The Cladosporium fulvum resistance gene Cf-ECP3 is part of the Orion cluster on the short arm of tomato Chromosome 1

The resistance of tomato to the pathogenic fungus Cladosporiumfulvum complies with the gene-for-gene relationship. Race specificresistance is based on Cf-gene mediated recognition ofsecreted avirulence products, resulting in a hypersensitive response (HR).Besides the avirulence gene products, C. fulvum secretes anumber of extra cellular proteins (ECPs) into the apoplast. Two L.esculentum accessions have previously been identified that reactedwith a HR upon injection with purified ECP3. The corresponding resistance genedesignated Cf-ECP3 was mapped by using an F₂population composed of 192 plants from the cross of susceptible MoneyMaker toresistant L. esculentum G1.1153.Cf-ECP3 inherited monogenically, cosegragated with theChromosome 1 Cleaved Amplified Polymorphic Sequence (CAPS) marker CT116 and wasmapped accurately at Orion, a locus harbouring Cf-ECP2 inother genotypes. RFLP anaysis with a Cf-9 probe furtherdemonstrated cosegregation of Cf-ECP3 with anHcr9 (Homologue of Cladosporiumfulvumresistance gene Cf-9) indicating that this gene is likelyan Hcr9. Thus in addition to the Milky Way locusharbouringthe Cf-4, Cf-4A andCf-9 resistance genes targeted against AVR4, AVR4A andAVR9, Orion is another complex locus on the short arm of Chromosome 1 thatharbours at least two functional Cf-genes,Cf-ECP2 and Cf-ECP3, targeted againstthe fungal excreted proteins ECP2 and ECP3.     

The Solanum pimpinellifolium Cf-ECP1 and Cf-ECP4 genes for resistance to Cladosporium fulvum are located at the Milky Way locus on the short arm of chromosome 1

The interaction between tomato and the leaf mould pathogen Cladosporium fulvum is an excellent model to study gene-for-gene interactions and plant disease resistance gene evolution. Most Cf genes were introgressed into cultivated tomato (Solanum lycopersicum) from wild relatives such as S. pimpinellifolium and novel Cf-ECP genes were recently identified in this species. Our objective is to isolate Cf-ECP1, Cf-ECP2, Cf-ECP4 and Cf-ECP5 to increase our understanding of Cf gene evolution, and the molecular basis for recognition specificity in Cf proteins. The map locations of Cf-ECP2 and Cf-ECP5 have been reported previously and we report here that Cf-ECP1 and Cf-ECP4 map to a different locus on the short arm of chromosome 1. The analysis of selected recombinants and allelism tests showed both genes are located at Milky Way together with Cf-9 and Cf-4. Our results emphasise the importance of this locus in generating novel Cf genes for resistance to C. fulvum. Candidate genes for Cf-ECP1 and Cf-ECP4 were also identified by DNA gel blot analysis of bulked segregant pools. In addition, we generated functional cassettes for expression of the C. fulvum ECP1, ECP2, ECP4 and ECP5 proteins using recombinant Potato Virus X, and three ECPs were also expressed in stable transformed plants. Using marker-assisted selection we have also identified recombinants containing Cf-ECP1, Cf-ECP2, Cf-ECP4 or Cf-ECP5 in cis with a linked T-DNA carrying the non-autonomous Zea mays transposon Dissociation. Using these resources it should now be possible to isolate all four Cf-ECPs using transposon tagging, or a candidate gene strategy. Eleni Soumpourou and Michael Iakovidis contributed equally to this work.     

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. Received: 15 June 1999 / Accepted: 24 August 1999     

The tomato Orion locus comprises a unique class of <Emphasis Type="Italic">Hcr9</Emphasis> genes

Resistance against the tomato fungal pathogen Cladosporium fulvum is often conferred by Hcr9 genes (Homologues of the C. fulvum resistance gene Cf-9) that are located in the Milky Way cluster on the short arm of chromosome 1. These Hcr9 genes mediate recognition of fungal avirulence gene products. In contrast, the resistance gene Cf-Ecp2 mediates recognition of the virulence factor Ecp2 and is located in the Orion (OR) cluster on the short arm of chromosome 1. Here, we report the map- and homology-based cloning of the OR Hcr9 cluster. A method was optimised to generate clone-specific fingerprint data that were subsequently used in the efficient calculation of genomic DNA contigs. Three Hcr9s were identified as candidate Cf-Ecp2 genes. By PCR-based cloning using specific OR sequences, orthologous Hcr9 genes were identified from different Lycopersicon species and haplotypes. The OR Hcr9s are very homologous. However, based on the relative low sequence homology to other Hcr9s, the OR Hcr9s are classified as a new subgroup.Data deposition: The sequence of the Cf-Ecp2 Hcr9 gene cluster and the orthologous Hcr9 sequences have been deposited in the GenBank database (accession No. AY639600..AY639604)     

RFLP linkage analysis of the Cf-4 and Cf-9 genes for resistance toCladosporium fulvum in tomato

Four different populations segregating for one of the two closely linked (possibly allelic) tomato disease resistance genes to the fungusCladosporium fulvum,Cf-4 andCf-9, were generated and analysed for recombination frequencies between theCf-genes and restriction fragment length polymorphism (RFLP) loci. The population consisting of F₂ progeny from the interspecific crossLycopersicon esculentum carryingCf-9 ×L. pennellii was identified as the most useful for RFLP mapping of theCf-4/9 locus and an RFLP map around this locus was constructed mainly using this population. The two closest markers identified were CP46, 2.6 cM distal, and a group of 11 markers including TG236, 3.7 cM proximal toCf-4/9. A polymerase chain reaction (PCR)-based procedure for the rapid identification of recombination events between these two markers was developed. The regions of foreign DNA introgression surroundingCf-4 andCf-9 in near-isogenic lines were delimited.     

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

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 F₁ seedlings was found to differ significantly. Macroscopic observation revealed that in F₁ 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.     

Novel Mutations Detected in Avirulence Genes Overcoming Tomato Cf Resistance Genes in Isolates of a Japanese Population of Cladosporium fulvum

Leaf mold of tomato is caused by the biotrophic fungus Cladosporium fulvum which complies with the gene-for-gene system. The disease was first reported in Japan in the 1920s and has since been frequently observed. Initially only race 0 isolates were reported, but since the consecutive introduction of resistance genes Cf-2, Cf-4, Cf-5 and Cf-9 new races have evolved. Here we first determined the virulence spectrum of 133 C. fulvum isolates collected from 22 prefectures in Japan, and subsequently sequenced the avirulence (Avr) genes Avr2, Avr4, Avr4E, Avr5 and Avr9 to determine the molecular basis of overcoming Cf genes. Twelve races of C. fulvum with a different virulence spectrum were identified, of which races 9, 2.9, 4.9, 4.5.9 and 4.9.11 occur only in Japan. The Avr genes in many of these races contain unique mutations not observed in races identified elsewhere in the world including (i) frameshift mutations and (ii) transposon insertions in Avr2, (iii) point mutations in Avr4 and Avr4E, and (iv) deletions of Avr4E, Avr5 and Avr9. New races have developed by selection pressure imposed by consecutive introductions of Cf-2, Cf-4, Cf-5 and Cf-9 genes in commercially grown tomato cultivars. Our study shows that molecular variations to adapt to different Cf genes in an isolated C. fulvum population in Japan are novel but overall follow similar patterns as those observed in populations from other parts of the world. Implications for breeding of more durable C. fulvum resistant varieties are discussed.     

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.     

The Cf-4 and Cf-9 resistance genes against Cladosporium fulvum are conserved in wild tomato species

The Cf-4 and Cf-9 genes originate from the wild tomato species Lycopersicon hirsutum and L. pimpinellifolium and confer resistance to strains of the leaf mold fungus Cladosporium fulvum that secrete the Avr4 and Avr9 elicitor proteins, respectively. Homologs of Cf-4 and Cf-9 (Hcr9s) are located in several clusters and evolve mainly through sequence exchange between homologs. To study the evolution of Cf genes, we set out to identify functional Hcr9s that mediate recognition of Avr4 and Avr9 (designated Hcr9-Avr4s and Hcr9-Avr9s) in all wild tomato species. Plants responsive to the Avr4 and Avr9 elicitor proteins were identified throughout the genus Lycopersicon. Open reading frames of Hcr9s from Avr4- and Avr9-responsive tomato plants were polymerase chain reaction-amplified. Several Hcr9s that mediate Avr4 or Avr9 recognition were identified in diverged tomato species by agroinfiltration assays. These Hcr9-Avr4s and Hcr9-Avr9s are highly identical to Cf-4 and Cf-9, respectively. Therefore, we conclude that both Cf-4 and Cf-9 predate Lycopersicon speciation. These results further suggest that C. fulvum is an ancient pathogen of the genus Lycopersicon, in which Cf-4 and Cf-9 have been maintained by selection pressure imposed by C. fulvum.     

A second gene at the tomato Cf-4 locus confers resistance to cladosporium fulvum through recognition of a novel avirulence determinant

The tomato Cf-4 and Cf-9 genes confer resistance to the leaf mould pathogen Cladosporium fulvum and map at a complex locus on the short arm of chromosome 1. It was previously shown that the gene encoding Cf-4, which recognizes the Avr4 avirulence determinant, is one of five tandemly duplicated homologous genes (Hcr9-4s) at this locus. Cf-4 was identified by molecular analysis of rare Cf-4/Cf-9 disease-sensitive recombinants and by complementation analysis. The analysis did not exclude the possibility that an additional gene(s) located distal to Cf-4 may also confer resistance to C. fulvum. We demonstrate that a number of Dissociation-tagged Cf-4 mutants, identified on the basis of their insensitivity to Avr4, are still resistant to infection by C. fulvum race 5. Molecular analysis of 16 Cf-4 mutants, most of which have small chromosomal deletions in this region, suggested the additional resistance specificity is encoded by Hcr9-4E. Hcr9-4E recognizes a novel C. fulvum avirulence determinant that we have designated Avr4E.     

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.     

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.     

Induction of Defense-Related Responses in Cf9 Tomato Cells by the AVR9 Elicitor Peptide of Cladosporium fulvum Is Developmentally Regulated

The AVR9 elicitor from the fungal pathogen Cladosporium fulvum induces defense-related responses, including cell death, specifically in tomato (Lycopersicon esculentum Mill.) plants that carry the Cf-9 resistance gene. To study biochemical mechanisms of resistance in detail, suspension cultures of tomato cells that carry the Cf-9 resistance gene were initiated. Treatment of cells with various elicitors, except AVR9, induced an oxidative burst, ion fluxes, and expression of defense-related genes. Agrobacterium tumefaciens-mediated transformation of Cf9 tomato leaf discs with Avr9-containing constructs resulted efficiently in transgenic callus formation. Although transgenic callus tissue showed normal regeneration capacity, transgenic plants expressing both the Cf-9 and the Avr9 genes were never obtained. Transgenic F₁ seedlings that were generated from crosses between tomato plants expressing the Avr9 gene and wild-type Cf9 plants died within a few weeks. However, callus cultures that were initiated on cotyledons from these seedlings could be maintained for at least 3 months and developed similarly to callus cultures that contained only the Cf-9 or the Avr9 gene. It is concluded, therefore, that induction of defense responses in Cf9 tomato cells by the AVR9 elicitor is developmentally regulated and is absent in callus tissue and cell-suspension cultures, which consists of undifferentiated cells. These results are significant for the use of suspension-cultured cells to investigate signal transduction cascades.     

Identification and Ds‐tagged isolation of a new gene at the Cf‐4 locus of tomato involved in disease resistance to Cladosporium fulvum race 5

Leaf mould disease in tomato is caused by the biotrophic fungus Cladosporium fulvum. An Ac/Ds targeted transposon tagging strategy was used to isolate the gene conferring resistance to race 5 of C. fulvum, a strain expressing the avirulence gene Avr4. An infection assay of 2-week-old seedlings yielded five susceptible mutants, of which two had a Ds element integrated in the same gene at different positions. This gene, member of a gene family, showed high sequence homology to the C. fulvum resistance genes Cf-9 and Cf-2. The gene is predicted to encode an extracellular transmembrane protein containing a divided domain of 25 leucine-rich repeats. Three mutants exhibited a genomic deletion covering most of the Lycopersicon hirsutum introgressed segment, including the Cf-4 locus. Southern blot analysis revealed that this deletion includes the tagged gene and five homologous sequences. To test whether the tagged gene confers resistance to C. fulvum via Avr4 recognition, the Avr4 gene was expressed in planta. Surprisingly, expression of the Avr4 gene still triggered a specific necrotic response in the transposon-tagged plants, indicating that the tagged resistance gene is not, or is not the only gene, involved in Avr4 recognition. Mutants harbouring the genomic deletion did not show this Avr4-specific response. The deleted segment apparently contains, in addition to the tagged gene, one or more other genes, which play a role in the Avr4 responses. The tagged gene is present at the Cf-4 locus, but it does not necessarily recognize Avr4 and is therefore designated Cf-4A.     

The tomato Cf-9 disease resistance gene functions in tobacco and potato to confer responsiveness to the fungal avirulence gene product avr 9

The Cf-9 gene encodes an extracytoplasmic leucine-rich repeat protein that confers resistance in tomato to races of the fungus Cladosporium fulvum that express the corresponding avirulence gene Avr 9. We investigated whether the genomic Cf-9 gene functions in potato and tobacco. Transgenic tobacco and potato plants carrying Cf-9 exhibit a rapid hypersensitive cell death response (HR) to Avr 9 peptide injection. Cf 9 tobacco plants were reciprocally crossed to Avr 9-producing tobacco. A developmentally regulated seedling lethal phenotype occurred in F1 progeny when Cf9 was used as the male parent and Avr 9 as the female parent. However, when Cf9 was inherited in the maternal tissue and a heterozygous Avr 9 plant was used as the pollen donor, a much earlier reaction was caused, leading to no germination of any F1 seed. Detailed analysis of the Avr 9-induced responses in Cf 9 tobacco leaves revealed that (1) most mesophyll cells died within 3 hr (compared with 12 to 16 hr in tomato); (2) the macroscopic HR was visible at an Avr 9 titer five times lower than that which caused visible symptoms in tomato; (3) the HR invariably extended into noninjected panels of the tobacco leaf; (4) no HR occurred in leaves of young tobacco plants; (5) in older plants, the HR was dramatically enhanced by sequential Avr 9 challenges; and (6) coexpression of a salicylate hydroxylase transgene (nahG) from Pseudomonas putida reduced the severity of the macroscopic leaf HR and also restored germination to Cf 9 x 35S:Avr 9 F1 seedlings. Simultaneous introduction of Cf-9 homologs (Hcr 9-9 genes A and B or D) along with the native Cf-9 gene did not alter the responses that were specifically induced by Avr 9. Various ways to use the Cf-9-Avr 9 gene combination to engineer broad-spectrum disease resistance in several solanaceous species are discussed.     

Molecular communication between host plant and the fungal tomato pathogenCladosporium fulvum

Host genotype specificity in interactions between biotrophic fungal pathogens and plants in most cases complies with the gene-for-gene model. Success or failure of infection is determined by absence or presence of complementary genes, avirulence and resistance genes, in the pathogen and the host plant, respectively. Resistance, expressed by the induction of a hypersensitive response followed by other defence responses in the host, is envisaged to be based on recognition of the pathogen, mediated through direct interaction between products of avirulence genes of the pathogen (the so-called race-specific elicitors) and receptors in the host plant, the putative products of resistance genes. The interaction between the biothrophic fungusCladosporium fulvum and its only host tomato is a model system to study fungus-plant gene-for-gene relationships. Here we report on isolation, characterization and biological function of putative pathogenicity factors ECP1 and ECP2 and the race-specific elicitors AVR4 and AVR9 ofC. fulvum and cloning and regulation of their encoding genes. Disruption ofecp1 andecp2 genes has no clear effect on pathogenicity ofC. fulvum. Disruption of theavr9 gene, which codes for the race-specific 28 amino acid AVR9 elicitor, in wild type avirulent races, leads to virulence on tomato genotypes carrying the complementary resistance geneCf9. The avirulence geneavr4 encodes a 105 amino acid race-specific elicitor. A single basepair change in the avirulence geneavr4 leads to virulence on tomato genotypes carrying theCf4 resistance gene.