An effector-targeted protease contributes to defense against Phytophthora infestans and is under diversifying selection in natural hosts

Since the leaf apoplast is a primary habitat for many plant pathogens, apoplastic proteins are potent, ancient targets for apoplastic effectors secreted by plant pathogens. So far, however, only a few apoplastic effector targets have been identified and characterized. Here, we discovered that the papain-like cysteine protease C14 is a new common target of EPIC1 and EPIC2B, two apoplastic, cystatin-like proteins secreted by the potato (Solanum tuberosum) late blight pathogen Phytophthora infestans. C14 is a secreted protease of tomato (Solanum lycopersicum) and potato typified by a carboxyl-terminal granulin domain. The EPIC-C14 interaction occurs at a wide pH range and is stronger than the previously described interactions of EPICs with tomato defense proteases PIP1 and RCR3. The selectivity of the EPICs is also different when compared with the AVR2 effector of the fungal tomato pathogen Cladosporium fulvum, which targets PIP1 and RCR3, and only at apoplastic pH. Importantly, silencing of C14 increased susceptibility to P. infestans, demonstrating that this protease plays a role in pathogen defense. Although C14 is under conservative selection in tomato, it is under diversifying selection in wild potato species (Solanum demissum, Solanum verrucosum, and Solanum stoliniferum) that are the natural hosts of P. infestans. These data reveal a novel effector target in the apoplast that contributes to immunity and is under diversifying selection, but only in the natural host of the pathogen.     

Cladosporium fulvum overcomes Cf-2-mediated resistance by producing truncated AVR2 elicitor proteins

The Cf-2 gene of tomato confers resistance to strains of the biotrophic pathogenic fungus Cladosporium fulvum carrying avirulence gene Avr2. To allow dissection of the biochemical mechanism of perception of AVR2 by Cf-2, we set out to clone the Avr2 gene. Here, we report the functional cloning of Avr2 cDNA, based on the induction of a hypersensitive response (HR) by the encoded AVR2 protein in Cf2 tomato plants. Analysis of strains of C. fulvum that are virulent on Cf2 tomato lines revealed various independent frameshift mutations in the Avr2 open reading frame (ORF) and a point mutation resulting in a premature stop codon. All modifications result in the production of truncated AVR2 proteins. Interestingly, an additional modification involves the insertion of a LINE-like element, Cfl1, in the Avr2 ORF. Cfl1 is the first LINE-like element identified in C. fulvum and provides the first example of loss of avirulence of a plant pathogen caused by insertion of a retrotransposable element in an Avr gene. Rcr3 represents an additional plant protein that is specifically required for Cf-2-mediated resistance. Analysis of two different rcr3 mutant Cf2 tomato plants revealed that their ability to respond to AVR2 with a HR correlates with their degree of resistance to AVR2-producing strains of C. fulvum. These data support a role for Rcr3 in the perception of AVR2 by Cf-2.     

Natural disulfide bond-disrupted mutants of AVR4 of the tomato pathogen Cladosporium fulvum are sensitive to proteolysis,circumvent Cf-4-mediated resistance,but retain their chitin binding ability

The extracellular AVR4 elicitor of the pathogenic fungus Cladosporium fulvum induces defense responses in the tomato genotype Cf-4. Here, the four disulfide bonds of AVR4 were identified as Cys-11-41, Cys-21-27, Cys-35-80, and Cys-57-72 by partial reduction with Tris-(2-carboxyethyl)-phosphine hydrochloride, subsequent cyanylation, and base-catalyzed chain cleavage. The resulting peptide fragments were analyzed by mass spectrometry. Sequence homology and the disulfide bond pattern revealed that AVR4 contains an invertebrate (inv) chitin-binding domain (ChBD). Binding of AVR4 to chitin was confirmed experimentally. The three disulfide bonds encompassing the inv ChBD motif are also required for protein stability of AVR4. Independent disruption of each of the three conserved disulfide bonds in AVR4 resulted in a protease-sensitive protein, whereas the fourth disulfide bond appeared not to be required for protein stability. Most strains of C. fulvum virulent on Cf-4 tomato contain Cys to Tyr substitutions in AVR4 involving two (Cys-11-41, Cys-35-80) of the three disulfide bonds present in the inv ChBD motif. These natural Cys to Tyr mutant AVR4 proteins did retain their chitin binding ability and when bound to chitin were less sensitive to proteases. Thus, the widely applied tomato Cf-4 resistance gene is circumvented by C. fulvum by amino acid substitutions affecting two disulfide bonds in AVR4 resulting in the absence of the corresponding AVR4 isoforms in apoplastic fluid. However, these natural isoforms of AVR4 appear to have retained their intrinsic function, i.e. binding to chitin present in the cell wall of C. fulvum, most likely to protect it against the deleterious effects of plant chitinases.     

The AVR4 elicitor protein of Cladosporium fulvum binds to fungal components with high affinity

The interaction between tomato and the fungal pathogen Cladosporium fulvum complies with the gene-for-gene system. Strains of C. fulvum that produce race-specific elicitor AVR4 induce a hypersensitive response, leading to resistance, in tomato plants that carry the Cf-4 resistance gene. The mechanism of AVR4 perception was examined by performing binding studies with 125I-AVR4 on microsomal membranes of tomato plants. We identified an AVR4 high-affinity binding site (KD = 0.05 nM) which exhibited all the characteristics expected for ligand-receptor interactions, such as saturability, reversibility, and specificity. Surprisingly, the AVR4 high-affinity binding site appeared to originate from fungi present on infected tomato plants rather than from the tomato plants themselves. Detailed analysis showed that this fungus-derived, AVR4-specific binding site is heat- and proteinase K-resistant. Affinity crosslinking demonstrated that AVR4 specifically binds to a component of approximately 75 kDa that is of fungal origin. Our data suggest that binding of AVR4 to a fungal component or components is related to the intrinsic virulence function of AVR4 for C. fulvum.     

Activity‐based proteomics reveals nine target proteases for the recombinant protein‐stabilizing inhibitor SlCYS8 in Nicotiana benthamiana

Co‐expression of protease inhibitors like the tomato cystatin SlCYS8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity‐based protein profiling to identify proteases that are inhibited by SlCYS8 in agroinfiltrated Nicotiana benthamiana. We discovered that SlCYS8 selectively suppresses papain‐like cysteine protease (PLCP) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar‐processing enzyme and serine hydrolase activity. A robust concentration‐dependent inhibition of PLCPs occurred in vitro when purified SlCYS8 was added to leaf extracts, indicating direct cystatin–PLCP interactions. Activity‐based proteomics revealed that nine different Cathepsin‐L/‐F‐like PLCPs are strongly inhibited by SlCYS8 in leaves. By contrast, the activity of five other Cathepsin‐B/‐H‐like PLCPs, as well as 87 Ser hydrolases, was unaffected by SlCYS8. SlCYS8 expression prevented protein degradation by inhibiting intermediate and mature isoforms of granulin‐containing proteases from the Resistant‐to‐Desiccation‐21 (RD21) PLCP subfamily. Our data underline the key role of endogenous PLCPs on recombinant protein degradation and reveal candidate proteases for depletion strategies.     

Specific HR-associated recognition of secreted proteins from Cladosporium fulvum occurs in both host and non-host plants

The resistance of tomato (Lycopersicon esculentum) to the pathogenic fungus Cladosporium fulvum complies with the gene-for-gene concept. Host resistance is based on specific recognition of extracellular fungal proteins, resulting in a hypersensitive response (HR). Five proteins secreted by C. fulvum were purified and the encoding cDNA clone was obtained from two novel ones among them. Various tomato breeding lines and accessions of Lycopersicon pimpinellifolium were tested for their recognitional specificity by injection of the purified proteins or potato virus X-based expression of the cDNA. We found that HR-associated recognition of one or more of these proteins, in addition to recognition of the race-specific elicitors AVR4 and AVR9 of C. fulvum, occurs among Lycopersicon species. Studies on the inheritance of this recognition confirmed that single dominant genes are involved. Furthermore, one of the extracellular proteins of C. fulvum is specifically recognized by Nicotiana paniculata, which is not a host for C. fulvum. These results indicate that plants have a highly effective surveillance system for the presence of 'foreign' proteins, which, together with the high mutation rate of pathogens, can explain the complex gene-for-gene relationships frequently observed in pathosystems.     

Expression of the Avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is regulated by the global nitrogen response factor NRF1

Here we describe the role of the Cladosporium fulvum nitrogen response factor 1 (Nrf1) gene in regulation of the expression of avirulence gene Avr9 and virulence on tomato. The Nrf1 gene, which was isolated by a polymerase chain reaction-based strategy, is predicted to encode a protein of 918 amino acid residues. The protein contains a putative zinc finger DNA-binding domain that shares 98% amino acid identity with the zinc finger of the major nitrogen regulatory proteins AREA and NIT2 of Aspergillus nidulans and Neurospora crassa, respectively. Functional equivalence of Nrf1 to areA was demonstrated by complementation of an A. nidulans areA loss-of-function mutant with Nrf1. Nrf1-deficient transformants of C. fulvum obtained by homologous recombination were unable to utilize nitrate and nitrite as a nitrogen source. In contrast to what was observed in the C. fulvum wild-type, the Avr9 gene was no longer induced under nitrogen-starvation conditions in Nrf1-deficient strains. On susceptible tomato plants, the Nrf1-deficient strains were as virulent as wild-type strains of C. fulvum, although the expression of the Avr9 gene was strongly reduced. In addition, Nrf1-deficient strains were still avirulent on tomato plants containing the functional Cf-9 resistance gene, indicating that in planta, apparently sufficient quantities of stable AVR9 elicitor are produced. Our results suggest that the NRF1 protein is a major regulator of the Avr9 gene.     

Attenuation of Cf-mediated defense responses at elevated temperatures correlates with a decrease in elicitor-binding sites

The interaction between the fungal pathogen Cladosporium fulvum and its only host, tomato, is a well-described gene-for-gene system and several resistance (Cf) genes of tomato and matching fungal avirulence (Avr) genes have been characterized. Transgenic tobacco suspension cells expressing Cf genes respond to matching elicitors with typical defense responses, such as medium alkalization and an oxidative burst. We found that this response is attenuated at elevated ambient temperatures. Tomato seedlings expressing both a Cf and the matching Avr gene rapidly die as a result of systemic necrosis at normal temperatures, but are rescued at 33 degrees C. We demonstrate that, at 33 degrees C, the Cf/Avr-mediated induction of defense-related genes is reversibly suppressed. Furthermore, in cell suspensions, the AVR-induced medium alkalization response is slowly suppressed upon incubation at 33 degrees C, but is quickly restored after transfer to lower temperatures. A high-affinity binding site (HABS) for AVR9 is present on plasma membranes isolated from solanaceous plants and has been suggested to act as a co-receptor for AVR9. The amount of AVR9-HABS is 80% reduced in tobacco cell suspensions incubated at 33 degrees C, as compared with cell suspensions incubated at 20 degrees C. Our data suggest that the temperature sensitivity of Cf-mediated defense responses resides at the level of perception of the fungal avirulence factors.     

Molecular and biochemical basis of the interaction between tomato and its fungal pathogen Cladosporium fulvum

The interaction between the biotrophic fungal pathogen Cladosporium fulvum and tomato complies with the genefor-gene model. Resistance, expressed as a hypersensitive response (HR) followed by other defence responses, is based on recognition of products of avirulence genes from C. fulvum (race-specific elicitors) by receptors (putative products of resistance genes) in the host plant tomato. The AVR9 elicitor is a 28 amino acid (aa) peptide and the AVR4 elicitor a 106 aa peptide which both induce HR in tomato plants carrying the complementary resistance genes Cf9 and Cf4, respectively. The 3-D structure of the AVR9 peptide, as determined by 1H NMR, revealed that AVR9 belongs to a family of peptides with a cystine knot motif. This motif occurs in channel blockers, peptidase inhibitors and growth factors. The Cf9 resistance gene encodes a membrane-anchored extracellular glycoprotein which contains leucine-rich repeats (LRRs). 125I labeled AVR9 peptide shows the same affinity for plasma membranes of Cf9+ and Cf9- tomato leaves. Membranes of solanaceous plants tested so far all contain homologs of the Cf9 gene and show similar affinities for AVR9. It is assumed that for induction of HR, at least two plant proteins (presumably CF9 and one of his homologs) interact directly or indirectly with the AVR9 peptide which possibly initiates modulation and dimerisation of the receptor, and activation of various other proteins involved in downstream events eventually leading to HR. We have created several mutants of the Avr9 gene, expressed them in the potato virus X (PVX) expression system and tested their biological activity on Cf9 genotypes of tomato. A positive correlation was observed between the biological activity of the mutant AVR9 peptides and their affinity for tomato plasma membranes. Recent results on structure and biological activity of AVR4 peptides encoded by avirulent and virulent alleles of the Avr4 gene (based on expression studies in PVX) are also discussed as well as early defence responses induced by elicitors in tomato leaves and tomato cell suspensions.     

Identification of Two Genes Required in Tomato for Full Cf-9-Dependent Resistance to Cladosporium fulvum

Mutagenesis was used to identify and characterize plant genes required for fungal disease resistance gene function in tomato. Seed of a stock homozygous for the Cf-9 gene for resistance to Cladosporium fulvum were treated with ethyl methanesulfonate, and 568 M2 families were screened for mutations to C. fulvum sensitivity. Eight mutants with reduced resistance were isolated. Four mutations, all of which mapped to the Cf-9 gene, lost both resistance and response to the race-specific AVR9 elicitor. The other four mutations partially lost resistance and response to the AVR9 elicitor. Cytological analysis revealed that a unique host cell staining pattern accompanied the reduced-resistance phenotype in three mutants. Two of the mutants with reduced resistance mapped to Cf-9, and two mapped to two distinct loci designated Rcr-1 and Rcr-2 (Required for Cladosporium resistance) that are unlinked to Cf-9.     

Papain-like cysteine proteases: key players at molecular battlefields employed by both plants and their invaders

Papain-like cysteine proteases (PLCPs) play crucial roles in plant–pathogen/pest interactions. During these parasitic interactions, PLCPs act on non-self substrates, provoking the selection of counteracting inhibitors and other means to evade proteolysis. We review examples of PLCPs acting on molecular battlefields in the extracellular space, plant cytoplasm and herbivore gut. Examples are maize Mir1 ( M aize i nbred r esistance 1 ), tomato Rcr3 ( R equired for C ladosporium r esistance- 3 ), Pseudomonas AvrRpt2 and AurPphB, insect DvCAL1 ( D iabrotica v irgifera c athepsin L -like p rotease- 1 ) and nematode MiCpl1 ( M eloidogyne i ncognita c athepsin L -like p rotease 1 ). The data suggest that PLCPs cleave specific proteins and that their translocation, activation and inhibition of PLCPs are tightly regulated.     

The AVR9 race-specific elicitor of Cladosporium fulvum is processed by endogenous and plant proteases.

The avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum encodes a race-specific peptide elicitor that induces a hypersensitive response in tomato plants carrying the complementary resistance gene Cf9. The avr9 gene is highly expressed when C. fulvum is growing in the plant and the elicitor accumulates in infected leaves as a 28-amino acid (aa) peptide. In C. fulvum grown in vitro, the peptide elicitor is not produced in detectable amounts. To produce significant amounts of the AVR9 elicitor in vitro, the coding and termination sequences of the avr9 gene were fused to the constitutive gpd-promoter (glyceraldehyde 3-phosphate dehydrogenase) of Aspergillus nidulans. Transformants of C. fulvum were obtained that highly expressed the avr9 gene in vitro and produced active AVR9 peptide elicitors. These peptides were partially sequenced from the N terminus and appeared to consist of 32, 33, and 34 aa's, respectively, and are the precursors of the mature 28-aa AVR9 peptide. We demonstrated that plant factors process the 34-aa peptide into the mature 28-aa peptide. We present a model for the processing of AVR9 involving cleavage of a signal peptide during excretion and further maturation by fungal and plant proteases into the stable 28-aa peptide elicitor.     

Balancing selection at the tomato RCR3 Guardee gene family maintains variation in strength of pathogen defense

Coevolution between hosts and pathogens is thought to occur between interacting molecules of both species. This results in the maintenance of genetic diversity at pathogen antigens (or so-called effectors) and host resistance genes such as the major histocompatibility complex (MHC) in mammals or resistance (R) genes in plants. In plant-pathogen interactions, the current paradigm posits that a specific defense response is activated upon recognition of pathogen effectors via interaction with their corresponding R proteins. According to the "Guard-Hypothesis," R proteins (the "guards") can sense modification of target molecules in the host (the "guardees") by pathogen effectors and subsequently trigger the defense response. Multiple studies have reported high genetic diversity at R genes maintained by balancing selection. In contrast, little is known about the evolutionary mechanisms shaping the guardee, which may be subject to contrasting evolutionary forces. Here we show that the evolution of the guardee RCR3 is characterized by gene duplication, frequent gene conversion, and balancing selection in the wild tomato species Solanum peruvianum. Investigating the functional characteristics of 54 natural variants through in vitro and in planta assays, we detected differences in recognition of the pathogen effector through interaction with the guardee, as well as substantial variation in the strength of the defense response. This variation is maintained by balancing selection at each copy of the RCR3 gene. Our analyses pinpoint three amino acid polymorphisms with key functional consequences for the coevolution between the guardee (RCR3) and its guard (Cf-2). We conclude that, in addition to coevolution at the "guardee-effector" interface for pathogen recognition, natural selection acts on the "guard-guardee" interface. Guardee evolution may be governed by a counterbalance between improved activation in the presence and prevention of auto-immune responses in the absence of the corresponding pathogen.     

Subclassification and biochemical analysis of plant papain-like cysteine proteases displays subfamily-specific characteristics

Papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes associated with development, immunity, and senescence. Although many properties have been described for individual proteases, the distribution of these characteristics has not been studied collectively. Here, we analyzed 723 plant PLCPs and classify them into nine subfamilies that are present throughout the plant kingdom. Analysis of these subfamilies revealed previously unreported distinct subfamily-specific functional and structural characteristics. For example, the NPIR and KDEL localization signals are distinctive for subfamilies, and the carboxyl-terminal granulin domain occurs in two PLCP subfamilies, in which some individual members probably evolved by deletion of the granulin domains. We also discovered a conserved double cysteine in the catalytic site of SAG12-like proteases and two subfamily-specific disulfides in RD19A-like proteases. Protease activity profiling of representatives of the PLCP subfamilies using novel fluorescent probes revealed striking polymorphic labeling profiles and remarkably distinct pH dependency. Competition assays with peptide-epoxide scanning libraries revealed common and unique inhibitory fingerprints. Finally, we expand the detection of PLCPs by identifying common and organ-specific protease activities and identify previously undetected proteases upon labeling with cell-penetrating probes in vivo. This study provides the plant protease research community with tools for further functional annotation of plant PLCPs.     

A Genetic Mechanism for Emergence of Races in Fusarium oxysporum f. sp. lycopersici: Inactivation of Avirulence Gene AVR1 by Transposon Insertion

Compatible/incompatible interactions between the tomato wilt fungus Fusarium oxysporum f. sp. lycopersici (FOL) and tomato Solanum lycopersicum are controlled by three avirulence genes (AVR1-3) in FOL and the corresponding resistance genes (I-I3) in tomato. The three known races (1, 2 and 3) of FOL carry AVR genes in different combinations. The current model to explain the proposed order of mutations in AVR genes is: i) FOL race 2 emerged from race 1 by losing the AVR1 and thus avoiding host resistance mediated by I (the resistance gene corresponding to AVR1), and ii) race 3 emerged when race 2 sustained a point mutation in AVR2, allowing it to evade I2-mediated resistance of the host. Here, an alternative mechanism of mutation of AVR genes was determined by analyses of a race 3 isolate, KoChi-1, that we recovered from a Japanese tomato field in 2008. Although KoChi-1 is race 3, it has an AVR1 gene that is truncated by the transposon Hormin, which belongs to the hAT family. This provides evidence that mobile genetic elements may be one of the driving forces underlying race evolution. KoChi-1 transformants carrying a wild type AVR1 gene from race 1 lost pathogenicity to cultivars carrying I, showing that the truncated KoChi-1 avr1 is not functional. These results imply that KoChi-1 is a new race 3 biotype and propose an additional path for emergence of FOL races: Race 2 emerged from race 1 by transposon-insertion into AVR1, not by deletion of the AVR1 locus; then a point mutation in race 2 AVR2 resulted in emergence of race 3.     

The C-terminal dilysine motif for targeting to the endoplasmic reticulum is not required for Cf-9 function

The tomato resistance gene Cf-9 encodes a membrane-anchored, receptor-like protein that mediates specific recognition of the extracellular elicitor protein AVR9 of Cladosporium fulvum. The C-terminal dilysine motif (KKRY) of Cf-9 suggests that the protein resides in the endoplasmic reticulum. Previously, two conflicting reports on the subcellular location of Cf-9 were published. Here we show that the AARY mutant version of Cf-9 is still functional in mediating AVR9 recognition, suggesting that functional Cf-9 resides in the plasma membrane. The data presented here and in reports by others can be explained by masking the dilysine signal of Cf-9 with other proteins.     

Disulfide bond structure of the AVR9 elicitor of the fungal tomato pathogen Cladosporium fulvum: evidence for a cystine knot

Disease resistance in plants is commonly activated by the product of an avirulence (Avr) gene of a pathogen after interaction with the product of a matching resistance (R) gene in the host. In susceptible plants, Avr products might function as virulence or pathogenicity factors. The AVR9 elicitor from the fungus Cladosporium fulvum induces defense responses in tomato plants carrying the Cf-9 resistance gene. This 28-residue beta-sheet AVR9 peptide contains three disulfide bridges, which were identified in this study as Cys2-Cys16, Cys6-Cys19, and Cys12-Cys26. For this purpose, AVR9 was partially reduced, and the thiol groups of newly formed cysteines were modified to prevent reactions with disulfides. After HPLC purification, the partially reduced peptides were sequenced to determine the positions of the modified cysteines, which originated from the reduced disulfide bridge(s). All steps involving molecules with free thiol groups were performed at low pH to suppress disulfide scrambling. For that reason, cysteine modification by N-ethylmaleimide was preferred over modification by iodoacetamide. Upon (partial) reduction of native AVR9, the Cys2-Cys16 bridge opened selectively. The resulting molecule was further reduced to two one-bridge intermediates, which were subsequently completely reduced. The (partially) reduced cysteine-modified AVR9 species showed little or no necrosis-inducing activity, demonstrating the importance of the disulfide bridges for biological activity. Based on peptide length and cysteine spacing, it was previously suggested that AVR9 isa cystine-knotted peptide. Now, we have proven that the bridging pattern of AVR9 is indeed identical to that of cystine-knotted peptides. Moreover, NMR data obtained for AVR9 show that it is structurally closely related to the cystine-knotted carboxypeptidase inhibitor. However, AVR9 does not show any carboxypeptidase inhibiting activity, indicating that the cystine-knot fold is a commonly occurring motif with varying biological functions.