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.     

Gene shuffling-generated and natural variants of the tomato resistance gene Cf-9 exhibit different auto-necrosis-inducing activities in Nicotiana species

Tomato Cf genes encode membrane-bound proteins with extracellular leucine-rich repeats, and confer resistance to the fungal tomato pathogen Cladosporium fulvum, and a hypersensitive response (HR) to C. fulvum-derived race-specific elicitors. Several Cf genes, including Cf-4 and Cf-9, are members of the highly homologous Hcr9 (homologues of C. fulvumresistance gene Cf-9) gene family. Hcr9s evolve mainly by sequence exchange between paralogues, by which novel Cf genes may be generated. To mimic this aspect of natural evolution, we generated chimeras between multiple Hcr9s in vitro by gene shuffling. The shufflants were tested for novel specificities by transient expression in Nicotiana benthamiana. Many shufflants induced an HR in the absence of fungal elicitors and were designated auto-activators. We also identified two natural Hcr9 auto-activators in the wild tomato species Lycopersicon peruvianum, which induced an HR upon expression in N. benthamiana. The Hcr9 auto-activators exhibit different auto-necrosis-inducing specificities in five selected species of the Nicotiana genus, and they were shown to function in the same signalling pathway as Cf-9. Auto-activating alleles of nucleotide binding site-leucine-rich repeat genes and the protein kinase Pto were previously described. The auto-activators described here, belonging to the Cf-like structural class of resistance genes, shed light on this important phenotype and may be used as tools to unravel the mechanisms by which this class of resistance proteins function.     

Molecular analysis of the avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum fully supports the gene-for-gene hypothesis

The interaction between the fungal pathogen Cladosporium fulvum and tomato is supposed to have a gene-for-gene basis. Races of C. fulvum which have 'overcome' the resistance gene Cf9 of tomato, lack the avirulence gene avr9 which encodes a race-specific peptide elicitor. Races avirulent on tomato genotypes carrying the resistance gene Cf9 produce the race-specific peptide elicitor, which induces the hypersensitive response (HR) on those genotypes. The causal relationship between the presence of a functional avr9 gene and avirulence on tomato genotype Cf9 was demonstrated by cloning of the avr9 gene and subsequent transformation of C. fulvum. A race virulent on tomato genotype Cf9 was shown to become avirulent by transformation with the cloned avr9 gene. These results clearly demonstrate that the avr9 gene is responsible for cultivar specificity on tomato genotype Cf9 and fully support the gene-for-gene hypothesis. The avr9 gene is the first fungal avirulence gene to be cloned.     

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.     

Cladosporium fulvum circumvents the second functional resistance gene homologue at the Cf-4 locus (Hcr9-4E ) by secretion of a stable avr4E isoform

Introgression of resistance trait Cf-4 from wild tomato species into tomato cultivar MoneyMaker (MM-Cf0) has resulted in the near-isogenic line MM-Cf4 that confers resistance to the fungal tomato pathogen Cladosporium fulvum. At the Cf-4 locus, five homologues of Cladosporium resistance gene Cf-9 (Hcr9s) are present. While Hcr9-4D represents the functional Cf-4 resistance gene matching Avr4, Hcr9-4E confers resistance towards C. fulvum by mediating recognition of the novel avirulence determinant Avr4E. Here, we report the isolation of the Avr4E gene, which encodes a cysteine-rich protein of 101 amino acids that is secreted by C. fulvum during colonization of the apoplastic space of tomato leaves. By complementation we show that Avr4E confers avirulence to strains of C. fulvum that are normally virulent on Hcr9-4E-transgenic plants, indicating that Avr4E is a genuine, race-specific avirulence determinant. Strains of C. fulvum evade Hcr9-4E-mediated resistance either by a deletion of the Avr4E gene or by production of a stable Avr4E mutant protein that carries two amino acid substitutions, Phe(82)Leu and Met(93)Thr. Moreover, we demonstrate by site-directed mutagenesis that the single amino acid substitution Phe(82)Leu in Avr4E is sufficient to evade Hcr9-4E-mediated resistance.     

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.     

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.     

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.     

Agrosuppression: a bioassay for the hypersensitive response suited to high-throughput screening

We describe a novel method, agrosuppression, that addresses the need for an assay of the hypersensitive response (HR) in intact plants that is rapid and adapted to high-throughput functional screening of plant and pathogen genes. The agrosuppression assay is based on inoculation of intact plants with a mixture of Agrobacterium tumefaciens strains carrying (i) a binary plasmid with one or more candidate HR-inducing genes and (ii) a tumor-inducing (oncogenic) T-DNA. In the absence of HR induction, tumor formation is initiated, resulting in a typical crown gall phenotype. However, upon induction of the HR, tumor formation by the oncogenic T-DNA is suppressed, resulting in a phenotype that can be readily scored. We tested and optimized agrosuppression in Nicotiana benthamiana using the inf1 elicitin gene from the oomycete pathogen Phytophthora infestans, which specifically induces the HR in Nicotiana spp., and the gene-for-gene pair Avr9/Cf-9 from the fungal pathogen Cladosporium fulvum and Lycopersicon pimpinellifolium (currant tomato), respectively. Agrosuppression protocols that can be rapidly performed using simple mechanical wounding of petioles of intact N. benthamiana plants were developed and appeared particularly adapted to intensive high-throughput screening. This assay promises to greatly facilitate the cloning of novel plant R genes and pathogen Avr genes and to accelerate functional analyses and structure-function studies of these genes.