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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Functional expression of Cf9 and Avr9 genes in Brassica napus induces enhanced resistance to Leptosphaeria maculans

The tomato Cf9 resistance gene induces an Avr9-dependent hypersensitive response (HR) in tomato and transgenic Solanaceae spp. We studied whether the Cf9 gene product responded functionally to the corresponding Avr9 gene product when introduced in a heterologous plant species. We successfully expressed the Cf9 gene under control of its own promoter and the Avr9 or Avr9R8K genes under control of the p35S1 promoter in transgenic oilseed rape. We demonstrated that the transgenic oilseed rape plants produced the Avr9 elicitor with the same specific necrosis-inducing activity as reported for Cladosporium fulvum. An Avr9-dependent HR was induced in Cf9 oilseed rape upon injection of intercellular fluid containing Avr9. We showed Avr9-specific induction of PR1, PR2, and Cxc750 defense genes in oilseed rape expressing CJ9. Cf9 x Avr9 oilseed rape did not result in seedling death of the F1 progeny, independent of the promoters used to express the genes. The F1 (Cf9 x Avr9) plants, however, were quantitatively more resistant to Leptosphaeria maculans. Phytopathological analyses revealed that disease development of L. maculans was delayed when the pathogen was applied on an Avr9-mediated HR site. We demonstrate that the CJ9 and Avr9 gene can be functionally expressed in a heterologous plant species and that the two components confer an increase in disease resistance.     

CITRX thioredoxin interacts with the tomato Cf-9 resistance protein and negatively regulates defence

To identify proteins involved in tomato Cf-9 resistance protein function, a yeast two-hybrid screen was undertaken using the cytoplasmic C-terminus of Cf-9 as bait. A thioredoxin-homologous clone, interacting specifically with Cf-9, was identified and called CITRX (Cf-9-interacting thioredoxin). Virus-induced gene silencing (VIGS) of CITRX resulted in an accelerated Cf-9/Avr9-triggered hypersensitive response in both tomato and Nicotiana benthamiana, accompanied by enhanced accumulation of reactive oxygen species, alteration of protein kinase activity and induction of defence-related genes. VIGS of CITRX also conferred increased resistance to the fungal pathogen Cladosporium fulvum in the otherwise susceptible Cf0 tomato. CITRX acts as a negative regulator of the cell death and defence responses induced through Cf-9, but not Cf-2. Recognition of the Cf-9 C-terminus by CITRX is necessary and sufficient for this negative regulation. This is the first study that implicates thioredoxin activity in the regulation of plant disease resistance.     

An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins

Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1(D481V), which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protein that also functions in cell death signalling pathways triggered by other R proteins.     

The F-box protein ACRE189/ACIF1 regulates cell death and defense responses activated during pathogen recognition in tobacco and tomato

Virus-induced gene silencing identified the Avr9/Cf-9 RAPIDLY ELICITED gene ACRE189 as essential for the Cf-9- and Cf-4-mediated hypersensitive response (HR) in Nicotiana benthamiana. We report a role for ACRE189 in disease resistance in tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum). ACRE189 (herein renamed Avr9/Cf-9-INDUCED F-BOX1 [ACIF1]) encodes an F-box protein with a Leu-rich-repeat domain. ACIF1 is widely conserved and is closely related to F-box proteins regulating plant hormone signaling. Silencing of tobacco ACIF1 suppressed the HR triggered by various elicitors (Avr9, Avr4, AvrPto, Inf1, and the P50 helicase of Tobacco mosaic virus [TMV]). ACIF1 is recruited to SCF complexes (a class of ubiquitin E3 ligases), and the expression of ACIF1 F-box mutants in tobacco compromises the HR similarly to ACIF1 silencing. ACIF1 affects N gene-mediated responses to TMV infection, including lesion formation and salicylic acid accumulation. Loss of ACIF1 function also reduced confluent cell death induced by Pseudomonas syringae pv tabaci. ACIF1 silencing in Cf9 tomato attenuated the Cf-9-dependent HR but not Cf-9 resistance to Cladosporium fulvum. Resistance conferred by the Cf-9 homolog Cf-9B, however, was compromised in ACIF1-silenced tomato. Analysis of public expression profiling data suggests that Arabidopsis thaliana homologs of ACIF1 (VFBs) regulate defense responses via methyl jasmonate- and abscisic acid-responsive genes. Together, these findings support a role of ACIF1/VFBs in plant defense responses.     

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.