7. Advances in host-pathogen molecular interactions: rust effectors as targets for recognition

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1 7. Advances in host-pathogen molecular interactions: rust effectors as targets for recognition Peter Dodds 1, Greg Lawrence 1, Rohit Mago 1, Michael Ayliffe 1, Narayana Upadhyaya 1, Les Szabo 2, Robert Park 3, Jeff Ellis 1. Abstract Rust fungi can cause devastating diseases in agriculture and are particularly important pathogens of wheat. We have been using the flax (Linum usitatissimum) and flax rust (Melampsora lini) model system to study disease resistance mechanisms to this important class of pathogens. Rust resistance in flax and other plants is mediated by the plant innate immunity system in which highly polymorphic resistance (R) proteins act as receptors that recognize specific avirulence (Avr) proteins produced by the pathogen. This race-specific resistance is characterised by Flor s gene-for-gene model, first proposed based on the flax rust system. In gene-for-gene resistance, recognition between the R and Avr proteins initiates defense responses leading to host resistance to infection, including a localised necrosis or hypersensitive response. Nineteen different rust resistance genes have been cloned from flax, including 11 allelic variants of the L locus, which all encode cytosolic proteins with conserved nucleotide-binding (NB) and Leucine-rich repeat (LRR) domains. Four families of Avr genes, AvrL567, AvrM, AvrP123 and AvrP4, have been identified in the flax rust pathogen and all encode small secreted proteins. Rust Avr proteins are secreted from haustoria, specialized infection structures that penetrate the host cell wall, and are translocated across the host plasma membrane and into the host cytoplasm. These proteins are probably members of a suite of disease effectors involved in manipulating host cell biology to facilitate infection, but have become targeted for recognition by the host immune system. As yet the mechanism of Avr protein transport is unknown, but could prove to be a useful target for novel disease control strategies. Recognition of at least two of these Avr proteins is based on direct interaction with the cytoplasmic NB-LRR R proteins. One interesting observation from the flax rust system is that all of the virulent rust strains retain 1 CSIRO Plant Industry, PO Box 1600, Canberra, ACT 2601 Australia; 2 USDA- ARS Cereal Disease Laboratory, University of Minnesota, St Paul, MN 55108, USA: 3 Plant Breeding Institute, University of Sydney, PMB 11, Camden, NSW 2570, Australia. peter.dodds@csiro.au intact copies of the Avr genes, but have altered their sequences sufficiently to escape recognition. Thus it may be possible to re-engineer R genes to recognise new Avr gene variants. We are currently identifying haustorially expressed secreted proteins from wheat stem rust as candidate Avr/effector proteins. Keywords: haustoria, avirulence, resistance, secreted proteins, effectors Introduction In order to successfully establish disease, plant pathogens such as the wheat rusts, must first overcome the natural defences of the plant. These include preformed barriers, such as the waxy cuticle, as well as inducible responses triggered by the plant innate immunity system (Takemoto and Jones 2004). The first layer of the immune system involves recognition of pathogen associated molecular patterns (PAMPs) such as chitin or flagellin (Jones and Dangl 2006). Recognition of these factors by cell surface receptors leads to PAMP-triggered immunity (PTI) which is effective in preventing infection by non-adapted pathogens. Bacterial pathogens of plants are known to overcome these defences through the use of effector proteins that are delivered into host cells by the Type III secretion system. However, many of these effectors are recognized by a second layer of the plant defense system that involves intracellular receptors that are the products of the classically defined resistance (R) genes of the gene-for-gene system, first defined in the flax rust disease system (Flor 1971). In this context pathogen effectors are known as avirulence (Avr) proteins and their recognition leads to rapid activation of a localized cell death termed the hypersensitive response (HR), which is thought to limit the spread of the pathogen from the infection site (Chisholm et al. 2006). This layer of defense has been termed effector triggered immunity (ETI), and involves direct or indirect recognition of pathogen effector proteins by plant R proteins. Recent advances in the study of biotrophic oomycete and fungal pathogens, including rusts, indicate that this general picture of pathogen effector/host immunity interactions also holds true for these eukaryotic pathogens (Ellis et al. 2007; Tyler 2009). Among studies of biotrophic fungi, work on the flax (Linum usitatissimum) and flax rust (Melampsora lini) disease system has so far yielded the most information (Lawrence et al. 2007). Rust fungi are obligate biotrophs, meaning that they are completely dependent on nutritional resources obtained from living host cells Oral Papers 2009 Technical Workshop 49

2 for their growth and reproduction. During infection of host plants, fungal hyphae grow in the intercellular spaces of the leaf, but form a close association with host mesophyll cells through haustoria. These specialized infection structures penetrate the plant cell wall and invaginate the plant cell plasma membrane, and are thought to be the primary sites of nutrient acquisition from the plant (Voegele and Mendgen 2003). The flax rust system has been an enduring model in plant disease resistance, having been the basis for Flor s gene-for-gene model. In gene-for-gene resistance, the products of host resistance (R) genes determine recognition of pathogen avirulence (Avr) gene products to initiate defense responses leading to resistance. One of the hallmarks of this system is the high degree of specificity between corresponding R and Avr genes. Previous isolation of flax resistance genes, including 11 alleles of the L locus and representatives of the M, N and P loci, has provided insights into resistance gene specificity. The recent identification of flax rust Avr proteins has now allowed more detailed analysis of the recognition events that trigger rust resistance. Flax R genes and their products Genetic studies of the interaction between the flax plant and flax rust have identified about 30 flax resistance (R) genes, which occur as series of closely linked or allelic genes at 5 loci, and about 30 corresponding flax rust avirulence (Avr) genes that are mostly dispersed in the flax rust genome. Nineteen different rust resistance genes have now been cloned from flax, including 11 allelic variants of the L locus and representatives of the M, N and P loci (Ellis et al. 1999; Anderson et al. 1997; Dodds et al. 2001a, b). These genes all encode predicted cytosolic resistance proteins containing nucleotide binding (NB) and leucine rich repeat (LRR) domains, as found for the majority of known R genes in plants. The flax R proteins belong to a major subclass of this family which contain an N-terminal domain related to the Drosophila Toll and human interleukin-like receptor intracellular signaling domains (TIR domain). The precise roles of these domains and the mechanism by which recognition is linked to the activation of defense signaling is not well understood. However, the LRR domain appears to be the major determinant of recognition specificity, since most amino acid variation occurs in this domain as a result of strong positive selection (Dodds et al. 2000), and domain swaps between alleles of either the L or P loci of flax, show that this region controls recognition specificity (Ellis et al. 1999; Dodds et al. 2001a). LRR domains occur in a wide range of proteins and are generally implicated in protein-protein interactions (Kobe and Kajava 2001). The TIR domain is likely to be involved in signaling, as suggested by the functions of mammalian homologues and the observation that deletion and point mutations in this region of the N gene disrupt signaling events that lead to tobacco mosaic virus resistance (Dinesh-Kumar et al. 2000). Indeed we have found that overexpression of the L6 TIR domain leads to activation of the HR (Frost et al. 2004). However, the TIR may also play a role in pathogen recognition (Ellis et al. 1999; Luck et al. 2000; Burch- Smith et al. 2007). The NB domain is presumed to bind and hydrolyse ATP, as has now been shown for two tomato R proteins (Tameling et al. 2002, 2006), and it is likely that ATP/ADP exchange plays a role in controlling R protein activation. Studies on several NB-LRR proteins demonstrated that domains within the proteins interact (Bendahmane et al. 2002; Moffett et al. 2002; Ueda et al. 2006) and some constitutive gain-of-function mutations have been identified in the NB domain. These data support the notion that R proteins may be held in an inactive state through intramolecular interactions that are released by the presence of the Avr protein. Flax rust Avr genes and their products Until recently isolation of avirulence (Avr) genes from biotrophic fungi and oomycetes has been difficult because these organisms cannot be readily cultured or transformed. However, four families of Avr genes, AvrL567, AvrM, AvrP123 and AvrP4 have now been identified in flax rust (Table 1; Dodds et al. 2004, Catanzariti et al. 2006). The first of these (AvrL567) was Table 1 Cloned Avr gene families from flax rust Avirulence locus Product size (aa) Cys rich # gene family members Cognate R genes AvrL no 12 L5, L6, L7 AvrM no 6 M AvrP4 95 yes 3 P4 AvrP yes >2 P, P1, P2, P3 50 Advances in host-pathogen molecular interactions: rust effectors as targets for recognition

3 isolated by a subtractive hybridisation screen for rust genes expressed during infection followed by genetic mapping in a rust family segregating for multiple Avr specificities. The subsequent three were isolated by screening a cdna library from rust haustoria by ConAaffinity chromatography (Hahn and Mendgen 1992) for genes encoding secreted proteins. All four Avr gene families encode small secreted proteins that are expressed in haustoria and are apparently translocated into host cells during infection. Evidence for this translocation comes from the observation that transient expression of these Avr proteins as cytoplasmic proteins (i.e. lacking the signal peptide) in plants can trigger a defense response dependent on the corresponding R genes. This shows that Avr protein recognition occurs inside plant cells, implying that these proteins are translocated during infection. Indeed, our recent work has detected the flax rust AvrM protein inside infected host cells by immunolocalisation. Kemen et al. (2005) also showed that a protein (UfRTP1) secreted from broad-bean rust (Uromyces fabae) haustoria is translocated into host cells. It seems likely that these proteins are part of a larger suite of proteins (probably including the other 20 or so flax rust Avr gene products) that are secreted from rust haustoria and translocated into the plant cytoplasm. These proteins represent a set of host-targeted effector proteins that are presumed to play roles in promoting the infection process. Biotrophic and hemibiotrophic oomycetes also secrete large arrays of effector proteins that are directed into the host cytoplasm during infection (Kamoun et al. 2006). These oomycete proteins are characterised by a conserved RxLR motif that is related to a transport signal responsible for uptake of secreted proteins of the malaria parasite (Plasmodium falciparum) across the erythrocyte vacuolar membrane (Hiller et al. 2004; Marti et al. 2004, Bhattarcharjee et al. 2006). Thus there appears to be a conserved translocation mechanism used by these distantly related plant and animal pathogens. These motifs apparently direct uptake of the oomycete effectors into host cells in the absence of the pathogen, implicating a transport mechanism involving host plant components (Dou et al. 2008). Although the flax rust Avr proteins do not contain such a highly conserved motif, initial experimental data indicate that their likely route of uptake is also via a host encoded system rather than a specialized rust secretory system. For example, transient expression of the AvrM protein with or without the signal peptide (SP) induces an M gene specific HR, but addition of the HDEL endoplasmic retention signal prevents recognition of the secreted, but not the cytoplasmic, version (Catanzariti et al. 2006). This is consistent with recognition of the secreted form by the cytoplasmic M protein after secretion and re-entry into the plant cell. More recently we have shown that full length AvrL567- and AvrM-GFP fusion proteins, including the signal peptide, accumulate inside host cells after transient expression in plants. However, fusion of just the SPs of these proteins to GFP results in GFP accumulation outside the plant cell confirming that these signals do correctly direct secretion of the fusion protein in plants. Furthermore, addition of an HDEL endoplasmic reticulum retention signal to the full length Avr-GFP fusion results in accumulation of GFP in the endoplasmic reticulum, confirming that these proteins do enter the secretory pathway. Thus, these results indicate that uptake of the rust effectors into host cells from the apoplast can occur in the absence of the pathogen, implicating a host-derived transport mechanism. We have tested several truncation constructs using smaller regions of AvrL567 and AvrM, and narrowed down the uptake signal to the N-terminal regions of AvrL567 and AvrM. These regions do not contain an obviously conserved motif such as the RxLR motif in oomycete effector proteins, but are rich in the positively charged amino acids arginine and lysine which are common to several proteins known to transport across membranes. The molecular basis of Avr protein recognition and gene-for-gene specificity The co-localization of Avr and R proteins in the flax cytoplasm and the genetics of gene-for-gene interactions are consistent with direct interaction between these proteins. This hypothesis has been investigated experimentally using the yeast two hybrid system to detect R-Avr protein interactions. In these experiments the full length L5, L6 alleles and a chimeric construct L6L11RV that differs from L6 by 11 amino acid differences derived from L11 in the 3 C-terminal LRR units, and 12 AvrL567 variants (AvrL567-A to AvrL567-L) were co-expressed in yeast two hybrid assays. Protein-protein interactions were detected in yeast for the same combinations of L and AvrL567 genes as induced HR in transient expression assays in planta (Dodds et al. 2006). The close correspondence between the detection of a protein interaction in yeast and the induction of HR in planta indicates that direct R-Avr protein interaction is the basis for recognition specificity. For example, L6 but not L5, interacts with AvrL567-D in yeast, and co-expression of L6, but not L5, with AvrL567-D induces HR in planta. Furthermore, the L6L11RV chimera interacts with only AvrL567-J in yeast and again induces HR with only this Avr gene in planta. The observation that L6L11RV and L6 differ only Oral Papers 2009 Technical Workshop 51

4 in the last 3 LRR units indicates that both the resistance and interaction specificities are controlled by the LRR domain. No interactions were detected in yeast between the resistance proteins and the proteins encoded by the virulence alleles that do not induce HR in flax lines. In the flax rust system, the observation of direct interaction between L5 and L6 proteins and corresponding Avr proteins has now been extended to M and AvrM (PN Dodds, unpublished results). However, whereas M is approximately 80% identical to L5 and L6, the AvrL567 and AvrM proteins are unrelated. Similarly, while L6 and L11 differ by only 32 LRR polymorphisms, their corresponding Avr proteins are also apparently unrelated. In addition, all the other distinct L alleles interact with genetically independent avirulence genes and these are not sufficiently related in DNA sequence to be detected by AvrL567 DNA probes. If as seems likely, all these R proteins directly interact with their corresponding Avr proteins, the picture that is emerging is that NBS-LRR proteins can interact with diverse ligands and that the LRR region is highly flexible in an evolutionary sense with the capacity to recognize by direct interaction diverse pathogen ligands when coupled with the NBS domain. Co-evolution of Avr and R genes in the flax rust system Recognition by direct interaction has led to a high level of sequence diversity in rust Avr genes as a consequence of strong diversifying selection to escape recognition and host resistance. The AvrL567 genes are highly variable, with 12 different sequence variants (A-L) found in six rust strains of diverse origin. The 127 amino acid sequence of the mature AvrL567 protein contains 35 polymorphic sites, with nine sites showing multiple polymorphisms. These variants have arisen through positive selection, as indicated by the excess of non-synonymous nucleotide substitutions over synonymous changes in their coding sequences. This suggests that there has been a co-evolutionary arms race between the corresponding Avr and R genes in this system. Evidence that the diversification of the AvrL567 genes is driven by R gene-mediated selection comes from the observation that the sequence differences between the AvrL567 proteins lead to differences in recognition specificity by the corresponding L5, L6 and L7 resistance proteins. The structures of AvrL567-A and D have been determined by X-ray crystallography (Wang et al. 2007) and structural modeling indicates that avirulence and virulence variants of this protein have very similar structures and physical properties. The polymorphic residues map to the surface of the protein and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single and multiple amino acid substitutions in AvrL567 proteins has confirmed the role of individual residues in conferring differences in recognition, but also suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The fact that naturally occurring virulence forms are expressed and encode products highly related to the avirulence variants suggests that there has been selection for Avr variants that escape detection by R proteins but retain a selective value for the pathogen, most likely through a virulence effector function. AvrL567 proteins show no similarity to any known or predicted proteins in current data bases and do not contain any known functional motifs, so the identification of their postulated virulence function is an important target of continuing research. Transgenic flax expressing the rust avirulence genes show no obvious phenotype in the absence of the corresponding resistance gene, and are not compromised in their expression of resistance to otherwise avirulent rust strains, which could have indicated a suppression of defense activity. Diversifying selection is also evident in the other flax rust Avr genes, and most particularly the AvrP123 gene, which like AvrL567, encodes an array of allelic variants with diverse recognition specificities for the corresponding P, P1, P2 and P3 resistance genes. Co-expression of the AvrP123 alleles with the P or P2 resistance genes in tobacco shows a conservation of the recognition and HR induction in this heterologous host, which is also consistent with a direct recognition event that does not require conservation of other host recognition factors. Thus it seems likely that direct R-Avr protein recognition prevails in this disease system, which contrasts with Arabidopsis-Pseudomonas disease resistance interactions. Part of the evolutionary explanation for this discrepancy may lie in the obligate parasitic and narrow host range characteristics of flax rust compared to bacterial pathogens. Mechanistically, the rust effectors may influence host target proteins through binding interactions rather than enzymatic modifications that can be detected indirectly. Towards isolating stem rust effectors and Avr proteins Wheat stem rust (caused by Puccinia graminis f.sp. tricini Pgt) is one of the most destructive diseases of wheat, but has until recently been recalcitrant to molecular analysis. However, the recent advances in rust effector identification as well as the increased power and 52 Advances in host-pathogen molecular interactions: rust effectors as targets for recognition

5 availability of genome sequencing technologies have provided new approaches to the previously intractable system. A draft genome sequence for Pgt has now been developed ( genome/puccinia_graminis/home.html) which will serve as a scaffold for understanding variation between rust fungal strains and identifying genes associated with virulence differences. We have now extended the haustorial secreted protein screen that was successful in isolating flax rust avirulence proteins to this system. We used the ConA affinitiy binding approach to isolate haustoria from wheat leaves infected with the Australian stem rust race This strain was chosen because it is avirulent on a large number of wheat stem rust resistance (Sr) genes. It also represents the founder line of a lineage of Australian field isolates that were derived by sequential mutation to overcome specific Sr genes employed in agriculture. 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