Mycosphaerellaceae - Chaos or clarity?

The Mycosphaerellaceae represent thousands of fungal species that are associated with diseases on a wide range of plant hosts. Understanding and stabilising the taxonomy of genera and species of Mycosphaerellaceae is therefore of the utmost importance given their impact on agriculture, horticulture and forestry. Based on previous molecular studies, several phylogenetic and morphologically distinct genera within the Mycosphaerellaceae have been delimited. In this study a multigene phylogenetic analysis (LSU, ITS and rpb2) was performed based on 415 isolates representing 297 taxa and incorporating ex-type strains where available. The main aim of this study was to resolve the phylogenetic relationships among the genera currently recognised within the family, and to clarify the position of the cercosporoid fungi among them. Based on these results many well-known genera are shown to be paraphyletic, with several synapomorphic characters that have evolved more than once within the family. As a consequence, several old generic names including Cercosporidium, Fulvia, Mycovellosiella, Phaeoramularia and Raghnildiana are resurrected, and 32 additional genera are described as new. Based on phylogenetic data 120 genera are now accepted within the family, but many currently accepted cercosporoid genera still remain unresolved pending fresh collections and DNA data. The present study provides a phylogenetic framework for future taxonomic work within the Mycosphaerellaceae.

How plants recognize pathogens and defend themselves.

Plants have an innate immunity system to defend themselves against pathogens. With the primary immune system, plants recognize microbe-associated molecular patterns (MAMPs) of potential pathogens through pattern recognition receptors (PRRs) that mediate a basal defense response. Plant pathogens suppress this basal defense response by means of effectors that enable them to cause disease. With the secondary immune system, plants have gained the ability to recognize effector-induced perturbations of host targets through resistance proteins (RPs) that mediate a strong local defense response that stops pathogen growth. Both primary and secondary immune responses in plants depend on germ line-encoded PRRs and RPs. During induction of local immune responses, systemic immune responses also become activated, which predispose plants to become more resistant to subsequent pathogen attacks. This review gives an update on recent findings that have enhanced our understanding of plant innate immunity and the arms race between plants and their pathogens.

Efficient 13C/15N double labeling of the avirulence protein AVR4 in a methanol-utilizing strain (Mut+) of Pichia pastoris.

Cost effective 13C/15N-isotope labeling of the avirulence protein AVR4 (10 kDa) of the fungal tomato pathogen Cladosporium fulvum was achieved with the methylotrophic yeast Pichia pastoris in a fermentor. The 13C/15N-labeled AVR4 protein accumulated to 30 mg/L within 48 h in an initial fermentation volume of only 300 mL, while prolonged optimized overexpressions yielded 126 mg/L. These protein yields were 24-fold higher in a fermentor than in flask cultures. In order to achieve these protein expression levels, we used the methanol-utilizing strain (Mut+) of Pichia pastoris which has a high growth rate while growing on methanol as the only carbon source. In contrast, the methanol-sensitive strain (MutS) could intrinsically yield comparable protein expression levels, but at the expense of additional carbon sources. Although both strains are generally used for heterologous protein expression, we show that the costs for 13C-isotope labeling can be substantially reduced using the Mut+ strain compared to the MutS strain, as no 13C3-glycerol is required during the methanol-induction phase. Finally, nitrogen limitations were precluded for 15N-labeling by an optimal supply of 10 g/L (15NH4)2SO4 every 24 h.

Intragenic recombination generated two distinct Cf genes that mediate AVR9 recognition in the natural population of Lycopersicon pimpinellifolium.

Resistance gene Cf-9 of cultivated tomato (Lycopersicon esculentum) confers recognition of the AVR9 elicitor protein of the fungal pathogen Cladosporium fulvum. The Cf-9 locus, containing Cf-9 and four homologs (Hcr9s), originates from Lycopersicon pimpinellifolium (Lp). We examined naturally occurring polymorphism in Hcr9s that confer AVR9 recognition in the Lp population. AVR9 recognition occurs frequently throughout this population. In addition to Cf-9, we discovered a second gene in Lp, designated 9DC, which also confers AVR9 recognition. Compared with 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 sequences of Cf-9 and 9DC suggest that Cf-9 evolved from 9DC by intragenic recombination between 9DC and another Hcr9. The fact that the 9DC and Cf-9 proteins differ in 61 aa residues, and both mediate recognition of AVR9, shows that in nature Hcr9 proteins with the same recognitional specificity can vary significantly.

No evidence for binding between resistance gene product Cf-9 of tomato and avirulence gene product AVR9 of Cladosporium fulvum.

The gene-for-gene model postulates that for every gene determining resistance in the host plant, there is a corresponding gene conditioning avirulence in the pathogen. On the basis of this relationship, products of resistance (R) genes and matching avirulence (Avr) genes are predicted to interact. Here, we report on binding studies between the R gene product Cf-9 of tomato and the Avr gene product AVR9 of the pathogenic fungus Cladosporium fulvum. Because a high-affinity binding site (HABS) for AVR9 is present in tomato lines, with or without the Cf-9 resistance gene, as well as in other solanaceous plants, the Cf-9 protein was produced in COS and insect cells in order to perform binding studies in the absence of the HABS. Binding studies with radio-labeled AVR9 were performed with Cf-9-producing COS and insect cells and with membrane preparations of such cells. Furthermore, the Cf-9 gene was introduced in tobacco, which is known to be able to produce a functional Cf-9 protein. Binding of AVR9 to Cf-9 protein produced in tobacco was studied employing surface plasmon resonance and surface-enhanced laser desorption and ionization. Specific binding between Cf-9 and AVR9 was not detected with any of the procedures. The implications of this observation are discussed.

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.

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.

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.

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.

Specific recognition of AVR4 and AVR9 results in distinct patterns of hypersensitive cell death in tomato, but similar patterns of defence-related gene expression.

Summary Hypersensitive cell death occurs in tomato seedlings that are derived from a cross between plants that express a resistance (Cf) gene against the pathogenic fungus Cladosporium fulvum and plants that contain the matching avirulence (Avr) gene originating from this fungus. The pattern of Cf-9/Avr9- and Cf-4/Avr4-induced necrosis in these F(1) seedlings was found to differ significantly. Macroscopic observation revealed that in F(1) tomato seedlings containing both Cf-9 and Avr9, numerous necrotic spots developed that were scattered over the entire cotyledon, while the midvein and primary veins remained unaffected. In seedlings containing both Cf-4 and Avr4, however, initially only one or a few necrotic spots developed on each cotyledon, in most cases in the midvein and occasionally in primary veins. Subsequently, these spots turned rapidly into lesions that enlarged along the midvein and primary veins, eventually causing the cotyledons to wilt and abscise. These observations were confirmed by detailed histological studies. Production of the AVR proteins in adult tomato plants carrying the matching Cf gene, employing potato virus X, resulted in similar patterns of necrosis. RNA gel blot analysis demonstrated that both Avr4 and Avr9, controlled by the CaMV 35S promoter, were highly expressed in seedlings already at one day post-emergence, indicating that the distinct necrotic patterns are not due to differences in Avr expression levels. We have analysed the expression of many genes involved in defence signalling pathways and the defence response itself, during the onset of the Cf/Avr-initiated hypersensitive response (HR). Although most of the genes were expressed stronger and faster in Cf-4/Avr4 seedlings than in Cf-9/Avr9 seedlings at the onset of HR, no significant qualitative differences in the expression of genes involved in downstream signalling were observed when Cf-4/Avr4- and Cf-9/Avr9-induced defence responses were compared.

A functional cloning strategy, based on a binary PVX-expression vector, to isolate HR-inducing cDNAs of plant pathogens.

We have devised a novel, high-throughput functional cloning method to isolate cDNAs from plant pathogens of which the products elicit a hypersensitive response (HR) in plants. Copy DNA, made from RNA isolated from the tomato pathogen Cladosporium fulvum grown under nutrient-limiting conditions in vitro, was cloned into a binary, potato virus X (PVX)-based expression vector and transformed to Agrobacterium tumefaciens. 9600 colonies were individually toothpick-inoculated onto leaflets of tomato plants resistant to C. fulvum. Four cDNAs were identified whose expression induced formation of a necrotic lesion around the inoculation site. One of these clones, specifically inducing HR on tomato plants carrying the Cf-4 resistance gene, encodes race-specific elicitor AVR4. The other three cDNAs, inducing a non-genotype-specific HR, encode a protein highly homologous to bZIP, basic transcription factors. To determine whether this approach has general applicability, part of the library was also inoculated onto Nicotiana tabacum var. Samsun NN, which is not a host for C. fulvum. Four independent HR-inducing cDNAs were identified which all encode ECP2, an extracellular protein of C. fulvum known to induce necrosis in certain Nicotiana species. These observations confirm that this functional screening method is a versatile strategy to identify cDNAs of pathogens that encode (race-specific) elicitors and other HR-inducing proteins.

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.

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.

Avirulence and resistance genes in the Cladosporium fulvum-tomato interaction.

The fungus Cladosporium fulvum infects tomato and secretes various proteins that are recognized by resistant plants that respond with a hypersensitive response. Strains of the fungus that escape recognition by tomato are virulent. Resistance genes in tomato, either directly or indirectly involved in recognition of the fungal proteins, encode extracellular membrane-anchored, leucine-rich repeat proteins, which occur in gene clusters. Much progress has been made in our understanding of the evolution of recognitional specificities in the host plant.

Folding and conformational analysis of AVR9 peptide elicitors of the fungal tomato pathogen Cladosporium fulvum.

The race-specific elicitor AVR9, produced by the phytopathogenic fungus Cladosporium fulvum, is a 28-residue beta-sheet peptide containing three disulfide bridges. The folding of this peptide to its native conformation was examined in the presence of oxidized (GSSG) and reduced (GSH) glutathione at concentrations resembling those present in the endoplasmic reticulum. The concentrations of GSH and GSSG, and the applied temperature strongly affected the folding efficiency. The effect of temperature appeared reversible. The conditions for in vitro folding were optimized and a maximum yield of 60-70% of correctly folded peptide was obtained. In vitro folded AVR9 is equally as active as native fungal AVR9. They both display similar NMR characteristics, indicating that they have the same 3D structure and identical disulfide bridges. Thus, AVR9 can be folded correctly in vitro. This folding can be described by disulfide bridge formation leading to scrambled three-disulfide species, followed by disulfide reshuffling to acquire the native structure. The presence of urea significantly affected the folding of AVR9, indicating that noncovalent interactions play a role in directing correct folding. Protein disulfide isomerase increased the folding rate at least 15-fold, but had no effect on the yield. The folding procedure has also been applied successfully to two mutant AVR9 peptides, (K23A)AVR9 and biotinylated AVR9. We conclude that the 28-residue sequence, without the preprosequence (as present in vivo), contains sufficient information to direct correct folding and disulfide bridge formation in vitro.

Transcription of the avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is regulated by a GATA-type transcription factor in Aspergillus nidulans.

The avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is highly induced during infection of tomato plants. Expression of the Avr9 gene can also be induced in vitro when cells are grown on synthetic liquid medium containing little or no nitrogen. The Avr9 promoter contains six copies of the sequence TAGATA and six additional copies of the core sequence GATA within 0.4 kb upstream of the translation start site. In the filamentous fungi Aspergillus nidulans and Neurospora crassa, these promoter sequences have been identified as the binding sites for a wide-domain GATA-type regulator (AREA in A. nidulans and NIT2 in N. crassa) involved in nitrogen utilization. Quantification of GUS activity of A. nidulans transformants containing a single copy of the fully active Avr9 promoter-uidA (GUS) reporter gene fusion in different areA backgrounds, following starvation for nitrogen, showed that induction of the Avr9 promoter is regulated similarly in A. nidulans and C. fulvum. This suggests that AREA can regulate the Avr9 promoter and that C. fulvum contains an AREA-like regulator that can bind to these specific sequence motifs. Comparison of the induction profiles of Avr9 and niaD showed that Avr9 expression is independent of NIRA, as is niaD expression upon nitrogen starvation. Studies with Avr9 promoter-uidA fusions in which all or most of these sequences had been deleted, showed that Avr9 promoter activity is dependent on the presence of these specific cis-regulatory elements, suggesting that they do indeed function in transcriptional regulation of the Avr9 gene.

The Cf-ECP2 gene is linked to, but not part of, the Cf-4/Cf-9 cluster on the short arm of chromosome 1 in tomato.

A gene has been identified in tomato, which confers resistance to Cladosporium fulvum through recognition of the pathogenicity factor ECP2. Segregation analysis of F2 and F3 populations showed monogenic dominant inheritance, as for previously reported Cf resistances. The gene has been designated Cf-ECP2. Using several mapping populations, Cf-ECP2 was accurately mapped on chromosome 1, 7.7 cM proximal to TG236 and 6.0 cM distal to TG184. Although Cf-ECP2 is linked to Cf-4, it is not located in the Hcr9 cluster "Milky Way". Therefore, Cf-ECP2 is the first functional Cf homologue on chromosome 1 that does not belong to this Hcr9 cluster. No recombination events between Cf-ECP2 and CT116 have been observed in three populations tested, representing 282 individuals. The low value for the physical distance per cM around CT116 reported previously and the high probability that Cf-ECP2 is also a member of a Hcr9 cluster will facilitate cloning of the locus.

Buffer zones for reducing pesticide drift to ditches and risks to aquatic organisms.

Pesticide drift from field sprayers fitted with different types of spray nozzles was investigated under various wind speed conditions. Droplet drift was measured adjacent to the sprayed field, on the ditch bank, and in the ditch. Measurements were carried out in the normal sprayed situation and with an unsprayed buffer zone 3 or 6 m wide. The results indicate that there are major differences between spray nozzles. Drift deposition increases with wind speed. In the sprayed situation and with a wind speed of 0.5 m/s, there was a maximum of 6.0% drift deposition halfway down the ditch bank and no drift deposition in the ditch. At 3 m/s wind speed these figures are 25.1 and 2.2%, respectively. At 5 m/s wind speed, 7.2% drift deposition was measured in the ditch. Risk assessment (cf. SLOOTBOX model) carried out with 17 pesticides used in the study area indicated that at this wind speed, 8 of the 17 pesticides investigated posed a risk to aquatic organisms. Creation of a 3-m buffer zone decreases drift deposition in the ditch by a minimum of 95%. Adjacent to the buffer zone only 4 of the 17 pesticides investigated posed a (minor) risk to aquatic organisms. With a 6-m buffer zone no drift deposition in the ditch could be measured (wind speed maximum, 4.5 m/s). Creating unsprayed crop edges offers good possibilities for the protection of aquatic ecosystems. Socioeconomic research among farmers indicates that buffer zones, such as unsprayed cereal edges and unsprayed grass strips, could well be adopted in agricultural practice.

Fungal avirulence genes: structure and possible functions.

Avirulence (Avr) genes exist in many fungi that share a gene-for-gene relationship with their host plant. They represent unique genetic determinants that prevent fungi from causing disease on plants that possess matching resistance (R) genes. Interaction between elicitors (primary or secondary products of Avr genes) and host receptors in resistant plants causes induction of various defense responses often involving a hypersensitive response. Avr genes have been successfully isolated by reverse genetics and positional cloning. Five cultivar-specific Avr genes (Avr4, Avr9, and Ecp2 from Cladosporium fulvum; nip1 from Rhynchosporium secalis; and Avr2-YAMO from Magnaporthe grisea) and three species-specific Avr genes (PWL1 and PWL2 from M. grisea and inf1 from Phytophthora infestans) have been cloned. Isolation of additional Avr genes from these fungi, but also from other fungi such as Uromyces vignae, Melampsora lini, Phytophthora sojae, and Leptosphaeria maculans, is in progress. Molecular analyses of nonfunctional Avr gene alleles show that these originate from deletions or mutations in the open reading frame or the promoter sequence of an Avr gene. Although intrinsic biological functions of most Avr gene products are still unknown, recent studies have shown that two Avr genes, nip1 and Ecp2, encode products that are important pathogenicity factors. All fungal Avr genes cloned so far have been demonstrated or predicted to encode extracellular proteins. Current studies focus on unraveling the mechanisms of perception of avirulence factors by plant receptors. The exploitation of Avr genes and the matching R genes in engineered resistance is also discussed.

Successful search for a resistance gene in tomato targeted against a virulence factor of a fungal pathogen.

The interaction between tomato and its fungal pathogen Cladosporium fulvum complies with the gene-for-gene system, in which specific recognition of fungal proteins by plant genotypes with matching resistance genes results in host resistance. Two proteins, ECP1 and ECP2, secreted by C. fulvum during infection, are required for full virulence of the fungus on tomato. We chose the most important virulence factor, ECP2, for a targeted search for hypersensitive response (HR)-based resistance among a collection of tomato genotypes. By screening with recombinant potato virus X that expresses the Ecp2 gene, we identified four lines that respond with HR toward ECP2. The capacity to recognize ECP2 and induce HR is sufficient to confer resistance in tomato against C. fulvum producing ECP2. Resistance is based on a single dominant gene, which we have designated Cf-ECP2, for resistance to C. fulvum through recognition of ECP2. Accordingly, an Ecp2-minus strain created by gene replacement is pathogenic on Cf-ECP2 plants. However, due to lack of ECP2 the mutant strain is only weakly virulent. All strains of a worldwide collection of C. fulvum strains that were tested were found to produce a HR-inducing ECP2 protein. Because the Cf-ECP2 gene operates through recognition of an important virulence factor, we expect it will confer durable resistance against C. fulvum. A similar targeted approach should allow the discovery of new valuable resistance genes in other pathosystems.