The Cf-4 and Cf-9 resistance proteins of tomato: molecular aspects of specificity and elicitor perception

To feed the increasing world population, agricultural production needs continuous improvement. Especially protection of crops from disastrous diseases is crucial. The interaction between the pathogenic fungus Cladosporium fulvum and its host, tomato, serves as a model system for plant-pathogen interactions. Some tomato plants carry resistance ( R ) genes that confer recognition of fungal strains carrying complementary avirulence ( Avr ) genes. A number of these R genes have been cloned, as well as their complementary Avr genes. The aim of the research described in this thesis was to examine how R gene products confer recognition of fungal strains carrying the matching Avr genes. Profound understanding of the molecular basis of this interaction might help us to improve the protection of other crop plants against economically important diseases.Chapter 1 introduces the state of the art on interaction between Cladosporium fulvum and tomato at the time the research described in this thesis was initiated. C. fulvum is a specialised, biotrophic pathogen, causing tomato leaf mold. The fungus infects tomato leaves by entering stomata at the lower side of the leaf. The infection will proceed if no resistance R genes of the plant match any of the Avr genes of the fungus. However, the plant recognises the fungus when it carries an R gene that matches an Avr gene present in the fungus. This recognition results in the induction of plant defence responses, including a rapid death of cells surrounding the infection site, called the hypersensitive response (HR). Further fungal growth is prohibited by these defence responses. During its lifecycle on susceptible plants, C. fulvum is restricted to the extracellular space of the tomato leaves and secretes many proteins that potentially play a role in virulence. Also the elicitor proteins encoded by the Avr9 and Avr4 are secreted. Injection of these proteins is sufficient to trigger HR in tomato plants carrying Cf-9 and Cf-4 resistance genes, respectively. Both AVR9 and AVR4 are small, stable, cysteine-rich proteins. The complementary Cf-9 and Cf-4 genes encode highly similar, membrane-anchored, receptor-like proteins with extracytoplasmic leucine-rich repeats (LRRs) and a short cytoplasmic tail. Differences between Cf-9 and Cf-4 proteins are located in the N-terminal half, predominantly in amino acid residues at putative solvent-exposed positions of the LRRs, which is thought to form the 'recognition surface' of these proteins.To examine the role of the various domains of Cf proteins in perception of AVR proteins of C. fulvum in more detail, a functional, transient expression system was developed for the Cf-4 and Cf-9 resistance genes ( chapter 2 ). This expression system is based on infiltration of tobacco leaves with Agrobacterium strains that carry Cf genes on the T-DNA of binary plasmids (agroinfiltration). The AVR proteins are delivered either by injection, agroinfiltration, Potato Virus X-mediated expression or by using Avr -transgenic tobacco plants. This chapter also describes differences between Avr9/Cf-9 - and Avr4/Cf-4 -induced necrosis, which are mainly due to a difference in Avr gene activity upon expression in the plant. Finally, it is shown that the signal transduction pathway leading to HR is conserved in solanaceous plants, but likely not in non-solanaceous plant species. An exception is the non-solanaceous plant lettuce, in which the Avr4/Cf-4 gene pair is functional. The agroinfiltration assay is an excellent expression system to study the effect of mutations in Cf genes. In chapter 3 , agroinfiltration was used to determine specificity determinants in Cf proteins by exchanging domains between Cf-4 and Cf-9 and subsequently examining the effect of these mutations on specificity of perception of AVR proteins. Cf-4 differs from Cf-9 at 67 amino acid positions and also contains three deletions. Significantly, Cf-4 lacks two LRRs compared to Cf-9, which appears essential for Cf-4 function. The two additional LRRs in Cf-9 are required for Cf-9 function. Specificity determinants in Cf-4 reside not only in the LRR domain but also in the B-domain. In contrast, specificity determinants in Cf-9 reside entirely in the LRR domain and are likely scattered throughout this domain. The specificity determinants in the LRRs of Cf-4 cluster in a few adjacent LRRs and reside in only three amino acid residues at putative solvent-exposed positions. Thus, most of the 67 amino acids that vary between Cf-4 and Cf-9 appear not to be required for specificity, but probably serve as a source to generate new specificities. To learn more about specificity determinants of Cf-9 proteins occurring in natural populations, we examined the molecular variation of Cf-9 in Lycopersicon pimpinellifolium (Lp) , from which the Cf-9 locus has been introgressed into cultivated tomato ( chapter 4 ). It appears that AVR9 recognition occurs frequently throughout the Lp population. In addition to Cf-9 , a second gene, designated 9DC , confers AVR9 recognition in Lp . Compared to Cf-9 , 9DC is more polymorphic, occurs more frequently and is more widely spread throughout the Lp population, suggesting that 9DC is older than Cf-9 . The second half of the 9DC gene is nearly identical to the second half of Cf-9 , whereas the first half is nearly identical to Hcr9-9D , a Cf homolog adjacent to Cf-9 at the Cf-9 locus. This suggests that Cf-9 has evolved by intragenic recombination between 9DC and another Cf homolog. The fact that 9DC and Cf-9 proteins both confer recognition of AVR9 but differ in 61 amino acid residues shows that Hcr9 proteins can be highly variable, without affecting their recognitional specificity.After having examined their specificity determinants, we subsequently focused on the cellular location of Cf proteins. The presence of a dilysine motif in the G-domain of Cf-9 ( KK RY) suggests that the protein resides in the endoplasmic reticulum (ER) instead of the plasma membrane (PM). Previously, two conflicting reports on the subcellular location of Cf-9 were published. One report showed that Cf-9 accumulates in the ER and is absent in the plasma membrane, whereas the other showed that Cf-9 resides in the plasma membrane. In chapter 5 we have mutated the dilysine motif and show that the mutant Cf-9 protein remains functional in AVR9 recognition and mediation of HR. The data presented in this chapter, in combination with the two previous reports on Cf-9 localisation, can be explained by assuming that proteins that interact with Cf-9 mask the dilysine motif. This theory suggests that functional Cf-9 protein resides in small quantities in the plasma membrane, where it mediates recognition of the extracellular AVR9 protein as a component of a receptor complex. AVR9 recognition in tomato plants carrying Cf-9 most likely involves the high-affinity binding site (HABS) for AVR9 that was identified in plasma membranes. However, the HABS is not encoded by Cf-9 because it is also present in tomato plants that lack Cf-9 and in many other plant species. As it is likely that both the HABS and the Cf-9 protein reside in the plasma membrane and may be present in the same receptor complex, it is essential to isolate the HABS in order to get more insight in the molecular mechanism of AVR9 perception. In chapter 6 , a procedure is described that allows solubilisation of the HABS without affecting its AVR9-binding activity. Of the 19 detergents that were tested, only octyl glucoside appeared to be suitable for solubilisation of the HABS. Removal of the detergent is crucial in this procedure, as it interferes with AVR9 binding. The described procedure may become an essential tool to study the AVR9 receptor complex at the biochemical level. In the final chapter ( chapter 7 ), the experimental data presented in the previous chapters are discussed. In addition to AVR9/Cf-9 there are many other examples of gene-for-gene interactions where no direct interaction was found between R and Avr gene products. In many cases, there are indications for the involvement of an additional host protein, which may represent the virulence target of the Avr protein. The prevalence of R proteins that 'guard' virulence targets can be explained by natural selection for R genes that are maintained in the plant population through 'trench-warfare', resulting in recognition events that cannot be circumvented by the pathogen without taking a virulence penalty. The 'guard' hypothesis significantly changes the focus of current research to the role of virulence targets of Avr proteins, and might explain absence of functionality of R genes in heterologous plant species, despite the fact that they belong to conserved gene families.</font

Subunit-selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections

The proteasome is a nuclear‐cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions, but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit‐selective inhibitors and dual‐color fluorescent activity‐based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant–microbe interactions. Our data reveal that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both β1 and β5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1‐1 [PtoDC3000(ΔhQ)] whilst the activity profile of the β1 subunit changes. Infection with wild‐type PtoDC3000 causes proteasome activities that range from strongly induced β1 and β5 activities to strongly suppressed β5 activities, revealing that β1 and β5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community, and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species.Bio-organic Synthesi

The plant proteolytic machinery and its role in defence

The diverse roles of plant proteases in defence responses that are triggered by pathogens or pests are becoming clearer. Some proteases, such as papain in latex, execute the attack on the invading organism. Other proteases seem to be part of a signalling cascade, as indicated by protease inhibitor studies. Such a role has also been suggested for the recently discovered metacaspases and CDR1. Some proteases, such as RCR3, even act in perceiving the invader. These exciting recent reports are probably just the first examples of what lies beneath. More roles for plant proteases in defence, as well as the regulation and substrates of these enzymes, are waiting to be discovered

Balancing selection favors guarding resistance proteins

The co-evolutionary arms race model for plant–pathogen interactions implies that resistance (R) genes are relatively young and monomorphic. However, recent reports show R gene longevity and co-existence of multiple R genes in natural populations. This indicates that R genes are maintained by balancing selection, which occurs when loss of the matching avirulence (Avr) gene in the pathogen is associated with reduced virulence. We reason that balancing selection favors R proteins that function as guards, monitoring changes in the virulence target mediated by the Avr factor, rather than recognizing the Avr factor itself. Indeed, the available experimental data support the notion that guarding is prevalent in gene-for-gene interaction

Identification of distinct specificity determinants in resistance protein Cf-4 allows construction of a Cf-9 mutant that confers recognition of avirulence protein AVR4

The tomato resistance genes Cf-4 and Cf-9 confer specific, hypersensitive response-associated recognition of Cladosporium carrying the avirulence genes Avr4 and Avr9, respectively. Cf-4 and Cf-9 encode type I transmembrane proteins with extracellular leucine-rich repeats (LRRs). Compared with Cf-9, Cf-4 lacks two LRRs and differs in 78 amino acid residues. To investigate the relevance of these differences for specificity, we exchanged domains between Cf-4 and Cf-9, and mutant constructs were tested for mediating the hypersensitive response by transient coexpression with either Avr4 or Avr9. We show that the number of LRRs is essential for both Cf-4 and Cf-9 function. In addition, Cf-9 specificity resides entirely in the LRR domain and appears to be distributed over several distant LRRs. In contrast, Cf-4 specificity determinants reside in the N-terminal LRR-flanking domain and three amino acid residues in LRRs 13, 14, and 16. These residues are present at putative solvent-exposed positions, and all are required for full Cf-4 function. Finally, we show that Cf-9 carrying the specificity determinants of Cf-4 has recognitional specificity for AVR4. The data indicate that diversifying selection of solvent-exposed residues has been a more important factor in the generation of Cf-4 specificity than has sequence exchange between Cf-4 progenitor genes. The fact that most variant residues in Cf-4 are not essential for Cf-4 specificity indicates that the diverse decoration of R proteins is not fully adapted to confer recognition of a certain avirulence determinant but likely provides a basis for a versatile, adaptive recognition system

Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis

The avirulence genes Avr9 and Avr4 from the fungal tomato pathogen Cladosporium fulvum encode extracellular proteins that elicit a hypersensitive response when injected into leaves of tomato plants carrying the matching resistance genes, Cf-9 and Cf-4, respectively. We successfully expressed both Avr9 and Avr4 genes in tobacco with the Agrobacterium tumefaciens transient transformation assay (agroinfiltration). In addition, we expressed the matching resistance genes, Cf-9 and Cf-4, through agroinfiltration. By combining transient Cf gene expression with either transgenic plants expressing one of the gene partners, Potato virus X (PVX)-mediated Avr gene expression, or elicitor injections, we demonstrated that agroinfiltration is a reliable and versatile tool to study Avr/Cf-mediated recognition. Significantly, agroinfiltration can be used to quantify and compare Avr/Cf-induced responses. Comparison of different Avr/Cf-interactions within one tobacco leaf showed that Avr9/Cf-9-induced necrosis developed slower than necrosis induced by Avr4/Cf-4. Quantitative analysis demonstrated that this temporal difference was due to a difference in Avr gene activities. Transient expression of matching Avr/Cf gene pairs in a number of plant families indicated that the signal transduction pathway required for Avr/Cf-induced responses is conserved within solanaceous species. Most non-solanaceous species did not develop specific Avr/Cf-induced responses. However, co-expression of the Avr4/Cf-4 gene pair in lettuce resulted in necrosis, providing the first proof that a resistance (R) gene can function in a different plant family

Activity profiling of papain-like cysteine proteases in plants

Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in plants. This technology is based on the use of biotinylated, irreversible protease inhibitors that react with active proteases in a mechanism-based manner. Using a biotinylated derivative of the Cys protease inhibitor E-64, we display simultaneous activities of many papain-like Cys proteases in extracts from various tissues and from different plant species. Labeling is pH dependent, stimulated with reducing agents, and inhibited specifically by Cys protease inhibitors but not by inhibitors of other protease classes. Using one-step affinity capture of bintinylated proteases followed by sequencing mass spectrometry, we identified proteases that include xylem-specific XCP2, desiccation-induced RD21, and cathepsin B- and aleurain-like proteases. Together, these results demonstrate that this technology can identify differentially activated proteases and/or characterize the activity of a particular protease within complex mixtures