The Cladosporium fulvum Avr2 protein behaves both as a virulence and an avirulence factor

Plants are not able to move or escape and have to confront environmental challenges like nutrient and water deprivation, low and high temperatures, and biotic stress imposed by pathogens like viruses, bacteria, fungi, nematodes and insects that all compete for plant nutrient sources. The outcome of a plant-pathogen interaction can vary from mild symptoms that are hardly harmful to the host to complete destruction of the host plant. Plants have evolved various mechanisms to counter-attack infections by pathogens. Mechanisms of evasion or suppression of basal host defense by pathogens on the one hand, and specific recognition of a pathogen by its host and activation of downstream defense signaling on the other hand, are complex and both organisms have to come up with sophisticated strategies to survive their encounters. In principle these encounters have two possible outcomes: (i) a pathogen successfully infects the host plant, which is also referred to as a compatible interaction (the pathogen is virulent and the host plant is susceptible), or (ii) the pathogen cannot successfully infect the host plant which stays healthy, also referred to as an incompatible interaction (the pathogen is avirulent and the host plant is resistant).
Nearly 70 years ago, Harold Flor (1942) studied the genetics of the interaction between the flax rust fungus Melampsora lini and flax, Linum usitatissimum. Based on these studies he postulated the so-called gene-for-gene hypothesis (Flor 1942) which states that for each dominant resistance (R) gene in the host there is a matching dominant avirulence (Avr) gene in the pathogen. Co-occurrence and expression of both genes leads to an incompatible interaction that is often associated with a hypersensitive response (HR).
The interaction between the fungus Cladosporium fulvum (syn. Passalora fulva) and the host tomato (Solanum lycopersicum) is an excellent model to study plant-pathogen interactions and obeys to the gene-for-gene hypothesis. C. fulvum is a biotrophic pathogen that causes leaf mold of tomato, avoids breaching the cell wall and exclusively colonizes the tomato leaf apoplast while establishing a long-term feeding relationship with the living cells of the host. During the infection process, the fungus secretes several effector molecules, relatively small, cysteine–rich proteins. They are likely to contribute to pathogen fitness and play a role in pathogen virulence. According to the 'Zig-Zag' model that explains the evolutionary development of plant-pathogen interactions, effectors are required for ETS (effector -triggered susceptibility). Tomato plants that carry cognate Cf resistance genes recognize the effector and elicit a defense response known as the hypersensitive response (HR), nowadays known as effector-triggered immunity (ETI). In this thesis I have focused on several molecular and biochemical aspects of the Avr2 and Cf-2 gene pair and on an additional gene, Rcr3 (required for Cladosporium resistance), that is required for Cf-2-mediated resistance with an emphasis on the role of Avr2 in ETS and ETI in the C. fulvum-tomato interaction.

The gene-for-gene hypothesis postulated by Harrold Flor has inspired many plant pathologists and initiated numerous plant-pathogen studies as discussed in chapter 1. This hypothesis has lead to the characterization of various host plant R genes and cognate pathogen Avr genes from fungi, bacteria and oomycetes. Plant resistance proteins are the basic molecules that mediate a defense reaction, triggered by cognate effectors directed against the pathogens, are found extracellularly as well as intracellularly and are divided in classes based on the composition of different subdomains that may have various functions. Particularly the LRR domain(s) are involved in recognition, regulating protein activation and signal transduction and are highly adjustable in diverse binding specificities to self and non-self molecules. The nucleotide binding (NB) domain acts a switch for activation of downstream host defenses, often resulting in HR. Inappropriate R protein folding and activation is controlled by intramolecular interactions between the various domains and by hetero-multimeric protein complexes.
Studies on interactions of plants, especially Arabidopsis thaliana, with prokaryotic pathogens have resulted in major scientific breakthroughs with respect to the gene-for-gene hypothesis. Research on the bacterial Type Three Secretion System and the delivery of the effectors has indentified sophisticated mechanisms for perception and recognition of pathogens and regulation of host resistance.
The functions of effectors of eukaryotic plant pathogens remain largely unknown so far. Oomycete pathogens such as Phytophthora infestans produce various types of effectors during infection of their hosts. One class of oomycete effectors localizes to, and operates in, the extracellular matrix while the other class acts inside the host plant cell. Recent studies on the interaction of the flax rust fungus Melampsora lini with flax (Linum usitatissimum) has revealed a number of direct Avr-R protein interactions in vitro. These interactions are expected to occur in the haustorial matrix which is produced by the fungus during host infection. Secreted Avr proteins of C. fulvum interact exclusively with the corresponding extracellular Cf proteins of tomato. The C. fulvum-tomato pathosystem is one of the most well-studied plant pathogen interactions and revealed important insights in perception and recognition of Avr proteins. For many years it was assumed that the interactions beween C. fulvum Avrs and tomato R proteins occurred in a direct manner, but proof for such interactions has never been obtained. Indirect interactions were more likely and obeyed to the guard hypothesis wherein the Avr protein interacts with a host target and this interaction is monitored, or guarded, by the Cf- protein.


Chapter 2 reports on the avirulence function of Avr2 in the Cf-2-mediated resistance that also requires the extracellular tomato cysteine protease Rcr3. The interaction between Avr2, Cf-2 and Rcr3 obeys to the guard hypothesis. Purified heterologously expressed and affinity-tagged Rcr3 and Avr2 were applied in co-immunoprecipitation assays and revealed a physical interaction between Avr2 and Rcr3 independent of additional plant and or fungal factors. It is shown that Avr2 binds and inhibits Rcr3, and blocking of the active site of Rcr3 by the irreversible cysteine protease inhibitor E-64 eliminates this interaction. The interaction with and the inhibition of Rcr3 by Avr2 occurs in a pH-dependent fashion and the pH optimum for Rcr3 activity and its inhibition by Avr2 coincides with the pH of the tomato apoplast. Cysteine protease activity profiling showed that, in addition to Rcr3, Avr2 inhibits several other apoplastic cysteine proteases in tomato, but this inhibition did not lead to Cf-2-mediated HR. Infiltration of purified active Rcr3, or E-64-inactivated Rcr3, in combination with Avr2 in Cf-2/rcr3 tomato leaves revealed that only the Avr2-Rcr3 inhibition complex triggers Cf-2-dependent HR. It is proposed that Avr2 modifies Rcr3 which is recognized by Cf-2 and initiates the HR. This study represents the first indirect fungus-plant gene-for-gene interaction that obeys to the guard hypothesis.

In chapter 3 the focus is on the virulence function of Avr2, and it is demonstrated that Avr2 has an indisputable intrinsic biological virulence function for C. fulvum during infection of tomato. Silencing of the Avr2 gene in C. fulvum significantly compromised fungal virulence on tomato. Heterologous expression of Avr2 in tomato resulted in enhanced susceptibility towards natural Avr2-defective C.fulvum strains, but also towards Botrytis cinerea and Verticillium dahliae. In A. thaliana, Avr2 expression resulted in enhanced susceptibility to various extracellular fungal pathogens including Botrytis cinerea and Verticillium dahliae. Microarray analysis of unchallenged A. thaliana plants showed that Avr2 expression induced a global transcription profile that is comparable to the profile upon pathogen challenge. Cysteine protease activity profiling and LC-MS/MS analyses showed that Avr2 inhibits multiple extracellular A. thaliana cysteine proteases. Similar results were obtained for tomato, showing that Avr2 inhibits multiple cysteine proteases including Rcr3 and its close relative Pip1. This all shows that Avr2 is a genuine virulence factor of C. fulvum that inhibits several cysteine proteases that are required for basal host defense.






In chapter 4 the emphasis is on Avr2 protein features and the mode of inhibition of Rcr3. Like many other Avr genes, Avr2 lacks homology with sequences deposited in public databases. The mature Avr2 protein contains 8 cysteine residues and biochemical analyses revealed that all of these are involved in disulphide bridging, showing a unique disulphide bridge pattern. Based on a bioinformatics analysis, site-specific mutations were made in the Avr2 protein and affinity-tagged wild-type and mutant proteins were produced by heterologous expression in the yeast Pichia pastoris. After affinity purification, all proteins were infiltrated in tomato Cf-2 plants, and proteins with altered HR inducing activity were tested for their ability to inhibit Rcr3. From these assays it became evident that especially the C-terminal six amino acids that also include one disulphide bridge are essential for the interaction with and inhibition of Rcr3. All these data show that Avr2 is a novel type of cysteine protease inhibitor.

Chapter 5 is a general discussion about the role of plant cysteine proteases and cysteine protease inhibitors in plant-pathogen interactions. Microbial pathogens and host plants both employ cysteine proteases and cysteine protease inhibitors as weapons for attack and defence. This so-called arms race has led to multiple attacks and counter-attacks that have shaped co-evolution between pathogens and their host plants. Examples of some prokaryotic plant pathogens that employ cysteine proteases as effector proteins to suppress plant defense will be discussed, in addition to some eukaryotic pathogens that use cysteine protease inhibitors for the same purpose. Examples of plant cysteine proteases will be discussed that are involved in multiple processes including plant development, plant defense and processes in programmed cell death.