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.     

Metabolic changes in fruits of the tomato dx mutant

The tomato DWARF cytochrome P450 protein catalyzes the C-6 oxidation of 6-deoxo-castasterone to castasterone. The d(x) mutant does not produce a functional DWARF enzyme, and d(x) shoots display severe symptoms of brassinosteroid-deficiency. However, fruits express the CYP85A3 protein which compensates for the deficiency of the DWARF protein and produce bioactive brassinosteroids. Here, we report on the metabolic characterization of d(x) fruits. Fruit size, fresh weight, and pigment content were not altered. However, d(x) fruits showed reduced dry mass content. Levels of starch and various sugars were reduced, amino acid levels were elevated. BR application to d(x) leaves partially normalized dry mass content, sugar and amino acid levels in d(x) fruits. The data demonstrate that brassinosteroid in shoots is required for fruit development in tomato.     

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.     

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.     

A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics

We report a highly efficient protocol for the Agrobacterium-mediated genetic transformation of a miniature dwarf tomato (Lycopersicon esculentum), Micro-Tom, a model cultivar for tomato functional genomics. Cotyledon explants of tomato inoculated with Agrobacterium tumefaciens (Rhizobium radiobacter) C58C1Rif(R) harboring the binary vector pIG121Hm generated a mass of chimeric non-transgenic and transgenic adventitious buds. Repeated shoot elongation from the mass of adventitious buds on selection media resulted in the production of multiple transgenic plants that originated from independent transformation events. The transformation efficiency exceeded 40% of the explants. This protocol could become a powerful tool for functional genomics in tomato.     

Micro-Tom番茄矮化微型机制及其在植物功能基因组学研究中的应用

Micro-Tom番茄植株矮小, 生长密度高, 生命周期短, 容易被高效转化, 成为功能基因组学研究的新型模式植物。文章对Micro-Tom的由来和生物学特征作了概述, 重点阐述了Micro-Tom矮化微型的遗传机制以及该番茄在功能基因组学研究中的应用和遗传转化体系研究的进展。     

A new model system for tomato genetics

The purpose of this study was to develop a model system for studying tomato genetics. Agronomic, genetic, and molecular data are presented which show that the miniature Lycopersicon esculentum cultivar, Micro-Tom (Micro tomato), fulfills the requirements for such a model. It grows at high density (up to 1357 plants/m⁻²); it has a short life cycle (70–90 days from sowing to fruit ripening); and it can be transformed at frequencies of up to 80% through Agrobacterium -mediated transformation of cotyledons. Moreover, it differs from standard tomato cultivars by only two major genes. Therefore, any mutation or transgene can be conveniently studied in Micro-Tom's background and, when needed, transferred into a standard background. We took advantage of Micro-Tom's features to improve the infrastructure for mutagenesis in tomato. A screening of 9000 M1 and 20 000 M2 EMS mutagenized plants is described. Mutants with altered pigmentation or modified shape of leaves, flowers and fruits were found. In addition, an enhancer trapping and a gene trapping system, based on the Ac/Ds maize transposable elements, were transformed into Micro-Tom and found to be active. In summary, Micro-Tom opens new prospects to achieve saturated mutagenesis in tomato, and facilitates the application of transposon-based technologies such as gene tagging, trapping and knockout.     

Cloning and characterization of cDNA of avirulence gene avr9 of the fungal pathogen Cladosporium fulvum, causal agent of tomato leaf mold.

A race-specific peptide elicitor from Cladosporium fulvum induces a hypersensitive response on Cf9 tomato genotypes. We have hypothesized that the avirulence of fungal races on Cf9 genotypes is due to the production of this elicitor by an avirulence gene, avr9. To obtain cDNA clones of the avr9 gene, oligonucleotide probes were designed based on the amino acid sequence determined previously. In northern blot analysis, one oligonucleotide detected an mRNA of 600 nucleotides in tomato-C. fulvum interactions involving fungal races producing the elicitor. A primer extension experiment indicated that the probe hybridized to a region near position 270 of the mRNA. The probe was used to screen a cDNA library made from poly(A)+ RNA from an appropriate compatible tomato-C. fulvum interaction. One clone was obtained corresponding to the mRNA detected by the oligonucleotide probe. Sequence analysis revealed that this clone encoded the avr9 elicitor. By isolating longer clones and by RNA sequencing, the primary structure of the mRNA was determined. The mRNA contains an open reading frame of 63 amino acids, including the sequence of the elicitor at the carboxyterminus. A time course experiment showed that the avr9 mRNA accumulates in a compatible tomato-C. fulvum interaction in correlation with the increase of fungal biomass. The avr9 gene is a single-copy gene that is absent in fungal races which are virulent on tomato Cf9 genotypes. Possible functions of the avirulence gene are discussed.     

Genetic and molecular analysis of tomato Cf genes for resistance to Cladosporium fulvum.

In many plant-pathogen interactions resistance to disease is controlled by the interaction of plant-encoded resistance (R) genes and pathogen-encoded avirulence (Avr) genes. The interaction between tomato and the leaf mould pathogen Cladosporium fulvum is an ideal system to study the molecular basis of pathogen perception by plants. A total of four tomato genes for resistance to C. fulvum (Cf-2, Cf-4, Cf-5 and Cf-9) have been isolated from two genetically complex chromosomal loci. Their gene products recognize specific C. fulvum-encoded avirulence gene products (Avr2, Avr4, Avr5 and Avr9) by an unknown molecular mechanism. Cf genes encode extracellular membrane-anchored glycoproteins comprised predominantly of 24 amino acid leucine-rich repeats (LRRs). Cf genes from the same locus encode proteins which are more than 90% identical. Most of the amino-acid sequence differences correspond to the solvent-exposed residues within a beta-strand/beta-turn structural motif which is highly conserved in LRR proteins. Sequence variability within this motif is predicted to affect the specificity of ligand binding. Our analysis of Cf gene loci at the molecular level has shown they comprise tandemly duplicated homologous genes, and suggests a molecular mechanism for the generation of sequence diversity at these loci. Our analysis provides further insight into the molecular basis of pathogen perception by plants and the organization and evolution of R gene loci.     

Salicylic acid is not required for Cf-2- and Cf-9-dependent resistance of tomato to Cladosporium fulvum

Tomato leaves or cotyledons expressing the Cf-2 or Cf-9 Cladosporium fulvum resistance genes induce salicylic acid (SA) synthesis following infiltration with intercellular washing fluid (IF) containing the fungal peptide elicitors Avr2 and Avr9. We investigated whether SA was required for Cf gene-dependent resistance. Tomato plants expressing the bacterial gene nahG, encoding salicylate hydroxylase, did not accumulate SA in response to IF infiltration but remained fully resistant to C. fulvum. NahG Cf0 plants were as susceptible to C. fulvum as wild-type Cf0. Neither free nor conjugated salicylic acid accumulated in IF-infiltrated Cf2 and Cf9 NahG leaves and cotyledons but conjugated catechol did accumulate. The Cf-9-dependent necrotic response to IF was prevented in NahG plants and replaced by a chlorotic Cf-2-like response. SA also potentiated Cf-9-mediated necrosis in IF-infiltrated wild-type leaves. In contrast, the Cf-2-dependent IF response was retained in NahG leaves and chlorosis was more pronounced than in the wild-type. The distribution of cell death between different cell types was altered in both Cf2 and Cf9 NahG leaves after IF injection. IF-induced accumulation of three SA-inducible defence-related genes was delayed and reduced but not abolished in NahG Cf2 and Cf9 leaves and cotyledons. NahG Tm-22 tomato showed increased hypersensitive response (HR) lesion size upon TMV infection, as observed in TMV-inoculated N gene-containing NahG tobacco plants.     

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.     

Involvement of Reactive Oxygen Species, Glutathione Metabolism, and Lipid Peroxidation in the Cf-Gene-Dependent Defense Response of Tomato Cotyledons Induced by Race-Specific Elicitors of Cladosporium fulvum

The chronological order of responses to Cladosporium fulvum (Cooke) (Cf) race-specific elicitors was assessed in cotyledons of three near-isogenic tomato (Lycopersicon esculentum Mill.) lines carrying either Cf-9 or Cf-2 or no Cf gene. The responses observed were dependent on the presence of a Cf gene, Avr-gene product dose injected, and the relative humidity (RH) of the growth chamber. At ambient RH, superoxide formation and lipid peroxidation occurred after 2 h (Cf9) and 4 h (Cf2). At elevated RH (98%) and at lower avirulence elicitor dose, Cf-Avr-dependent lipid peroxidation was considerably attenuated. Significant electrolyte leakage occurred by 18 h but only at the lower RH. Total glutathione levels began to increase 2 to 4 h and 4 to 8 h after challenge of Cf9 and Cf2 cells, respectively, and by 48 h reached 665 and 570% of initial levels. A large proportion of this accumulation (87%) was as oxidized glutathione. When the RH was increased to 98%, increases in glutathione levels were strongly attenuated. Increased lipoxygenase enzyme activity was detected 8 h postchallenge in either incompatible interaction. These results indicate that the activation of the Cf-Avr-mediated defense response results in severe oxidative stress.     

Rearrangements in the Cf-9 disease resistance gene cluster of wild tomato have resulted in three genes that mediate Avr9 responsiveness

Cf resistance genes in tomato confer resistance to the fungal leaf pathogen Cladosporium fulvum. Both the well-characterized resistance gene Cf-9 and the related 9DC gene confer resistance to strains of C. fulvum that secrete the Avr9 protein and originate from the wild tomato species Lycopersicon pimpinellifolium. We show that 9DC and Cf-9 are allelic, and we have isolated and sequenced the complete 9DC cluster of L. pimpinellifolium LA1301. This 9DC cluster harbors five full-length Cf homologs, including orthologs of the most distal homologs of the Cf-9 cluster and three central 9DC genes. Two 9DC genes (9DC1 and 9DC2) have an identical coding sequence, whereas 9DC3 differs at its 3' terminus. From a detailed comparison of the 9DC and Cf-9 clusters, we conclude that the Cf-9 and Hcr9-9D genes from the Cf-9 cluster are ancestral to the first 9DC gene and that the three 9DC genes were generated by subsequent intra- and intergenic unequal recombination events. Thus, the 9DC cluster has undergone substantial rearrangements in the central region, but not at the ends. Using transient transformation assays, we show that all three 9DC genes confer Avr9 responsiveness, but that 9DC2 is likely the main determinant of Avr9 recognition in LA1301.     

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 Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection

Resistance against the leaf mold fungus Cladosporium fulvum is mediated by the tomato Cf proteins which belong to the class of receptor-like proteins and indirectly recognize extracellular avirulence proteins (Avrs) of the fungus. Apart from triggering disease resistance, Avrs are believed to play a role in pathogenicity or virulence of C. fulvum. Here, we report on the avirulence protein Avr4, which is a chitin-binding lectin containing an invertebrate chitin-binding domain (CBM14). This domain is found in many eukaryotes, but has not yet been described in fungal or plant genomes. We found that interaction of Avr4 with chitin is specific, because it does not interact with other cell wall polysaccharides. Avr4 binds to chitin oligomers with a minimal length of three N-acetyl glucosamine residues. In vitro, Avr4 protects chitin against hydrolysis by plant chitinases. Avr4 also binds to chitin in cell walls of the fungi Trichoderma viride and Fusarium solani f. sp. phaseoli and protects these fungi against normally deleterious concentrations of plant chitinases. In situ fluorescence studies showed that Avr4 also binds to cell walls of C. fulvum during infection of tomato, where it most likely protects the fungus against tomato chitinases, suggesting that Avr4 is a counter-defensive virulence factor.     

Cladosporium fulvum (syn. Passalora fulva), a highly specialized plant pathogen as a model for functional studies on plant pathogenic Mycosphaerellaceae

Taxonomy:  Cladosporium fulvum is an asexual fungus for which no sexual stage is currently known. Molecular data, however, support C. fulvum as a member of the Mycosphaerellaceae, clustering with other taxa having Mycosphaerella teleomorphs . C. fulvum has recently been placed in the anamorph genus Passalora as P. fulva . Its taxonomic disposition is supported by its DNA phylogeny, as well as the distinct scars on its conidial hila, which are typical of Passalora , and unlike Cladosporium s.s. , which has teleomorphs that reside in Davidiella , and not Mycosphaerella .
Host range and disease symptoms:  The presently known sole host of C. fulvum is tomato (members of the genus Lycopersicon ). C. fulvum is mainly a foliar pathogen. Disease symptoms are most obvious on the abaxial side of the leaf and include patches of white mould that turn brown upon sporulation. Due to stomatal clogging, curling of leaves and wilting can occur, leading to defoliation.
C. fulvum as a model pathogen:  The interaction between C. fulvum and tomato is governed by a gene-for-gene relationship. A total of eight Avr and Ecp genes, and for four of these also the corresponding plant Cf genes, have been cloned. Obtaining conclusive evidence for gene-for-gene relationships is complicated by the poor availability of genetic tools for most Mycosphaerellaceae – plant interactions. Newly developed tools, including Agrobacterium -mediated transformation and RNAi, added to the genome sequence of its host tomato, which will be available within a few years, render C. fulvum attractive as a model species for plant pathogenic Mycosphaerellaceae.
Useful websites:  http://www.sgn.cornell.edu/help/about/index.html ; http://cogeme.ex.ac.uk     

Small and remarkable: The Micro-Tom model system as a tool to discover novel hormonal functions and interactions

Hormones are molecules involved in virtually every step of plant development and studies in this field have been shaping plant physiology for more than a century. The model plant Arabidopsis thaliana, long used as a tool to study plant hormones, lacks significant important developmental traits, such as fleshy climacteric fruit, compound leaf and multicellular trichomes, suggesting the necessity for alternative plant models. An attractive option often used is tomato, a species also of major economic importance, being ideal to bring together basic and applied plant sciences. The tomato Micro-Tom (MT) cultivar makes it possible to combine the direct benefits of studying a crop species with the fast life cycle and small size required for a suitable biological model. However, few obscure questions are constantly addressed to MT, creating a process herein called “MT mystification”. In this work we present evidence clarifying these questions and show the potential of MT, aiming to demystify it. To corroborate our ideas we showed that, by making use of MT, our laboratory demonstrated straightforwardly new hormonal functions and also characterized a novel antagonistic hormonal interaction between jasmonates and brassinosteroids in the formation of anti-herbivory traits in tomato.Key words: Solanum lycopersicum, Arabidopsis thaliana, hormones, plant model, jasmonates, brassinosteroids     

Genetic variation at the tomato Cf-4/Cf-9 locus induced by EMS mutagenesis and intralocus recombination

The interaction between tomato (Lycopersicon esculentum) and the leaf mold pathogen Cladosporium fulvum is an excellent model for investigating disease resistance gene evolution. The interaction is controlled in a gene-for-gene manner by Cf genes that encode type I transmembrane extracellular leucine-rich repeat glycoproteins that recognize their cognate fungal avirulence (Avr) proteins. Cf-4 from L. hirsutum and Cf-9 from L. pimpinellifolium are located at the same locus on the short arm of tomato chromosome 1 in an array of five paralogs. Molecular analysis has shown that one mechanism for generating sequence variation in Cf genes is intragenic sequence exchange through unequal crossing over or gene conversion. To investigate this we used a facile genetic selection to identify novel haplotypes in the progeny of Cf-4/Cf-9 trans-heterozygotes that lacked Cf-4 and Cf-9. This selection is based on the ability of Avr4 and Avr9 to induce Cf-4- or Cf-9-dependent seedling death. The crossovers were localized to the same intergenic region defining a recombination hotspot in this cross. As part of a structure-function analysis of Cf-9 and Cf-4, nine EMS-induced mutant alleles have been characterized. Most mutations result in single-amino-acid substitutions in their C terminus at residues that are conserved in other Cf proteins.