147}(His)₆ seems to interact with sulfatide, Ptdlns(3)P, Ptdlns(4)P, Ptdlns(5)P,

phosphatidylglycerol and cardiolipin a titration of inositol 1 ,4 bisphosphate into 8 urea denatured AVR3a^{22' 47}(His)₆ was performed. Therefore, 500 μΙ of a 259 μ protein solution was dialysed 3x (2x3h final dialysis step ON) against 1 I of 8 M urea in 50 mM sodium phosphate pH 7.2. After dialysis the protein concentration was ca. 190 μΜ. The initial delay before the first titration step (0.4 μΙ) was set to 300 s and was followed by 19 steps with a 2 μΙ volume of 24 m inositol 1 ,4 bisphosphate.

(C) Titration of Fmoc-Tyr(S0₃)-OH to SpHtpl^{24 198}(His)₆ (25°C, 50 mM sodium phosphate pH 7.2).

The concentrations for the individual experiments are indicated in the figure. The obtained data were fitted according to a two site binding model and the results of the three individual experiments were averaged. The interaction parameters for this reaction are described by the following values: K_D = (159±44) μΜ, (0.93±0.38) binding sites, ΔΗ,το = (-15.5±7.1 ) kJ/mol and AS|_TC = (19.6±9.4) J/mol^"1-K^-1.

(D) Titration of Fmoc-Tyr(SO_)-OH to AVR3a^{22'1 7}-mRFP(His)₆. The titration was performed with 67 μΜ protein and a titrant concentration of 20 mM.

The initial delay was 300 s, the first titration step used 0.4 μΙ and was followed by 8 steps using 0.5 μΙ, 10 steps using 1 μΙ and 1 1 steps with 2 μΙ volume of the Fmoc- Tyr(S0₃)-OH stock solution. The delay interval was set to 150 s. Due to the complexity of the obtained data the thermogram was not quantified.

(E) , (F) Control aggregation measurements of the indicated proteins in the presence or absence of Fmoc-Tyr(S0₃)-OH and H-Tyr(S0₃)-OH.

Figure 13.

Effect of aryl-sulfatase treatment of onion-, RTG2- and HEK293 cells on the The cells were incubated with the respective media with or without 1.1 U of sulfatase type VI from Aerobacter aerogenes. After 3 hours of sulfatase treatment 3 μΜ of AVR3a^60"147-mRFP(His)₆ was added and the cells were further incubated for 1 h.

Non-sulfatase treated onion cells incubated with AVR3a^60"147-mRFP(His)₆ (A) showed mRFP fluorescence on the outline of the cells. In contrast, the sulfatase treated onion cells under identical conditions showed strongly reduced mRFP fluorescence (B).

The same observations were made when using RTG2 and HEK293 cells. There, untreated cells showed a binding and uptake of the mRFP protein (C,E) whereas sulfatase VI treatment nearly abolishes the binding of the protein (D,F). The viability control G shows that aryl-sulfatase treated HEK293 cells are still able to take up Alexa fluor 488 labelled transferrin.

The affinity of the AVR3a-mRFP constructs towards the cells can be regained if the sulfatase is washed away and the cells had time to recover as it is exemplarely shown in panel H for HEK293 cells. The viability control F shows a time lapse over 25 min after 1 h of incubation of AVR3a^22'147-mRFP(His)₆ on sulfatase treated RTG2 cells with an enhanced detector gain (900) for the mRFP fluorescence. The pictures show movement and structural rearrangements within the cells (movie available).

Figure 14.

Phytophtora infestans AVR3a sequences

The sequences shown are derived from the AVR3a sequence entry in the Uniprot® Database (http://www.uniprot.org), entry ID Q572D3.

(A) = Full sequence of AVR3a^1"147 Kl, including putative signal peptide (underlined) and showing the RxLR and EER motifs (bold);

(B) AVR3a^1'147 EM;

(C) AVR3a^22"147 Kl;

(D) AVR3a^22"147 EM;

(E) AVR3a^60"147 Kl;

(F) AVR3a^60"147 EM;

(G) AVR3a^22"59 Kl;

(H) AVR3a^22"59 EM;

(I) SpHtpl ^1'198;

(J) SpHtpl ^24'198. Figure 15.

Comparison of AVR3a^{22 59}(His)₆ with AVR3a^{22 59}(His)₆ KMIK and AVR3a^{22 59}(His)₆ A5.

A: Coomassie blue stained SDS-PAGE's of the respective AVR3a constructs.

B: Circular dichroism (CD) spectra of AVR3a^22'S9(His)₆ (black line— ), AVR3a^22' ⁵⁹(His)₆ KMIK (blue line— ) and AVR3a^{22 59}(His)₆ A5 (red line— )■ The secondary structure content was calculated using K2D (http://www.embl.de/~andrade/k2d/). Both AVR3a^{22 59}(His)₆ KMIK and AVR3a^{22 59}(His)₆ A5 contain ~6 % α-helical-, 32 % β-sheet- and 62 % random coil structure. AVR3a^22"59(His)₆ has ~6 % σ-helical-, 46 % β-sheet- and 48 % random coil structure. The spectra were recorded in 50 m sodium phosphate buffer pH 7.0 with 10 μΜ protein in a 1 cm cuvette.

C-E: Gel filtration run profiles of AVR3a^{22 59}(His)₆ (C), AVR3a²²-⁵⁹(His)₆ KMIK (D) and AVR3a^{2 59}(His)₆ A5 (E). Run conditions and calibration are given in the material and methods section. (^* this figure is already published in Wawra er a/. 2010)

Figure 16.

Reconstitution of AVR3a^{60 147}(His)₆ with either the WT RxLR-EER construct of A R3a, AVR3a^22'59(His)₆, or the mutants KMIK and A5.

Panel A shows the aggregation measured at 310 nm for 5 μΜ AVR3a^60"147(His)₆ (— ),5 μΜ AVR3a^{2 59}(His)₆ (— ) and the mix of 5 μΜ of each protein (— ) in 10 mM Tris/HCI pH 7.0. The addition of phosphate to a mixture of 5 μΜ AVR3a^{60'1 7}(His)₆ and 5 μΜ AVR3a^{22 59}(His)₆ reduces the light scattering signal of this solution but in contrast to full length AVR3a is not able to suppress the aggregation completely.

Panel B shows the light scattering signal measured at 310 nm for 5 μΜ

AVR3a⁶⁰-^{1 7}(His)₆ (— ), 24 μΜ AVR3a^22'59(His)₆ KMIK (— ) and 24 μΜ AVR3a^22'59(His)₆ A5 (— ). The dashed lines show the aggregation signals for the mixes of 5 μΜ AVR3a⁶⁰-¹⁴⁷(His)₆ with 24 μΜ AVR3a²²-⁵⁹(His)₆ KMIK (red dashed line) and 5 μΜ AVR3a^{6t 47}(His)₆ with 24 μΜ AVR3a^{22 59}(His)₆ A5 (blue dashed line). These absorption curves nearly represent the sum of the individual aggregation aggregation measurements (exemplarily shown for the sum of AVR3a^{60'1 7}(His)_e + 24 μΜ

AVR3a^22'59(His)₆ KMIK; dotted line).

C,D: Isothermal titrations profile obtained for the titration of AVR3a^22"59(His)_s (black AVR3a^{60' 7}(His)₆ was used with a bait concentration of 96 μ (200 μΙ) in 50 mM sodium phosphate pH 7.2. The concentration of AVR3a^22'59(His)₆ was 81 1 μΜ the concentration of AVR3a^{2 59}(His)₆ KMIK was 2.1 mM. Only weak signals were detected for both titrations. The titration utilising AVR3a^{22 59}(His)₆ shows an endothermic-, whereas the titration using AVR3a^{Z 59}(His)₆ KMIK has an exothermic profile. The titration of AVR3a^{22 9}(His)₆ into the dialysis buffer is shown by the red line in C. The initial delay for the experiments was set to 300 s. The first titration step used 0.4 μΙ and was followed by 19 steps with a 2 μΙ volume. The delay interval was set to 150 s.

E: Normalised CD spectra of AVR3a^{22 147}(His)₆ (— ), AVR3a^{60 7}(His)₆ (- -) and the spectra obtained immediately after mixing of AVR3a^60",47(His)₆ with AVR3a^{22 59}(His)₆ (— )■ The physical sum spectrum of AVR3a^{22 59}(His)₆ and AVR3a⁶°- ⁴⁷(His)₆ was recorded directly before mixing the two solutions (white circles). The spectra were recorded in 50 mM sodium phosphate buffer pH 7.0 with 5 μΜ of each protein in a 1 cm double chamber cuvette.

Figure 17.

A: CD-spectra of 5 μΜ AVR3a^{22' 47}(His)₆ with (— ) and without 50 mM phosphate (- - ) in 10 mM Tris/HCI pH 7.2 containing 500 mM KCI, 300 mM imidazol.

B: Gel filtration profile of AVR3a^{22 147}(His)₆ in 10 mM Tris/HCI pH 7.2 containing 500 mM KCI, 300 mM imidazol.

Figure 18.

AVR3a^{22'1 7}(His)₆ adopts different conformational states dependent on the protein concentration.

A,B: Unfolding transition of AVR3a^{2 "147}(His)₆ was followed using the change in fluorescence of CyrPro orange (Invitrogen) when released from unfolding protein. Each transition temperature is the average of 8 individual experiments.

A: Example of the 2^nd derivative derived from the fluorescence signal for selected concentrations showing the shift in the transition temperature (peak minima) with increasing protein concentration. B: Plot of the unfolding transition temperature against of the protein concentration of AVR3a^{22 147}(His)₆. A decrease of about 10°C in the concentration range between ~5 and -50 μΜ was observed.

C: ¹H-NOESY of AVR3a^22'147(His)₆ (250 μΜ, in 25 mM potassium phosphate pH 6.5), the red box shows the significant ¹H-¹H-cross peaks of the protein.

D: ⁵N-HSQC of two different concentrations of AVR3a^22"147 (His)_e. The spectrum depicted in green was recorded by using a ⁵N-labelled protein sample (~250 μ , in 25 mM potassium phosphate, pH 6.5), the spectrum shown in red was acquired with an ~1.2 mM, ¹³C¹⁵N-Iabelled protein sample in 25 mM potassium phosphate pH 6.5. The black box shows the part of the spectrum where peaks of residues, which belong to random-coil areas, are mostly found (Wishart et al., 1991 ).

Figure 19.

A: DnaK, unfolding and mutation of the RxLR-EER amino acids inhibit the uptake of AVR3a. The uptake of 2 μΜ AVR3a^22"147-mRFP(His)₆ (1) was strongly inhibited in the presence of 1 μΜ DnaK (2) or if the protein was pre-incubated in 6 M urea (3).

Refolding was initiated by a 1 :24 dilution of a 48 μΜ AVR3a^{22"1 7}-mRFP(His)₆ stock solution. The final urea concentration applied onto the cells was 0.25 M for all samples. The AVR3a-mRFP fusion construct were the RRxLR-EER amino acids were mutated to KKMIK-DDK is still able to translocate (4) but with clearly decreased efficiency compared to the WT construct. The fusion construct consisting of the AVR3a RxLR-EER-leader peptide and mRFP, AVR3a^{22 59}-mRFP(His)₆ was not translocated (5). Furthermore, the uptake of 2 μΜ AVR3a^60"147-mRFP(His)₆ (6) is also strongly inhibited in the presence of 1 μΜ DnaK (7). Lower magnifications are shown in supplementary figure S2.

B1 : Immuno-gold staining of RTG2 cells incubated with AVR3a^22'147(His)₆ in the process of refolding. B2: Immuno-gold staining of HEK293 cells exposed to refolding AVR3a^{22" 7}(His)₆. The RTG2 (HEK293) monolayers, grown in a 50 ml cell culture flask, were washed 3x and incubated with L15- (DMEM-) media containing 10 % FCS, 20 μΜ protein and 0.8 M urea for 30 min. After the incubation the cells were washed 5x with HBSS, scraped of the bottom of the culture flask and harvested by centrifugation for 3 min at 800 xg. The samples were than processed using high pressure freezing. Immuno-staining was carried out as described in material and secondary antibody (Gold anti mouse Igd from rabbit). Cells treated with refolding AVR3a^{22 147}(His)₆ showed gold particles mainly in the nucleus indicated by the black arrows and sometimes lumps of gold in the cytosol.

C indicates the cytosol; Mt are mitochondria; N indicates the nucleus; Ex is the extracellular space and the asterisks (*) indicate the nuclear border.

Figure 20.

A: Coomassie blue stained SDS-PAGE showing a time course of a partial proteolytic digest over 30 min for A R3a ^2'147(His)₆ using 0.04 mg/ml proteinase K (Invitrogen). The band indicated with the white asterisk has been subjected to N-terminal Edman sequencing revealing the N-terminal sequence (S)LNEEM indicating that the effector domain starts at amino acid (67)68.

B: Ponceau red stained membran and the subsequently developed western blott with the samples shown in A probed with the penta-H^'is antibody from Qiagen (1 :20,000).

C: Ponceau stain and developed western blott of RTG2 cells incubated with

AVR3a^{22 147}(His)₆ which were subsequently washed and subjected for 20 min to a 0.04 mg/ml proteinase K solution in cell culture media. After the digest the cells were washed 4x with PBS boiled in protein sample buffer and loaded onto the gel (lane 2). Lane 1 shows purified AVR3a ^2'147(His)₆ and lane 3 shows purified AVR3a^{60 147}(His)₆.

D: Ponceau stain and developed western blott of RTG2 cells incubated with denatured AVR3a^{22 1 7}(His)₆. The protein was denatured by dialysing against 6 M urea over night. Refolding was initiated by diluting the protein 1 : 10 in L15-media containing 10 % FCS. The RTG2 monolayer was imedeately incubated for 20 min with the refolding AVR3a ^{2 147}(His)₆ protein solution. Subsequently the cells have been washed 5x with HBSS, were subjected to proteinase K and scraped of the bottom of the culture flask and boiled in protein sample buffer and loaded onto the gel in lane 2. Lane 1 shows purified AVR3a ^{2' 7}(His)₆ and lane 3 shows purified AVR3a⁶⁰-¹⁴⁷(His)₆.

Figure 21.

DnaK, unfolding and mutation of the RxLR-EER amino acids inhibit the uptake of AVR3a. The uptake of 2 μΜ AVR3a^{2: 47}-mRFP(His)₆ (1 ) was strongly inhibited in the Refolding was initiated by a 1 :24 dilution of a 48 μΜ AVR3a^22'147-mRFP(His)₅ stock solution. The final urea concentration applied onto the cells was 0.25 M for all samples. The AVR3a-mRFP fusion construct were the RRxLR-EER amino acids were mutated to KKMIK-DDK is still able to translocate (4) but with clearly decreased efficiency compared to the WT construct. Furthermore, the uptake of 2 μΜ

AVR3a^{60 147}-mRFP(His)₆ (5) is also strongly inhibited in the presence of 1 μΜ DnaK (6).

Materials and Methods

Chemicals

Buffers were purchased from AppliChem (Darmstadt, Germany), Merck (Darmstadt, Germany). All other chemicals were purchased from Sigma (Munich, Germany) and of the highest purity available.

PCR/Primer/Cloning/protein expression conditions

PCRs were performed using the primer combinations listed below:

The clones for the GBP130 fragments are published by Boddey et al. (Boddey et al., 2009).

PCR was performed using KOD-Hot start DNA polymerase (Novagen; Lot# M00057770). The PCR-products were blunt end cloned into pETblue-2 or pUCl 9, digested out using the Nde and EcoR restriction sites embedded in the primers and sub cloned in pre-cut pET21b. The resulting fragments were in frame with the (His)₆ tag encoded by pET21 b.

The sequenced constructs in pET21 b were transformed in Rosetta garni B (DE3, pLys; Novagen) cells and over expressed under the control of the T7 promoter. For protein expression cells where grown in LB-media to an ODmo between 0.6 and 0.8 and induced with 1 mM IPTG for 6h at 37°C. 12 g cell pellet aliquots were resuspended in 40 ml 50 mM sodium-phosphate pH 7.1 and incubated for 20 min 11873580001) and 0.1 g lysozyme (Fluka, 62971 ). After the incubation the solution was French-pressed, diluted 1 :5 in the respective buffer and the soluble fraction was separated from the non-soluble via spinning at 50,000xg for 1 h.

Column specifications:

NTA column: 15 ml NTA Agarose (Invitrogen, #60-0441), column dimensions: 1 cm 0, 20 cm height.

S0₃ ^" column: 40 ml Fractogel-EMD-SO^3" (M) (Merck, 1.16882.0100), column dimensions: 2 cm 0, 20 cm height.

QAE column: 35 ml QAE-sephadex A25 (GE Healthcare, 17-0190-01), column dimensions: 2 cm 0, 20 cm height

Purification of AVR3a^{22 147} (His)_fi

Over-expression of AVR3a^{22 147} (His)₆ at 37°C resulted in the formation of inclusion bodies (IB). The French-press pellet was homogenised in 30 ml of 50 mM sodium- phosphate pH 7.1 containing 0.1 % triton X-100 to resolve membrane lipids. The solution was then centrifuged at 9000xg for 1h. The IB's where again homogenised in 30 ml of 50 mM sodium-phosphate pH 7.1 and pelleted. This step was repeated until the solution did not foam during homogenisation. The IB-pellet was then resuspended in 40 ml of 50 mM sodium-phosphate containing 10 M Urea (Fluka) adjusted to pH 7.1 for 30 min at RT and centrifuged for 30 min at 9000xg. The supernatant was applied (1 ml/min) to a pre equilibrated NTA column and washed with 5x volumes of urea buffer. Refolding was initiated by a slow solvent exchange (0.15 ml/min) against 50 mM sodium-phosphate pH 7.1 over 20 h. Subsequently, the column was washed with 100 ml 50 mM sodium-phosphate containing 30 mM imidazole. The protein was eluted with 50 mM sodium-phosphate containing 300 mM imidazole adjusted to pH 7.1 and fractions were analysed by SDS-PAGE.

Purification of AVR3a^{60 147} (His)«

The French-press supernatant was applied to the QAE column and washed with 25 mM Tris/HCI-buffer pH 7.5. The flow through of this column was applied to a SO^3' column. After washing the column the bound proteins were eluted with a gradient from 0-1.5 M KCI (in 25 mM Tris/HCI pH 7.5) and the fractions were analysed by SDS-PAGE. The fractions containing the AVR3a^{60" 47}(His)₆ were pooled and applied imidazole (pH 7.5). Additionally the protein was eluted with 25 mM Tris/HCI containing 300 mM imidazole adjusted to pH 7.1 and fractions were analysed by SDS-PAGE.

Purification of AVR3a^{22 59} (His)_R

The French-press supernatant was applied to a SO^3' column (20 mM Tris/HCI pH 7.5). The flow through of this step was pumped over the NTA Agarose column. Subsequently, the column was washed with 100 ml 25 mM Tris/HCI containing 30 mM imidazole (pH 7.5). The protein was eluted with 25 mM Tris/HCI containing 300 mM imidazole adjusted to pH 7.5 and fractions were analysed by SDS-PAGE. As a final purification step the fractions containing the AVR3a^{22 59}(His)₆ were pooled and the protein was centrifuged through a VIVASPIN centricon with a cut off of 10,000 Da. The flow through of this step was collected and concentrated.

After French-press the supernatant was applied in 50 mM sodium-phosphate buffer pH 7.2 to the QAE column and the flow through was directly pumped onto the SO^3" column. The cat ion exchange material was then washed with 10 volumes of 50 mM phosphate containing 300m KCI and 0.1% Tween 20 (pH 7.2) and additionally with 10 volumes of plain sodium phosphate buffer (pH 7.2). The protein was eluted onto the NTA column by applying 1 M KCI in phosphate buffer (pH 7.2) to the cat-ion exchange column. Subsequently, the column was washed with 100 ml 50 mM phosphate containing 30 mM imidazole (pH 7.5) and the protein was eluted with 50 mM phosphate buffer containing 300 mM imidazole adjusted to pH 7.1 and fractions were analysed by SDS-PAGE.

Purification of AVR3a^{22 147}-mRFP (His)_g

The French press supernatant was passed through a S0₃ ^" column and washed with 50 mM sodium phosphate buffer containing 300 mM KCI and 0.1 % Tween 20 (pH 7.2). The flow through was pumped over a NTA agarose column. Subsequently, the column was washed with 100 ml 50 mM phosphate buffer containing 30 mM imidazole (pH 7.5) and the protein was eluted with 50 mM phosphate buffer containing 300 mM imidazole adjusted to pH 7.1 and fractions were analysed by SDS-PAGE. This protein was purified from inclusion bodies (IB). The French press pellet was resuspended in 40 ml of 50 mM phosphate buffer containing 0.5 % triton X-100 at pH = 6.5 and homogenized. The solution was then centrifuged at 9000 g for 1 . The IB's were homogenised again in 30 ml of 50 mM sodium-phosphate pH 7.1 and pelleted. This step was repeated until the solution did not foam during homogenisation. The IB-pellet was then resuspendet in 40 ml of 50 mM sodium-phosphate containing 10 M Urea (Fluka) adjusted to pH 7.1 for 30 min at RT and centrifuged for 30 min at 9000xg. The supernatant was applied (1 m!/min) to a pre equilibrated NTA column and washed with 5x volumes of urea buffer. Refolding was initiated by a slow solvent exchange (0.15 ml/min) against 50 mM sodium-phosphate pH 7.1 over 20 h. Subsequently, the column was washed with 100 ml 10 mM Tris containing 300 mM NaCI, 0.1 % Tween 20 and 25 mM imidazole (pH 7.5). The protein was eluted with 10 mM Tris/HCI containing 300 mM imidazole and 300 mM NaCI adjusted to pH 7.5 and fractions were analysed by SDS-PAGE.

Purification of AVR3a^{22 59}-mRFP (His)_fi

The French press supernatant was adjusted to pH 5.4 and passed through the QAE column. The flow through of this column was applied to the S0₃ ^" column adjusted with 50 mM sodium phosphate buffer pH 5.4 and was washed with 5 volumes of the same buffer. From this column the protein was eluted with 200 mM phosphate buffer adjusted to pH 7.5. The eluted fractions containing the mRFP construct were passed through the NTA agarose column. Subsequently, the column was washed with 100 ml 50 mM phosphate buffer containing 30 mM imidazole (pH 7.5) and the protein was eluted with 50 mM phosphate buffer containing 300 mM imidazole adjusted to pH 7.1 and fractions were analysed by SDS-PAGE.

Purification of SpHtpl a^{24 198} (His)_fi and SpHtpla^69'198 (His)*

The French press supernatant was first applied to an SO^3' column and washed with 10 volumes of 25 mM sodium phosphate buffer pH 7.0 containing 25 mM potassium chloride. The flow through of this column was additionally applied to a QAE- column. After washing the column with 10 volumes 25 mM sodium phosphate buffer pH 7.0 containing 25 mM potassium chloride the bound proteins were eluted with a gradient from 0-1.5 M KCI (in 25 mM Tris/HCI pH 7.5) and the fractions where analysed by SDS-PAGE. The fractions containing the Htp1^24'198 (His)₆ where pooled and applied to an NTA column. The column was then washed with 100 ml 25 mM Tris/HCI containing 30 mM imidazole (pH 7.5). Additionally the protein was eluted with 25 mM Tris/HCI containing 300 mM imidazole adjusted to pH 7.1 and fractions were analysed by SDS-PAGE.

All proteins were concentrated using VIVASPIN 6 centricons ( WCO 5,000) before dialysis 2x against 3 litres of the respective storage buffer.

Immuno-localisation

RTG2 (HEK293) cells were washed three times with HBSS prior to 20 min incubation with 20 μ of the respective protein and 2 μΜ FM4-64FX (Invitrogen), as a membrane counter stain, in L15 (DMEM)-medium containing 10% FCS (BioSera) at RT (37°C). After washing the cells four times with PBS, the cells were fixed with 4 % paraformaldehyde diluted in the respective media. Fixed cells were washed three times with PBS, permeabilised for 15 min with PBS containing 0.1% Triton-X100 and washed again three times before incubation with the primary penta-His antibody at 37°C for 1h (Qiagen; titre 1 :300; diluted in media containing 10 % FCS). Subsequently, the samples were washed 3x with PBS, and incubated at 37°C for 1h with the secondary antibody (FITC-488 conjugated goat anti mouse IgG; Jackson ImmunoResearch) according to manufacturer's protocol. TO-PRO-3 iodine (Invitrogen) stain was carried out according to the protocol provided by the manufacture. Microscopy was carried out using a Zeiss LSM 510 META confocal microscope (1 pm slice, green channel: Exit.: 488 nm; Detector gain: 750; Filter setting: BP 505-530 nm) at the appropriate wavelength.

Isothermal calorimetric titration (ITC)

Titration experiments were performed with a MicroCal iTC₂₀o at the indicated temperatures. Before the experiment the instrument was heat-pulse-calibrated and the protein samples were extensively dialysed against the respective buffers. Titrant stock solutions were prepared with the same batch of buffer as used for dialysis. All solutions used were degassed before filling the sample cell and syringe. Titration steps, compound concentrations and volumes are given in the figure legends for the individual experiments. The ITC stirring speed was set to 1000 rpm; the feedback gain mode was set to high. Since the initial injection generally delivers inaccurate data, the first step was omitted from the analysis. The collected data were analyzed using the program "Origin" (MicroCal) and binding isotherms were fitted using the binding models provided by the supplier. Errors correspond to the SD of the nonlinear least-squares fit of the data points of the titration curve. Circular-dichroism (CD) spectroscopy and UV-Vis spectroscopy

CD-spectra were recorded on a Jasco J710-, whilst UV-Vis spectra and light scattering curves were recorded on an Agilent 5483 spectrometer. Conditions are given in the figure legends.

Phospholipid binding assays

The lipid spot membranes (Echelon Bioscience; #S-6000, #P-6001 , #P6002) were equilibrated for 10 min with PBS containing 0.1 % Tween 20 and 5 % milk powder (semi skimmed milk powder LIDL UK). The solution was then changed and the protein was added to a final concentration of 20 μΜ and incubated with the membranes for 20 min. Subsequently, the membranes were washed 3x with PBS Tween/Milk. Antibody detection was performed with a HRP coupled anti-His- antibody (Qiagen penta His) in PBS Tween/Milk at a titre of 1 :20,000. Detection was carried out using ECL.

Lipid binding under denaturing conditions: Lipid spot membranes where equilibrated in PBS containing 0.1 % Tween 20 and 5 % milk powder containing 8 M UREA or 6 M GdnHCI for 10 min. Protein samples where denatured in the same buffer for 30 min at RT. Subsequently, the membranes were incubated with the protein solutions for 20 min at RT. After incubation the membranes where extensively washed with PBS/Tween containing 8 M UREA or 6 M GdnHCI and were at least 3x 5 min re-equilibrated with PBS Tween. Antibody detection was performed as described above.

Alkaline phosphatase was purchased from Roche (#713023). Cell life imaging

The washed cover slips were transferred in optical Petri-dishes (0 3cm) and covered with the respective media containing 10 % FCS and the mRFP tagged proteins at the indicated concentrations and temperatures. Images were recorded using a ZeissLSM510 confocal microscope equipped with a dipping lens. The microscope settings were: optical slice = 2 μηι; red channel excitation = 543 nm; detector gain = 750; filter setting = LP 560 nm

Fmoc cleavage of Fmoc-Tyr(SC>3)-OH

100 mg of Fmoc-Tyr(S0₃)-OH (Novabiochem; #04-12-1251 ) was dissolved in 3 ml reaction product is not soluble in THF and was separated from the reactive solution by centrifugation. The pellets were washed extensively alternating with petrol ether and THF to remove the remaining piperidine until a neutral pH was reached. The raw product was resolved in 2 ml, 25 mM sodium phosphate buffer pH 7 and applied to a small QAE column. The column was washed extensively and the final product was eluted from the column using acetic acid and subsequently lyophilised. The quality of the product was checked by analytical HPLC and spectroscopic properties.

Thermal unfolding measurements

Thermal unfolding measurements were performed as described by Ericsson er al. (2006) and Yeh ef al. (2006) using a Light Cycler 480 (Roche) and SYPRO Orange (invitrogen #S6651) in a dilution of 1 :2000 (Ericsson et al., 2006; Yeh et al., 2006). The 96 well micro-titre plates were filled with 25 μΙ sample.

General methods: Gel filtration calibration and conditions are given in Figure 13. The maintenance of the RTG2 cell-line is described in van West et al. (2010). For cell desulfation sulfatase type VI from Aerobacter aerogenes (Sigma #S1629) was utilised. Experimental conditions are given in the respective figure legends. For the mixed heteromerisatton, the indicated concentration of the respective proteins were mixed and incubated for 30 minu on ice before application onto the cells.

TEM microscopy and immuno-gold labelling

Cells were frozen with a Leica Empact 2 High Pressure Freezer. The freeze was substituted with acetone using a Leica AFS2 and embedded in HM20 resin. For normal TEM, sections were stained with Uranyl acetate and lead citrate. For immuno- gold labelling the sections were cut onto nickel grids and blocked for 10 min with normal goat serum. Primary antibody incubation was carried out overnight (1.50) in blocking solution. The gold probe from Aurion (goat anti-mouse, 10nm) was incubated for 1 hr (1 :40) diluted in PBS/BSA. Subsequently the sections were lightly stained for 15 s in uranyl acetate and 5 s in lead citrate in the Leica AC20. TEM pictures were taken on an CM10 TEM.

Nuclear magnetic resonance spectroscopy

All NMR-experiments were performed on a 700 MHz NMR spectrometer equipped with a cryoprobe (Bruker Biospin) at 298 K. The NMR-samples contained -250 μΜ of unlabelled AVR3a²²-¹⁴⁷(His)₆, -250 μΜ of ¹⁵N-labelled AVR3a^22'147(His)₆ and -1 .2 mM mM potassium phosphate buffer, pH 6.5 or pH 7.0. A NOESY- (Jeener, 1979) and ¹H-¹⁵N-HSQC-spectra (Farrow et al., 1994; Kay et al., 1989) were recorded to get information about the folding of the protein. All spectra were recorded with water suppression, using the 3-9-19 WATERGATE pulse sequence (Piotto et al., 1992) with 2048 data points in F2 and 512 data points in F1 , 40 scans each for the homonuclear spectrum and 8 scans each for the heteronuclear spectra. Mixing times were 200 ms for the NOESY, the 90 degree high power pulse was 12.5 ps. The recorded data were processed with the software Topspin (Bruker). For apodisation of data a shifted sine-bell square window function was applied using zero-filling to 2048 data points in F2 and 1024 data points in F1 for all spectra.

Examples

Example 1 : Cell surface binding of the Phvtophthora infestans AVR3a protein is achieved by the effector domain and not the RxLR leader sequence of the protein. In order to investigate how the uptake of the RxLR-protein AVR3a from P. infestans is achieved the present inventors set up an RxLR protein uptake system for AVR3a. Recently the present inventors discovered that a His-tagged RxLR protein, SpHtpl , from the fish pathogenic oomycete Saprolegnia parasitica is able to self-translocate at concentrations of 20 μΜ (0.21 mg/ml) into fish cells (RTG2 cell line) within 20 min [van West et a/., submitted]. Therefore, the present inventors decided to follow a similar strategy and designed three different AVR3a constructs AVR3a^22"147,

AVR3a^{60 47} and AVR3a^22'59, and C-terminally fused either a simple (His)₆-tag or a combined mRFP(His)₆-tag. The respective proteins were synthesised in E coli and purified. The largest AVR3a fragment AVR3a^22'147 lacks only the putative signal peptide as it was predicted by signalP (http://www.cbs.dtu.dk/services/SignalP/), The AVR3a^{60' 47} constructs additionally lack the RxLR-EER motif and the third constructs, AVR3a^22"59 contain only the AA (amino acids) 22-59, which contains the RxLR-EER motif of AVR3a.

Circular dichroism (CD) spectroscopic experiments show that AVR3a^{22 59}(His)₆ adopts a flexible low structured fold, probably containing a β-sheet structure, and the protein does not show a thermal unfolding transition even at 70°C. In contrast, both AVR3a²²-¹⁴⁷(His)₆ and AVR3a⁶°-¹⁴⁷(His)₆ are typical a-helical proteins with thermal unfolding transition temperatures of more than 40°C (Figure 7). Compared to AVR3a^60"147(His)₆, the AVR3a ^{2" 47}(His)₆ protein has a higher random coil content and reduced T_M, which suggests that the attachment of the flexible N-terminal peptide destabilises the effector domain of AVR3a and introduces a higher structural flexibility. CD-spectra of the AVR3a-mRFP(His)_s constructs have not been recorded since the large mRFP domain masks the signal of polypeptides derived from AVR3a.

The AVR3a-mRFP(His)₆ fusion proteins were used to investigate the translocation process in living cells. When live onion cells were incubated with a 3 μΜ solution of AVR3a^22"147-mRFP(His)₆ for 30 min or up to 1 h a bright red fluorescence signal was observed in the outer cell membrane region (Figure 1A). These observations are to some extend comparable with some earlier results described by Dou et a). (2008), who performed localisation studies with a recombinant chimeric protein consistent of RxLR_Av ib-fusion protein was also observed on the membrane region of onion cells and is eventually taken up after 12 hours, when large quantities of protein (8 mg/ml) were given (Dou et al., 2008b).

However from both these and the present results it is unclear to what the fusion proteins are binding. Therefore the present inventors also decided to perform uptake experiments with animal cells as these lack a cell wall. When live cells from fish (RTG2 cell line) and human (HEK293 cell line) were used, a bright red fluorescent signal was found at the cell membrane (Figure 1 D,G). However red fluorescent dots were also seen inside the cells; these localised in the cytoplasm to several areas surrounding the nucleus (Figure 1 J,K). Comparable observations were made when 3 μ AVR3a^{60' 47}-mRFP(His)₆ was applied to the onion, fish and human cells, even though the fluorescence signals were less intense (Figure 1 B,E,H). The cell membranes of all three types of cells were red fluorescent and only in the animal cells were fluorescent spots visible. When the animal and plant cells were treated with AVR3a^{22 59}-mRFP(His)₆ no fluorescence could be detected inside or on the surface of the cells using identical conditions. Even with concentrations 5 times higher than used for AVR3a^{22 147}-mRFP(His)₆ and AVR3a⁶°-¹⁴⁷-mRFP(His)₆, no fluorescence was observed (Figure 1C,F,I).

These observations were confirmed by performing antibody based immuno- localisation on RTG2 and HEK293 cells (Figure 8). In these experiments

AVR3a^{22"1 7}(His)₆ and AVR3a ^{2 147}-mRFP(His)₆ localise to the outer surface of HEK293 cells (Figure 8A,D). The same localisation was found for AVR3a^{2 "147}(His)₆ on RTG2 cells (Figure 8I). These localisation patterns were similar to what the earlier findings of the present inventors for SpHtpl^24"198 (His)₆ using identical microscope settings (van West et al., 2010). The recombinant proteins AVR3a^60'147(His)₆ and AVR3a^{60 47}-mRFP(His)₆ showed the same localisation pattern as the

AVR3a^{22 1 7}(His)₆ and AVR3a^{2 147}-mRFP(His)₆ constructs but had a decreased signal intensity (Figure 8B,E,J). No fluorescent signal could be observed on untreated cells (Figure 8L,H) and cells that were incubated with AVR3a^22"59(His)₆ or

AVR3a^{22 S9}-mRFP(His)₆ (Figure 8C,F,K). Furthermore, a previously described recombinant protein fragment from the Plasmodium falciparum effector GBP130 containing a functional PEXEL motif, PfGBP130^65"196(His)i₈, was included in these experiments (Boddey et al., 2009). This PfGBP130 variant showed a localisation pattern similar to the constructs containing the AVR3a effector domain (Figure 8G). In contrast to the experiments utilising the AVR3a-mRFP(His)₆ constructs on living cells, the immuno-localisation did not show specific signals inside the RTG2 and HE 293 cells neither with the (His)₆- nor with the mRFP(His)₆-tagged protein constructs (Figure 8A,D). This discrepancy is, most likely caused by the chemical fixative used with this method. This is also reflected in the observations made by using transmission electron microscopy (TEM).

For TEM immuno-gold labelling HEK293 cells were used since these cells gave the best ultrastructure and immuno-gold signal. Sufficient immuno-gold labelling inside the HEK293 cells on TEM sections was only achieved when the samples were prepared without the fixative uranyl-acetate (Figure 2A-A,B)- When the fixative uranyl-acetate was applied, for better preservation of the cellular ultrastructure, immuno-gold labelling inside the cells was largely decreased and higher antibody titres were needed to detect the protein on the cell surfaces (Figure 2A, 8M-A.B). This indicates that the protein constructs after uptake might be in structures containing high protein concentration so that even mild fixative concentrations like 4 % paraformaldehyde crosslink the (His)₆-epitope to the surrounding proteins and therefore it is inaccessible for any antibody detection.

Immuno-gold stained TEM samples either incubated with AVR3a^22"147(His)6 or AVR3a^{60' 47}(His)₆ lacking the uranyl-acetate gold particles were often found close to the mitochondrial membrane (Figure 2A-C.D). In addition, life cell imaging on RTG2 cells with either AVR3a^{22 7}-mRFP(His)₆ or AVR3a⁶°- ⁴⁷-mRFP(His)₆ showed that these proteins partially co-localise with the mitochondrial stain MitoTracker green FM (Figure 2B). However, if this mitochondrial localisation of AVR3a is genuine or caused by an excessive amount of protein present in the cells needs further investigation. In conclusion, these experiments demonstrate that the binding and translocation of AVR3a to cells happens in a pathogen independent manner and requires the AA 60-147 and not the RxLR-leader peptide, although its presence strengthens the interaction.

Example 2: The RxLR-EER sequence of AVR3a contains a dimerisation motif that is also capable of forming heteromers with SpHtpl

The fusion of the AVR3a RxLR leader sequence (Aamino acids 22-59) to GUS has been shown to translocate into potato cells when leaves are infected with transgenic P. infestans strains expressing this construct (Whisson et al., 2007). Here the present not able to bind to any of the used cell types and cannot enter the RTG2 and HEK293 cells, which is apparently in disagreement with the above results.

The explanation for both findings came from the size exclusion chromatography runs of the recombinant fusion proteins (Figure 9). Following column calibration (Figure 9 J,K), the size exclusion runs clearly show that the AVR3a construct containing the RxLR-EER motif and the effector domain (AA22-147) fused to the His-tag is a dimeric protein whereas the construct lacking the RxLR-EER runs according to its monomeric size. The RxLR-EER sequence fused to the His-tag is capable of forming tetra-, di- and possible monomers under the same conditions. The mRFP construct AVR3a^{60 147}-mRFP(His)₆ is monomeric (Figure 9E), whereas the mRFP-contructs AVR3a²²-¹⁴⁷-mRFP(His)₆ (Figure 9D), and AVR3a^{22 59}-mRFP(His)₆ (Figure 9F) do not have retention times expected for dimeric proteins. However, AVR3a^22'147- mRFP(His)₆ and AVR3a^{22 59}-mRFP(His)₆ show an apparent mass addition of ca. 20- and 10 kDa, respectively and two additional bands on SDS-PAGE gels, which could not be separated from the full length construct in the size exclusion runs (Figure 7 D,E).

MALDI-TOF spectra showed that the sum of these fragments adds up to the full- length mass (see legend for Figure 7). This suggests that the dimer formation of two full-length RxLR leader sequence containing mRFP-fusion molecules is not completely efficient, probably due to a steric hindrance of two bulky mRFP-domains in close proximity to one another, thus possibly destabilising the protein and gives an explanation for the amount of protein found in the flow through. Therefore, the dimers of AVR3a ^{2 47}-mRFP(His)₆ and AVR3a^{22 59}-mRFP(His)₆ must consist of one full length molecule and one truncated product that is cleaved within the mRFP domain. SpHtp1 ^{< 98}(His)₆ shows an equilibrium between a monomeric- and tetrameric form. It is not clear if the construct is capable of forming a higher oligomeric form because a large amount of the protein was eluted within the exclusion volume of the column but these fractions did not show any light scattering (Figure 9G). SpHtp1^{6S 98}(His)₆ could not be applied due to the fact that the theoretical extinction coefficient of this fragment at the detector wavelength of 280 nm is null.

The observation that the RxLR-EER containing mRFP constructs of AVR3a and their RxLR containing fragments are able to form dimers that could be considered as hetero-dimers and the fact that SpHtp1^24"l98(His)₆ adopts a tetrameric structure led to To address the above point, size exclusion runs were performed of the protein mixes AVR3a²²-⁵⁹(His)₆ / AVR3a²²-¹⁴⁷ mRFP(His)₆ and AVR3a^22"59(His)₆ / SpHtp1² -¹⁹⁸(His)₆. These runs, the data from which is shown in full below, gave indications that interactions between the proteins in mixes have taken place:

MALDI-TOF mass data:

A VR3a²²-⁵⁹-mRFP(His)₆:

Expected M_w: 32105.0

Found: 6284.6 = of 12568.7

9725.5 = AA 45-131

12568.7 = AA 194-304

19535.2 = A A 22-193

31976.3 = full length construct without start 'Μ'

AVR3a²²-⁵⁹(His)₆:

Expected Mw- 6812.4

Found: 6807.9 = full length protein (Δ-4 Da)

6676.2 = full length construct without start 'M' (Δ-4 Da)

AVR3a²²-¹⁴⁷(His)₆:

Expected M_w: 16979. 1

Found: 16982.9 = full length protein (Δ+4 Da)

8492.5 = 2⁺ of 16982.9

AVR3a²²-¹⁴ -mRFP(His)₆.

Expected M_w: 42271.6

Found: 42274.6 full length protein (Δ+2 Da)

19558.2 either 22-193 or 223-394

22730.6 either 194-394 or 22-222

A mixture of AVR3a^{22 59}(His)₆ and AVR3a^22"147 mRFP(His)₆ was applied to the column (Figure 9H). The chromatogram shows all peaks expected for the sum of both individual proteins. However, the amount of protein in the exclusion volume seems to AVR3a^22"147-mRFP(His)₆, appears. Furthermore, since only about one third of the mRFP construct and about half the concentration of AVR3a^22'59(His)₆ was used, compared to the individual runs, this does not explain why both proteins are detectable over the whole separation range of the column shown on the SDS-PAGE. Thus, these findings can only be explained if an interaction between both AVR3a constructs has taken place. Furthermore, a mixture of AVR3a^22'59(His)₆ and

SpHtp1^24"198(His)₆ also showed a decreased protein concentration in the exclusion volume and a higher amount of the tetrameric state of the SpHtp1^{2 198}(His)₆ when compared with the individual run, suggesting that heterodimerisation has taken place (Figure 131).

The above findings were confirmed when native protein gels were run using

SpHtp1^{24 98}(His)₆, AVR3a^{22 147}(His)₆ and AVR3a^{22 59}-mRFP(His)₆. On the coomassie blue stained native PAGE (Figure 3A), AVR3a^{2 '59}-mRFP(His)₆ shows two bands (lane 1 ), whereas four bands are visible for SpHtp1^24'198(His)₆ (lane 2). Mixing of both proteins results in the loss of one band (a) that is only present in the

SpHtp1^{24 198}(His)₆ sample, whereas a new band appears in the mixed sample (b, lane 3), thus, directly showing that a heteromer is formed between both proteins.

Since AVR3a^{22 147}(His)₆ has the same retention on the native page as

AVR3a^22'59-mRFP(His)₆, no mixed products could be detected. However, an indication that both proteins interact can be seen on unstained native PAGE'S following the fluorescence of AVR3a^{22 59}-mRFP(His)₆. If solely AVR3a^22"59- mRFP(His)₆ is run on a gel, a clear binding of the loading dye bromophenol blue is observed (Figure 3B, lane 3). Neither AVR3a^{22 147}(His)₆ nor SpHtp1^{24 198}(His)₆ showed this behaviour (Figure 3B, lane 2 and 4) however, when these proteins are mixed with AVR3a^{22 69}-mRFP(His)₆ (Figure 3B, lane 1 ,5) they suppress this interaction.

The dimerisation property of the RxLR-leader sequences led the present inventors to hypothesise that the formation of heteromers with a full length AVR3a construct via the RxLR leader sequence could facilitate the translocation of, for example,

AVR3a^22"59-mRFP(His)_e, which has the RxLR leader sequence but cannot translocate by itself. Indeed, this is what was observed, with a mixture of AVR3a ^{2 59}-mRFP(His)₆ with either AVR3a^{22" 47}(His)₆ or SpHtp1^{24 l98}(His)₆ enabling a small amount of the RxLR-mRFP construct to bind to onion and animal cells and to enter within 1 h the Example 3: RxLR proteins bind to phospholipids

The observations of AVR3a binding to the outer surface of animal and plant cells are most likely one of the first steps in the uptake process. To enter different cell types the effector protein has to utilise an interaction with membrane compound such as lipids, surface proteins or a glycoside. An interaction of AVR3a with cell surface glycosides was considered less likely since these differ considerably between plant-, human- and fish cells (Wilson, 2002).

Potential phospholipid binding was examined via phospholipid binding tests carried out initially using commercially available lipid-spotted membranes from Echelon Biosciences (Figure 11a). A subset of phospholipids was identified that interacted with the SpHtpl , AVR3a and PfGBP constructs that also showed cell surface binding as described in the experiments above. All tested proteins were incubated with the pre-equilibrated membranes for 20 min prior to antibody detection. The phospholipids that interact were sulfatide, phosphatidylglycerol, cardiolipin, phosphatidylinositol (3)- phosphate (PIP3), phosphatidyl-inositol (4)-phosphate (PIP4) and

phosphatidylinositol (5)-phosphate (PIP5). Furthermore, SpHtpl ^{6i 98}(His)₆, which lacks the RxLR leader sequence, was unable to bind the phospholipids, suggesting that the binding ability is linked to the presence of the RxLR leader sequence.

However, AVR3a^{60 1 7}(His)₆, which lacks the RxLR-EER motif of AVR3a, shows the same lipid binding pattern as AVR3a^22"147(His)₆, which does contain the RxLR leader sequence (Figure 11a2, a5). These observations were confirmed by the fact that a polypeptide consisting only of the RxLR-leader sequence fused to a (His)₆-tag (AVR3a^{22 59}(His)₆) does not display any specificity towards phospholipids

(Figure 11a6).

Since the oomycete RxLR- and the Plasmodium PEXEL motifs are functionally interchangeable (Bhattacharjee et al., 2006; Grouffaud et al., 2008), constructs derived from the Plasmodium PEXEL effector GBP130 were also tested. Both PfGBP^65'196(His)₁₈ and PfGBP⁶⁵-¹⁹⁶(His)₁₈-R84A/L86A/E88A lack the N-terminal signal peptide and the C-terminal transmembrane domain of GBP130, and the second construct containing the alanine substituted PEXEL is unable to translocate (Boddey et al., 2009). However, both PfGBP130 variants show the same lipid binding profile as the SpHtpl ^{24' 98}(His)₆, AVR3a ^{2 147}(His)₆ and AVR3a^{60 4} (His)₆ proteins (Figure 11a3, a7). These results show that the phospholipid binding ability of AVR3a and PfGBP130 is not per se linked to the presence of an RxLR or PEXEL motif, For SpHTP1^{69 198}(His)₆ it could simply be that the truncation destabilised the lipid- binding site, as the truncation of the protein was based purely on bioinformatic predictions. Therefore, additional tests of these proteins phospholipid binding ability were conducted; one method employed was Isothermal titration calorimetry (ITC)

ITC is a method that measures directly the enthalpy of chemical reactions, such as protein ligand interactions, and therefore would reveal insights into the binding mechanism. On the basis that all phospholipids interacting with the tested proteins carry double phosphorylated inositols in their polar head, isothermal titrations employing AVR3a^{22' 47}(His)₆ were performed using the polar head groups inositol and inositol-1 ,4-bisphosphate as titrants. It was expected that some binding with inositol- 1 ,4-bisphosphate would take place, since the specificity correlates with the presence of double phosphorylated inositol head groups and not the lipid chains. However, physical interactions of the AVR3a^{22"1 7}(His)₆ with either one of the titrants even at high, non-physiological, concentrations could not be detected (Figure 12A).

There are three possible reasons for the observed lack of interaction between AVR3a²²-^{1 7}(His)₆ and the titrants:

i) The binding of the protein variants to the phospholipids may be mainly due to an interaction with the fatty acid chains. However, this possibility can be ruled out since binding to lipids of the same structure carrying a non charged or triple charged head group could not be observed on the membranes;

ii) A second possibility could be that the binding is not coupled to a gain or loss in ground state enthalpy (ΔΗ⁰) and is only determined by a change in entropy (AS⁰), or any gain in ΔΗ⁰ is exactly cancelled out by the binding enthalpy (ΔΗπ-c)· However, such reactions are rare in chemistry and are normally coupled to gas formation/fixation so this is highly unlikely;

iii) only a small fraction of molecules are interacting with the phospholipids and therefore the heat of the reaction is below the detection limit of the apparatus (under such circumstances the interaction on the lipid spot membranes would still be detectable because antibody detection methods allow the amplification of weak signals).

That only a small fraction of the protein molecules is able to bind can be only explained by variations of the protein's three-dimensional structure. Therefore, lipid equilibriums. Interestingly, AVR3a^{22 47}(His)₆ is still able to bind to the phospholipids when completely unfolded in the presence of 8 urea (Figure 11 b). However, no binding to the phospholipids can be detected in the presence of 6 M GdnHCI, a charged chemical denaturant (Figure 11 c). This suggests that the phospholipid binding of the highly positively charged AVR3a^{2 147}(His)₆ (and the other lipid binding constructs) is mainly based on charge-charge interactions with the negatively charged phosphate groups of the phosphoinositol-phosphates. However, the variants AVR3a^{22 59}(His)₆ and SpHtp1^{69 l9B}(His)₆, which have similar charged stretches and charge distributions to AVR3a^{2 1 7}(His)₆ did not show a lipid binding in the presence of 8 urea (Figure 11d). This suggests that a specific spacing of the charged residues must be present in the constructs that show phospholipid affinity and only a small, unfolded fraction within these protein samples can bind.

Further evidence to corroborate this observation was obtained by adding

substoichiometric amounts of DnaK (Uniprot accession number P0A6Y8), the E.coli Hsp70 chaperone, in the binding assay. Native DnaK in its high affinity state was purified from E.coli using a standard extraction method (Zylicz and Georgopoulos, 1984). This method produced DnaK in the absence of its co-chaperones DnaJ and GrpE (a hydrolysis factor and nucleotide exchange factor respectively) in an ADP bound state, also referred to as the high affinity state (Bukau and Horwich, 1998; Ellis and Hartl, 1999; Packschies et al., 1997; Russell et al., 1998; Slepenkov and Witt, 2002). In this form DnaK binds and releases peptides over a time scale of minutes to even hours (Packschies et al., 1997), thus, a nearly 1 : 1 binding with DnaK can be assumed for molecules exposing hydrophobic stretches.

The above described binding properties can be assumed under the experimental conditions used for the phospholipid interactions assay. Indeed, when DnaK was used at a concentration of 1 μΜ, it was able to completely suppress the lipid binding of a solution of 20 μΜ PfGBP^{65 ~196}(His)i ₈ and strongly reduced the binding of 20 μΜ AVR3a ¹⁴⁷(His)₆ where weak signals only appeared after several hours exposure of the X-ray film (Figure 11e). SpHtp1^2<M98(His)₆ was not used due to the fact that this protein is a native unfolded protein [van West et al., 2010]. Therefore, it can be concluded that only a small fraction of AVR3a protein molecules bind to the phospholipids. Furthermore, the interaction seems to be based upon a specific charge-charge interaction. However, the significance of this finding is as yet unknown and could be artificial. Example 4: AVR3a and SpHtpl bind to tyrosine-O-sulfate

The first indication about one factor that contributes to cell surface attachment of AVR3a was given by an intrinsic property of AVR3a^{22' 47}(His)₆. In order to stabilise this construct in our initial experiments, HP0₄ ^2" ions were required (Figure 4A). Interestingly, the same stabilisation effect was achieved by employing SO4^2" ions. The amount of sulfate needed to stabilise AVR3a ^2"147(His)₆ is about half the concentration needed when using phosphate at RT (Figure 4B). In addition, the thermal stability of AVR3a^{22'1 7}(His)₆ is also positively affected by the presence of either phosphate or sulfate. Again, approximately half the concentration of sulfate is sufficient to accomplish the same stabilisation effect that can be achieved with phosphate (Figure 4C).

One post-translational modification of proteins present on the outer surface of cells and involving sulfate, is the conversion of tyrosine to tyrosine-O-sulfate (Chang et al., 2009). This modification has been demonstrated to be essential for some protein- protein interactions on the surface of cells (Kehoe and Bertozzi, 2000; Yu et al., 2007). Therefore, the present inventors initially tested if AVR3a^22"147(His)₆ is able to interact with tyrosine-O-sulfate using Fmoc-Tyr(S0₃)-OH. Interestingly, this compound induced aggregation of AVR3a^{22 7}(His)₆ even at high phosphate concentrations (Figure 4D). This interaction was so homogeneous and effective that the amount of protein available can be limiting during the aggregation measurements (Figure 4D inlet).

In order to rule out that the hydrophobic fluorenylmethyloxycarbonyl (Fmoc) group might induce aggregation of AVR3a^22"147(His)₆, the same experiment was carried out using H-Tyr(S0₃)-OH, which yielded the same result (Figure 12F). A control ITC titration experiment confirmed the interaction of AVR3a ^2'147(His)₆ with Fmoc- Tyr(S0₃)-OH, whereas in contrast even an 8 M urea denatured AVR3a^{22 147}(His)₆ sample did not show a detectable binding to inositol-1 ,4-bisphosphate (Figure 12B). Unfortunately, due to the fact that AVR3a^22'147(His)₅ aggregates in the presence of Fmoc-Tyr(S0₃)-OH, the titration could not be quantified.

Interestingly, AVR3a^22"147-mRFP(His)₆ did not aggregate when exposed to Fmoc- Tyr(S0₃)-OH (Figure 12E). However, isothermal titration experiments confirmed that Fmoc-Tyr(S0₃)-OH binds to AVR3a^{22' 7}-mRFP(His)₆ (Figure 12D). The recorded thermograms for this reaction are complex and suggest that the interaction goes detectable amount of aggregates may be attributed to the additional solubility permitted by the mRFP domain. Likewise, isothermal titration experiments showed that SpHtp1^{24 198}(His)₆ also binds to Fmoc-Tyr(S0₃)-OH but it does not aggregate in its presence. (Figure 12C).

The thermograms could not be satisfactorily fitted by a function adequate for a simple protein-ligand interaction. However, the thermograms could be described with a model involving two successive reactions. These "two-reaction" fits showed that the parameters describing the 'first reaction' (the phase with the highest amplitude) are rather similar for all the individual experiments performed. The parameters for the 'second reaction' were inconsistent between individual measurements. Therefore, it is most likely that the first, constant phase reflects the binding of Fmoc-Tyr(S0₃)-OH to SpHtp1^{24 198}(His)₆, whereas the second, inconsistent phase is probably caused by the thermal noise resulting from conformational changes within the protein. The fitted parameters describing the constant phase for the individual experiments were averaged and subsequent calculation yielded in a binding constant K_D of

(159±44) μΜ with a AH_ITC of (-15.5±7.1) kJ/mol, a TAS*₂₅=c of (5.8±2.8) kJ/mol and a molar ratio of (0.92±3.8) binding sites.

Willkins et al. (1995) have shown that the binding strength of P-selectin glycoprotein ligand-1 (PSLG-1) to P-selectin is strongly reduced when the sulfate groups of PSLG-1 's tyrosines are cleaved off by an aryl-sulfatase from Aerobacter aerogenes (Wilkins et al., 1995). In order to test if AVR3a interacts with a cell surface protein that carries a tyrosine-O-sulfate, the present inventors compared the uptake and binding of AVR3a^{22 147}-mRFP(His)₆ and AVR3a⁵°-¹⁴⁷-mRFP(His)₆ to cells treated with aryl-sulfatase (sulfatase type VI) and to non-treated cells. For all tested cell systems, onion-, RTG2 and HEK293, this treatment led to a strongly decreased binding/uptake signal with both AVR3a ^{2 7}-mRFP(His)₆ and AVR3a⁶°-¹⁴⁷-mRFP(His)₆ as compared to untreated cells (Figure 5 / Figure 13).

On sulfatase-treated cells the signal became stronger with longer incubation times and the same fluorescence pattern was observed when compared with untreated cells (Figure 131). This observation is expected for a protein-protein interaction because the sulfate cleavage from the tyrosines should mainly reduce the affinity- but not affect much the specificity constant of the interaction. The binding affinity to the cells could be restored when the sulfatase was washed away and the cells had time SpHtp1^{2 "198}(His)₆ also showed a reduced cell binding affinity towards aryl-sulfatase treated RTG2 cells compared to non-treated cells (Figure 6).

To ensure that the aryl-sulfatase preparation does not exhibit any phosphatase activity, the above described lipid membranes were first incubated for 3 h with 1 U aryl-sulfatase and subsequently with AVR3a^{22 147}(His)₆. In this experiment the protein showed the expected affinity towards the phospholipids on the membrane

(Figure 11 f). In contrast, when 1 U of alkaline phosphatase is was used instead of aryl-sulfatase, the binding of AVR3a^{22 47}(His)₆ to the phospholipids on the membrane was almost abolished.

Therefore, one can conclude that AVR3a and SpHtpl most likely bind to a cell surface protein that carries a sulfate modification in at least one tyrosine residue and that this protein (class) is probably conserved in onion-, fish- and human cells.

Example 5: Mutation of the RLLR amino acids within the RxLR-EER leader of AVR3a changes the secondary structure but still allows dimerisation and translocation of the polypeptide

In 2005 it was discovered that several oomycete effectors feature a conserved tetrameric motif, the RxLR motif, C-terminal after their signal peptides (Armstrong et al., 2005). Two years later, Whisson et al. were the first to show that this motif is involved in the translocation of these proteins (Whisson et al., 2007). Since then, several studies were carried out to understand the role that this motif plays in the translocation mechanism of these proteins (Bhattacharjee et al., 2006; Dou et al., 2008b; Grouffaud et al., 2008). Most of these studies relied on assays based on host pathogen interactions utilizing transgenic manipulated parasites carrying chimeric reporter genes mutated within this conserved motif.

It is herein shown that the RxLR-EER motif of AVR3a is not required for the translocation of this protein but seems to increase the uptake efficiency. In addition, the present inventors have discovered that the polypeptide containing this motif (the "RxLR leader sequence") contains a dimerisation site. Thus, the present inventors set out to investigate on a protein level the consequences of mutations within the conserved RxLR-motif.

Polypeptide containing the RxLR-EER motif of AVR3a (AA22-59) were compared of the variants had a homologue replacement of the AA43-47 RRLLR to KK IK and is hereinafter referred to as AVR3a^22"59 KMIK, the second variant carried an alanine replacement of the same amino acids stretch of amino acids and will hereinafter be referred to as AVR3a^22"59 A5. These variant sequences were each fused separately to the (His)₆-tag of pET21 b, over expressed in E. coli, and the corresponding polypeptides purified. The coomassie blue stained SDS-PAGES of the purified protein constructs are shown in Figure 15A.

The circular dichroism (CD) spectra of the RxLR variants AVR3a^22'59(His)₆ KMIK and AVR3a^{22 5} (His)₆ A5 are very similar to each other and, in comparison to the WT peptide, show a higher content of random coil structure (Figure 15B). Despite these structural differences, the KMIK and A5 variants are still able to form dimers (Figure 15D.E); however, compared to AVR3a^{2 59}(His)₆ (Figure 15C), the KMIK and A5 variants have a decreased tendency for the formation of tetrameric structure.

To investigate the effect that mutations within the RxLR-EER leader of AVR3a have on the ability of this protein to translocate into a eukaryotic cell, experiments were performed on RTG2 cells using a AVR3a-mRFP fusion protein bearing the following sequence variations: amino acids 43-47 RRLLR to KKMIK and amino acids 57-59 EER to DDK. This alteration of the RxLR-leader of AVR3a has been shown to be a loss of phenotype mutation (Whisson et al., 2007). Interestingly however, AVR3a^22' ⁷-mRFP KKMIK-DDK is still able to translocate into RTG2 cells, but with clearly decreased efficiency compared to the WT-mRFP fusion construct (Figure 19A 4, 21 4).

Example 6; 'Wild-type' AVR3a^{22 59}(His)_fi interacts with the effector domain of AVR3a whereas 'KMIK' an Ά5' AVR3a²²-⁵⁹(Hisy constructs do not

The stability of the AVR3a protein containing the RxLR leader sequence and the effector domain (amino acids 22-147) is strongly dependent on the presence of either phosphate- or sulfate ions; in the presence of 25 mM phosphate the

AVR3a^{22 ,47}(His)₆ is stable at 25°C, whilst in the absence of phosphate or sulfate ions the protein rapidly aggregates. AVR3a^60"147(His)₆, lacking the RxLR-EER motif, exhibited a slight tendency at 25°C to aggregate, with the tendancy to aggregate being independent of whether phosphate was present. However, when 5 μΜ

AVR3a⁶°-¹⁴⁷(His)₆ was mixed with 5 μΜ AVR3a^22'59(His)₆ an increased aggregation reaction was observed which could be suppressed to some extend with high In contrast to the 'wild-type' AVR3a^22"59(His)₆ construct, the 'KMIK' or Ά5'

AVR3a^{22 59}(His)₆ variants were not able to restore the sensitivity of AVR3a to the presence of phosphate ions, even when concentrations of the 'KMIK' or Ά5' peptides were used that were 5x higher than the effective concentration of the WT peptide (Figure 16B). In these experiments the resulting aggregation reaction resembled nearly the sum of the individual light scattering traces.

This difference in the interaction of AVR3a^{60 147}(His)₆ with WT AVR3a^22'59(His)₆ and the KMIK variant AVR3a ^{2 59}(His)₆ was confirmed by isothermal titration calorimetry (ITC). The thermogram obtained during the addition of AVR3a^22'59(His)₆ to

AVR3a^60'147(His)₆ showed a weak endothermic reaction which overcomes the exothermic dilution heat of AVR3a^{2 59}(His)₆ (Figure 16C). However, this thermogram could not be quantified because of the aggregation reaction in this mixture. In contrast, when AVR3a^{22 59}(His)₆ KMIK was used as a titrant, the thermogram profile showed only an exothermic reaction, most likely the result of the dilution heat; no aggregation occurred (Figure 16D).

The aggregation measurements and ITC data show that the WT RxLR-EER motif of AVR3a is interacting with the effector domain of this protein. In contrast, the interactions between the effector domain and either of the KMIK or A5 variants is marginal at best.

In order to obtain a deeper insight into the interaction of the AVR3a RxLR-EER motif with the effector domain, normalised CD spectra for AVR3a^60'147(His)₆ and spectra recorded immediately after mixing of AVR3a^{60 147}(His)₆ with AVR3a ⁵⁹(His)₆ (1 :1) were compared to the spectrum obtained for AVR3a^{2Z'1 7}(His)₆ (Figure 16E). The comparison shows that, at the first instance, the spectrum of the AVR3a^60"147(His)₆ plus AVR3a ^2"59(His)₆ mixture resembles the CD-spectrum of the AVR3a^{22"1 7}(His)₆ fragment. Thus, the interaction of the RxLR-EER peptide with the AVR3a effector domain does not seem to involve changes within the secondary structure, so must be based on tertiary interactions.

Example 7: Phosphate binding to AVR3a^{22 1 7}(His)_e effects secondary structure of the dimer The role phosphate plays in stabilising AVR3a ¹ (His)₆ were investigated using CD- spectra and gel-filtation profiles recorded in the presence and absence of phosphate ions.

The CD-spectra shown in Figure 17A reveal that phosphate binding to

AVR3a^{22 147}(His)₅ stabilises a certain secondary structure. Unfortunately, details in the range of 190-215 nm could not be gained due to the buffer composition needed to stabilise this protein when phosphate was absent, and therefore no predictions could be made for this conformational state.

The gel filtration profile for AVR3a^{22 7}(His)₆ in the absence of phosphate showed that the protein is mainly present in its dimeric form (Figure 17B). However, the broad shoulder at higher volumes of the dimeric peak may indicate that some proportion of the protein might be present in its monomeric state under these conditions.

Example 8: AVR3a^{22 7}(His)fi can adopt three soluble conformational states

When the temperature stability of AVR3a^22"147(His)_e was observed, it was noted that the transition temperature (T_M) changes quite dramatically with altered protein concentrations. This phenomenon was analysed using a fluorescence assay based on the release of CyproOrange from unfolding protein molecules ((Ericsson et al., 2006; Yeh et al., 2006) exemplarily shown in Figure 18A). This assay allowed us to consistently monitor the thermal unfolding of this protein over a large concentration range.

Using a protein concentration of 5 μΜ the present inventors found a thermal unfolding transition temperature (T_M) of 42°C with this method. This value is consistent with the value previously reported for the same concentration using CD- spectroscopic measurements . At this concentration the secondary structure of AVR3a^{22"1 7}(His)₆ is mainly a-helical . However, the T_M drops over 10°C from 43.3°C at 5.9 μΜ to 32.2°C at 44.2 μ suggesting a 2^nd order equilibrium favouring a low melting temperature state at higher protein concentrations (Figure 18B).

¹H-NOESY N R-spectra show that at a 250 μΜ AVR3a^22"147(His)₆ sample adopts a distinct fold because ¹H-¹H-cross peaks were well dispersed and a great amount of peaks both in the ¹HN-¹HN and in the ¹HN-¹H_a/p-region could be seen (red box in cross peaks in Figure 18D) confirmed that the protein adopted an a-helical fold at this concentration because most of the peaks were found in an NH-region≥ 0.22 ppm upfield-shifted (lower ppm values) to values of ~8.1 ppm, which is typical for residues building a-helices. The region typical for values of residues in a random- coil-conformation are indicated by the black box in the spectrum shown in Figure 18D (Wishart et al., 1991 ). The green cross peaks in the spectrum in Figure 18D (250 μ sample) showed a clear and good dispersion of about 1 8 peaks, therefore of about 132 residues (additional peaks resulting from measuring NH-couplings of the side chains of the tryptophane-Νε and therefore these have to be subtracted). AVR3a^{22 147}(His)₆ consists of 148 residues; therefore 145 peaks should be visible under ideal conditions (1 8 residues for the whole protein with His-tag minus 3 prolines, which cannot be seen in such a spectrum). Here more than 90 % of the residues can be found, which shows, that the protein has to be well folded at a concentration of 250 μΜ.

The red spectrum in Figure 18D was recorded with a sample concentration of -1.2 mM. There only about 30 peaks were found, thus, a third conformational state was observed. At this concentration the protein has lost its a-helical character and appears to be randomly folded/unfolded. However, the fact that AVR3a^{22" 47}(His)₆ is still perfectly soluble at this concentration indicates that the protein is able to adopt an soluble native unfolded state. Unfortunately, at present there is no single method available which would allow a consistent assessment of the secondary- or tertiary structure over such a broad concentration range. Due to the law of mass action all three conformations have to be always in equilibrium. Further investigation will shed light on how biological function is linked to a the conformation of AVR3a.

Example 9: The B. coli HSP70 chaperone DnaK inhibits the uptake of AVR3a into eukarvotic cells

The present inventors have provided evidence that the initial step of the AVR3a translocation into cells involves a partially unfolding of the protein induced by the binding to tyrosine-O-sulfate. If it is assumed this partial unfolding is transient, chaperones specialised in the binding of partially unfolded proteins, i.e. E. coli DnaK, should be able to affect this process.

To investigate this possibility RTG2 cells were used, since the size and optical properties of these cells allows visualising the uptake in great detail. The uptake of of ADP bound DnaK was tested. RTG2 cells incubated for 30 min with either AVR3a²²-¹⁴⁷-mRFP(His)₆ or AVR3a^{60 147}-mRFP(His)₆ showed a strongly reduced fluorescence uptake in the presence of substoichometric amounts of DnaK (Figure 19A and 21). The localisation pattern of these samples did not differ from the samples were no DnaK was added.

Example 10: Refolding AVR3a^22',47(His)fi is directed into the nucleus, refolding AVR3a^{22 147}-mRFP(His)g is not

According to the law of mass action the three conformations of AVR3a^22~147(His)₆ which had been observed must be in equilibrium. Therefore, the effect of changing the folding equilibrium on the binding and uptake of the protein into cells was investigated.

Due to the fact that chemical fixation abolishes the recognition of the (His)₆ epitope of intracellular localised AVR3a^{22~1 7}(His)₆ transmission electron microscopy (TEM) in combination with immuno-gold labelling was chosen to follow the localisation of the protein. HEK293 and RTG2 cells were used for the TEM immuno-gold experiments for the following reasons: i) localisation and uptake of the AVR3a-mRFP constructs is comparable for both RTG2 and HEK293 cells ii) HEK293 cells show a better ultra- structural preservation than RTG2 cells after TEM sample preparation using high pressure freezing . Thus, the information obtained using both cell lines should give a more robust picture and a better insight into the localisation of the translocated protein.

The present inventors exposed cells to refolding AVR3a ^2"147(His)₆ and AVR3a^22"147- mRFP(His)₆. Interestingly, TEM immuno-gold staining showed that AVR3a^{22 "147}(His)₆ in the process of refolding is directed into the nucleus in both RTG2 and HEK293 cells (Figure 19B). Occasionally conglomerates of gold particles are seen in the cytosol (Figure 19B, 2b/d) and close to the nuclear membrane (Figure 19B, 2e). Unfortunately, the sample preparation lacks the structural perseveration of membranes therefore, it is not entirely clear if the protein is transported in vesicles through the cells. However, the shape of the conglomerates in the cytosol and the higher electron background density in the areas where gold particles were detected could indicate such a process.

In order to test if the above result is caused by proteolytic degradation of the protein subsequently analysed by western blotting. This was then compared to samples obtained from RTG2 cells exposed to refolding AVR3a^{22"1 7}(His)₆.

To analyse the proteolytic stability of folded AVR3a^22'147(His)₆, the protein was incubated for various times in the presence of proteinase K (Figure 20A). This resulted in a specific degradation pattern leaving one prominent band after a 30 min incubation. This band was subjected to N-terminal Edman-sequencing and showed that the stable AVR3a fragment starts with AA67. Western blotting of these samples and subsequent probing with the same anti-His antibody that was used as primary antibody for the TEM immuno-gold staining showed that at first a fragment from the N-terminus of the protein in concert with the His-tag is removed (Figure 20B).

The western blot obtained with the proteins from RTG2 cells that were incubated with native AVR3a^{22'1 7}(His)₆ and subsequently treated with proteinase K (to remove all epitopes from protein bound to the surface of the cells) showed only one band with the size of the full length protein construct (Figure 21C). When refolding

AVR3a^22"'⁴⁷(His)₆ was applied onto RTG2 cells some of the protein seemed to be N- terminal processed (Figure 21 D). However, the observed size difference between both AVR3a^{22 147}(His)₆ forms was small. In contrast to the TEM results obtained with refolding AVR3a^{22 147}(His)₆, life imaging of cells exposed to refolding AVR3a^22'147- mRFP(His)e showed the same localisation pattern obtained with non-urea treated protein but with a decreased fluorescence uptake. The present inventors did not observe any translocation of this protein construct into the nucleus (Figure 19A 3; 21, 3). Considering that AVR3a ^{" 7}(His)₆ can be considered as completely unfolded after a 30 min incubation in 6 M urea , whereas the structure of mRFP stays intact under the same condition (Broering and Bommarius, 2008), thus, exposing

AVR3a ^{2 47}-mRFP(His)₆ to 6 urea should selectively denature the domain derived from AVR3a and not the mRFP part. Therefore, the presence of mRFP prevents the nuclear localisation of refolding AVR3a. References

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Claims

1. Use of a recombinant polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to the amino acid sequence AVR3a^60'147, AVR3a^22"147 or SpHtpl^24"198 shown in Figure 14 to enhance translocation of a payload across the membrane of a eukaryotic cell, wherein said membrane comprises a surface molecule including an aryl-O- sulfate moiety,

whereby said motif interacts with said aryl-O-sulfate moiety of said surface molecule causing translocation of said polypeptide and payload.

2. The use according to claim 1 to selectively translocate a payload into members of a specific cell population in an organism, wherein members of the specific cell population have a higher concentration or number of aryl-O-sulfate moieties on their surface molecules than members of otherwise comparable cell populations

3. A composition comprising:

(i) a translocation sequence comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to the amino acid sequence AVR3a^{60 147}, AVR3a^22"147 or SpHtpl^{24 198} shown in Figure 14, wherein the polypeptide sequence directs translocation of the polypeptide into a eukaryotic cell; and

(ii) a payload coupled to the translocation sequence.

4. The composition according to claim 3 wherein the translocation sequence is derived from an AVR protein.

5. The composition according to any one of claims 3 to 4 wherein the translocation sequence further comprises at least one RxLR leader sequence such as to form a multimer with the payload which is translocated.

6. The composition according to any one of claims 3 to 5 wherein the translocation sequence and the payload both include at least one RxLR leader sequence such as to form a multimer which is translocated.

7. The composition according to any one of claims 3 to 4 wherein the translocation sequence and/or the payload does not comprise a RxLR motif within the N-terminal most 60 amino acids.

8. The composition according to any one of claims 3 to 7 wherein the payload is a therapeutic agent, a marker, marker for viability when leads out, a protective agent, a cytotoxin, or siRNA.

9. The composition according to any one of claim 5-8 wherein the RxLR leader sequence comprises at least one RxLR motif.

10. A method of producing a composition according to any one of claims 3 to 9, comprising the steps of:

(i) providing a translocation sequence comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to the amino acid sequence AVR3a^60"147, AVR3a^22'147 or SpHtpl^24"198 shown in Figure 14, wherein the translocation sequence directs translocation of the polypeptide into a eukaryotic cell, and at least one RxLR leader sequence;

(ii) providing a payload comprising at least one RxLR leader sequence;

(iii) combining the translocation sequences and the payload in vitro so as to form multimers comprising at least one copy of the translocation sequence and at least one payload.

1 1. The method according to claim 10 wherein the RxLR leader sequence comprises at least one RxLR motif.

12. A multimer having:

(i) a first subunit comprising a translocation sequence comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to the amino acid sequence AVR3a^60'147, AVR3a^22"147 or SpHtpl^{24 198} shown in Figure 14, wherein the translocation sequence directs translocation of the polypeptide into a eukaryotic cell, and at least one RxLR leader sequence;

(ii) a second subunit comprising a payload and at least one RxLR leader sequence.

13. The multimer according to claim 12 further comprising at least one additional and either (i) comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% identity to the amino acid sequence AVR3a^60"147, AVR3a^{22 147} or SpHtpl ^24-198 shown in Figure 14, wherein the

translocation sequence directs translocation of the polypeptide into a eukaryotic cell, or

(ii) a payload.

14. The multimer according to either one of claims 12 or 13 comprising two or more non-identical payload subunits.

15. The multimer according to any one of claim 12-14 wherein the RxLR leader sequence comprises at least one RxLR motif. 6. The composition according to any one of claims 3 to 9 or the multimer according to any one of claims 12 to 15 for use in a method of treatment of the human or animal body.

17. The composition for use according to claim 16 wherein the composition or multimer is used to selectively deliver a therapeutic agent, a marker, marker for viability when leads out, a protective agent, a cytotoxin, or siRNA into a target cell. 8. The use of a composition according to any one of claims 3 to 9 or a multimer according to any one of claims 12 to 15 in the manufacture of a medicament for the treatment of a disorder.

19. A method of treating a disorder comprising administering an effective amount of a composition according to any one of claims 3 to 9 or a multimer according to any one of claims 12 to 15 to a patient in need thereof.

20. A method of producing a multimer, the method comprising the steps of:

(i) providing a first polypeptide comprising at least one RxLR leader sequence;

(ii) providing a second polypeptide comprising at least one RxLR leader sequence;

(iii) combining the first and second polypeptide sequences so as to form multimers.

21. The method according to claim 20 wherein the first polypeptide and/or the second polypeptide are recombinant.

22. The method according to either one of claims 20 or 21 wherein the first and second polypeptide sequences are combined in vitro.

23. The method according to any one of claim 20-22 wherein the RxL leader sequence comprises at least one RxLR motif.

24. The use of a first polypeptide sequence comprising at least one RxLR leader sequence and a second polypeptide sequence comprising at least one RxLR leader sequence to form a multimer.

25. The use according to claim 24 wherein the RxLR leader sequence comprises at least one RxLR motif,

26. Use of a desulfating agent or a chaperone agent to inhibit or block

translocation of a polypeptide across the plasma membrane of a eukaryotic cell, wherein the polypeptide comprises an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22'147 or SpHtpl^24'198 shown in Figure 14.

27. The use according to claim 26 wherein the desulfating agent specifically reduces the sulfation of a surface molecule including an aryl-O-sulfate moiety,

whereby the amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^{22"1 7} or SpHtpl^24"198 shown in Figure 14 interacts with said aryl- O-sulfate moiety of said surface molecule to cause translocation of said polypeptide and payload.

28. The use according to either one of claim 26 or claim 27 wherein the desulfating agent is an aryl-sulfatase enzyme.

29. The use according to claim 26 wherein the chaperone agent is E.coli DnaK.

30. The use according to any one of claims 26 to 29 wherein the protein whose

31. A process for increasing the pathogen resistance of a eukaryotic cell, wherein said process comprises introducing a genetic modification into the cell such as to inhibit or block the translocation of an effector peptide of the pathogen across the cell membrane into cell cytoplasm, the process comprising:

(i) providing a genetically modified cell;

(ii) providing a construct comprising a translocation sequence, which sequence comprises an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl^24"198 shown in Figure 14;

(iii) exposing the genetically modified cell to said construct;

(iv) determining the presence or amount of the construct in the ER or cytoplasm of the genetically modified cell;

(v) correlating a reduction in the presence or amount of the construct in the cytoplasm of the genetically modified cell compared with a corresponding

determination in the wild-type cell with the ability of the genetic modification to inhibit or block the translocation of an effector peptide of the pathogen across of the cell membrane.

32. A process according to claim 31 wherein the genetic modification of the cell reduces the number or concentration of aryl-O-sulfate moieties on the cell surface relative to a wild-type cell.

33. A process according to claim 32 wherein the genetic modification of the cell causes, relative to the wild-type cell, (i) an increase in the activity or expression of one or more aryl sulfatase enzymes, or (ii) a decrease in the activity or expression of one or more ty rosy I protein sulfotransferase enzymes.

34. A process according to claim 31 wherein the genetic modification causes, relative to the wild-type cell, an increase in the activity or expression of an agent that either (i) competes with the amino acid sequence motif for binding aryl-O-sulfate moieties on the cell surface; (ii) mimics an aryl-O-sulfate moiety of a cell surface molecule; or (iii) is a chaperone agent.

35. A process according to claim 34 wherein the agent comprises an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60'147 _p AVR3a^22"147 or SpHtpl ^24"198 shown in Figure 14.

36. A process according to claim 34 wherein the agent is E.coli DnaK.

37. A process according to any one of claims 33 to 36 wherein the increase in the activity or expression of the agent is upregulated in response to the onset of infection.

38. A process as claimed in any one of claims 31 to 37 wherein the construct further comprises at least one RxLR leader sequence.

39. A process as claimed in claim 38 wherein the RxLR leader sequence comprises at least one RxLR motif.

40. A process as claimed in any one of claims 31 to 39 wherein the construct includes an effector peptide of the pathogen, optionally having its native signal peptide and\or translocation sequence.

41. A process as claimed in any one of claims 31 to 40 wherein the construct is an artificial fusion construct comprising:

(i) a payload;

(ii) a translocation sequence comprising an amino acid sequence motif having 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl^24'198 shown in Figure 14; and, optionally,

(iii) an N-terminal signal sequence.

42. A process as claimed in any one of claims 31 to41 wherein the construct includes a detectable group which is optionally a fluorescent protein, GUS or any other reporter gene.

43. A process as claimed in any one of claims 31 to 42 wherein the cell is a plant cell.

44. A desulfating agent or a chaperone agent for use in a method of treatment of or blocks translocation of a polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl ^24"198 shown in Figure 14 across the plasma membrane of a eukaryotic cell.

45. A desulfating agent or a chaperone agent for use in a method of treatment of a disorder associated with pathogenic cell invasion, wherein the desulfating agent or chaperone agent inhibits or blocks translocation of a polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147,

AVR3a^{22 147} or SpHtp ^24'198 shown in Figure 14 across the plasma membrane of a eukaryotic cell.

46. A desulfating agent or a chaperone agent for use in the manufacture of a medicament for the treatment of a disorder associated with pathogenic cell invasion, wherein the desulfating agent or chaperone agent inhibits or blocks translocation of a polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22'147 or SpHtpl ^24"198 shown in Figure 14 across the plasma membrane of a eukaryotic cell.

47. A method of treating a disorder associated with pathogenic cell invasion comprising administering to a patient in need thereof an effective amount of a desulfating agent or a chaperone agent that inhibits or blocks translocation of a polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^50"147, AVR3a^22'147 or SpHtpl ^{24 198} shown in Figure 14 across the plasma membrane of a eukaryotic cell.

48. Use of an agent which inhibits binding of an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^{60"1 7}, AVR3a^22"147 or SpHtpl^{24 198} shown in Figure 14 to a surface molecule having an aryl-O-sulfate moiety to inhibit or block translocation of a polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60'147, AVR3a^22'147 or SpHtpl ^{24 198} shown in Figure 14

49. The use according to claim 48 wherein the agent specifically reduces translocation across the membrane of polypeptides comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22'147 or SpHtpl²⁴-¹⁹⁸ shown in Figure 14.

50. The use as claimed in either one of claims 48 or 49 wherein the agent is either:

(i) intended to compete with the polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl^24"198 shown in Figure 14 for binding aryl-O-sulfate moieties on the cell surface, or

(ii) a aryl-O-sulfate moiety mimetic intended to compete with the aryl-O- sulfate moieties on the cell surface for binding the polypeptide comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl^24"198 shown in Figure 14.

51. The use as claimed in any one of claims 48 to 50 wherein the agent comprises an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence

AVR3a^{60 H7}, AVR3a^22"147 or SpHtpl^24"198 shown in Figure 14, of or a variant thereof.

52. The use according to any one of claims 48 to 51 wherein the protein whose translocation is inhibited or blocked is an exogenous pathogenic protein.

53. A process for providing a pathogen resistance composition for use in a eukaryotic cell, wherein the composition comprises an agent which inhibits or blocks the translocation of an effector peptide of the pathogen across the plasma membrane of the cell into the cell cytoplasm, the process comprising:

(i) providing a test agent and a eukaryotic cell;

(ii) providing a construct comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 00% sequence identity to the amino acid sequence AVR3a^60'147, AVR3a^{22'1 7} or SpHtpl^24"198 shown in Figure 14; (iv) determining the presence of amount of the construct in the ER or cytoplasm of the cell;

(v) correlating a reduction in the presence or amount of the construct in the cytoplasm of the cell with the ability of the agent to inhibit or block the translocation of an effector peptide of the pathogen across the membrane of the cell.

54. A process as claimed in claim 53 wherein the test agent is either:

(i) intended to compete with the construct for binding aryl-O-sulfate moities on the cell surface;

(ii) an aryl-O-sulfate moiety mimetic intended to compete with the i.e tyrosine-O- sulfate residues on the cell surface for binding the construct;

(iii) intended to specifically remove the sulfate group from aryl-O-sulfate moities of cell surface molecules; or (iv) specifically prevent the addition of sulfate groups to aryl moieties of cell surface molecules.

55. A process as claimed in either one of claims 53 or 54 wherein the test agent is a peptide or protein provided by expression from nucleic acid introduced into the plant cell.

56. An agent which inhibits binding of an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22'147 or SpHtpl ^24-198 shown in Figure 14 to a surface molecule having an aryl-O-sulfate moiety for use in a method of treatment of the human or animal body.

57. An agent which inhibits binding of amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 00% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtp ⁹⁸ shown in Figure 14 to a surface molecule having an aryl-O-sulfate moiety for use in a method of treatment of a disorder associated with pathogenic cell invasion.

58. The use of an agent which inhibits binding of amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22'147 or SpHtpl^24"198 shown in Figure 1 to a surface molecule having an aryl-O-sulfate moiety for use in the manufacture of a medicament for the treatment of a disorder associated with

59. A method of treating a disorder associated with pathogenic cell invasion comprising administering to a patient in need thereof an effective amount of an agent which inhibits binding of an amino acid sequence motif having at least 20, 30, 40, 50,

60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^{22' 7} or SpHtpl^24'198 shown in Figure 14 to a surface molecule having an aryl-O-sulfate moiety.

60. A process for producing a plant with enhanced resistance to pathogens that express an effector protein comprising an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"147, AVR3a^22"147 or SpHtpl^24'198 shown in Figure 14, which process comprises any one or both of:

(i) over-expressing an agent selected in the plant, wherein the agent is a desulfating agent as described herein or an agent as described in any one of claims 48 to 52;

(ii) introducing into the plant a genetic modification as described in any one of claims 31 to 43.

61. A process for producing a plant with enhanced resistance to plant pathogens which comprises over-expressing in said plant an agent that either (i) binds to or which comprises the an amino acid sequence motif having at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to the amino acid sequence AVR3a^60"'⁴⁷, AVR3a^22"147 or SpHtpl^{24 198} shown in Figure 14, or (ii) reduces the number or concentration of sulfate groups on aryl moieties of cell surface molecules.

62. The process according to claim 61 wherein said agent is secreted to the extra-cellular compartment of the cell.

63. A plant produced according to process of any one of claims 60 to 62.