Tomato Cf-9 Research Articles

Analysis of five near-complete genome assemblies of the tomato pathogen Cladosporium fulvum uncovers additional accessory chromosomes and structural variations induced by transposable elements effecting the loss of avirulence genes

BackgroundFungal plant pathogens have dynamic genomes that allow them to rapidly adapt to adverse conditions and overcome host resistance. One way by which this dynamic genome plasticity is expressed is through effector gene loss, which enables plant pathogens to overcome recognition by cognate resistance genes in the host. However, the exact nature of these loses remains elusive in many fungi. This includes the tomato pathogen Cladosporium fulvum, which is the first fungal plant pathogen from which avirulence (Avr) genes were ever cloned and in which loss of Avr genes is often reported as a means of overcoming recognition by cognate tomato Cf resistance genes. A recent near-complete reference genome assembly of C. fulvum isolate Race 5 revealed a compartmentalized genome architecture and the presence of an accessory chromosome, thereby creating a basis for studying genome plasticity in fungal plant pathogens and its impact on avirulence genes.ResultsHere, we obtained near-complete genome assemblies of four additional C. fulvum isolates. The genome assemblies had similar sizes (66.96 to 67.78 Mb), number of predicted genes (14,895 to 14,981), and estimated completeness (98.8 to 98.9%). Comparative analysis that included the genome of isolate Race 5 revealed high levels of synteny and colinearity, which extended to the density and distribution of repetitive elements and of repeat-induced point (RIP) mutations across homologous chromosomes. Nonetheless, structural variations, likely mediated by transposable elements and effecting the deletion of the avirulence genes Avr4E, Avr5, and Avr9, were also identified. The isolates further shared a core set of 13 chromosomes, but two accessory chromosomes were identified as well. Accessory chromosomes were significantly smaller in size, and one carried pseudogenized copies of two effector genes. Whole-genome alignments further revealed genomic islands of near-zero nucleotide diversity interspersed with islands of high nucleotide diversity that co-localized with repeat-rich regions. These regions were likely generated by RIP, which generally asymmetrically affected the genome of C. fulvum.ConclusionsOur results reveal new evolutionary aspects of the C. fulvum genome and provide new insights on the importance of genomic structural variations in overcoming host resistance in fungal plant pathogens.

New Honorary Member of the BSPP

Jonathan D. G. Jones grew up in London and graduated in Botany from Cambridge University followed by graduate work at the PBI, Cambridge. His career in plant–microbe interactions began with Ausubel at Harvard, studying Rhizobium–legume associations. He then worked at Advanced Genetic Sciences Inc. where his expertise using broad host-range plasmids underpinned the development of plant genetic engineering tools using Agrobacterium. Since 1988, he has led a research team at The Sainsbury Lab (TSL), Norwich, where he isolated the tomato Cf-9 gene for resistance to the fungus Cladosporium fulvum. Cf-9 encodes a pioneer cell surface “receptor-like protein” immune receptor, with extracellular leucine-rich repeats (LRRs). He isolated six such genes, each recognizing different fungal virulence factors. These preceded the Nobel prize-winning discoveries of similar Toll-like receptors in animal innate immunity. He went on to characterize complex immune receptor-encoding haplotypes with multiple paralogs and infer their evolutionary trajectories. He discovered that solvent-exposed amino acids in plant immune receptor LRRs are hypervariable and under diversifying selection for specific recognition of distinct pathogen molecules, and that unequal crossing over between paralogous genes at complex loci also contributes to receptor diversification. In a Cf-2 resistance gene suppressor screen, he discovered Rcr3, a cysteine protease required for Cf-2 to recognize the Avr2. In collaboration with the de Wit laboratory in Wageningen, he showed that Cf-2 “guards” Rcr3, activating defence when Rcr3 is inhibited by the Avr2 protease inhibitor. The resulting guard hypothesis was the subject of an influential paper with Dangl in 2001. He also identified several protein kinases and E3 ubiquitin ligases involved in Cf-dependent immunity, verifying their requirement for defence activation, and was first to show the role of receptor-like cytoplasmic kinases in cell surface receptor-mediated immunity. Most plant resistance genes encode intracellular nucleotide-binding LRR (NLR) immune receptors. He cloned one of the earliest (RPP5) and has defined >12 more. He was first to reveal the extreme haplotype diversity at the RPP5 locus between Arabidopsis accessions, again verifying extreme diversity in solvent-exposed amino acids of the LRRs of different paralogs, and unequal crossovers between paralogs in their evolution. In an influential 2006 paper with Dangl, he articulated the roles of cell surface “pattern-triggered immunity” and intracellular “effector-triggered immunity” in plant defence, integrating them for the first time. He has led discoveries on cross-talk between various hormone signalling pathways, in pathogen effector mechanisms, and in the role of variation in detection capacity in population resistance to disease. Some NLR receptors carry integrated domains (IDs) and are encoded adjacent in the genome to another NLR gene. Arabidopsis RPS4 and RRS1 are a paradigmatic example; RRS1 carries a WRKY transcription factor domain and the pair creates an immune receptor that detects pathogen effectors that act on WRKY domains. His work provided the first demonstration that NLRs can evolve to be immune receptors that contain IDs that mimic authentic pathogen targets. His fascination with genetic variation in host–pathogen interactions led him to develop sequence capture-based methods to interrogate plant immune receptor diversity. Resistance gene enrichment sequencing (RenSeq) has greatly accelerated our understanding of plant immune receptor repertoires. He also developed capture-based methods to investigate the genetic diversity of obligate parasites growing on hosts (PenSeq). He used RenSeq to address nonhost resistance (NHR) and defined the genetic basis of Arabidopsis NHR to Brassica-infecting races of white rust, revealing the crucial role of NLR receptors. Solanum americanum shows NHR to the potato late blight pathogen Phytophthora infestans. By isolating two NLR immune receptors, Rpi-amr1 and Rpi-amr3, from S. americanum, defining their recognized effectors from P. infestans, and verifying the efficacy of Rpi-amr1, Rpi-amr3, and Rpi-vnt1 in transgenic potato field experiments, he showed that their deployment might enable NHR to control blight in the field. He has played a key role in making the case that this approach to controlling crop disease is useful, safe, and sustainable. His discoveries in plant molecular genetics earned him election as Professor at UEA (1996), EMBO member (1998), Royal Society Fellow (2003), and International Member of US National Academy of Sciences (2015). He served in Royal Society working groups on food security and coauthored the Royal Society’s 2009 “Reaping the Benefits” report.

Retracted: "CITRX thioredoxin interacts with the tomato Cf-9 resistance protein and negatively regulates defence".

The authors regret to announce they would like to withdraw this paper, for two main reasons: Since the paper was published, it has become clear that the thioredoxin that interacts in yeast 2-hybrid with the Cf-9 C-terminus is in fact localized in the chloroplast, rendering a role in Cf-9 signalling unlikely. Close scrutiny of the figures suggests several duplications. - In Fig 3A, the Anti-MBP band in lane 4 closely resembles the antiMBP band in Fig 3B lane 1, though slightly rotated. - In Fig 6A, the leaf disc in the panel labelled TRV:00, -Avr9, 30min looks identical to the leaf disc in Fig S5, panel labelled Cf2 TRV:CITRX, -Avr2, 1h. - In Fig 6C, multiple bands appear duplicated. For example, GlucA, TRV:00, -Avr9, 0h duplicated with 6h; and GlucB, TRV:00, +Avr9, 0h duplicated with Hin1, TRV:00, +Avr9, 0h. Source data for these figures are not available. All the authors agree that this paper should be withdrawn from the scientific literature.

An EFR-Cf-9 chimera confers enhanced resistance to bacterial pathogens by SOBIR1- and BAK1-dependent recognition of elf18.

SummaryThe transfer of well‐studied native and chimeric pattern recognition receptors (PRRs) to susceptible plants is a proven strategy to improve host resistance. In most cases, the ectodomain determines PRR recognition specificity, while the endodomain determines the intensity of the immune response. Here we report the generation and characterization of the chimeric receptor EFR‐Cf‐9, which carries the ectodomain of the Arabidopsis thaliana EF‐Tu receptor (EFR) and the endodomain of the tomato Cf‐9 resistance protein. Both transient and stable expression of EFR‐Cf‐9 triggered a robust hypersensitive response (HR) upon elf18 treatment in tobacco. Co‐immunoprecipitation and virus‐induced gene silencing studies showed that EFR‐Cf‐9 constitutively interacts with SUPPRESSOR OF BIR1‐1 (SOBIR1) co‐receptor, and requires both SOBIR1 and kinase‐active BRI1‐ASSOCIATED KINASE1 (BAK1) for its function. Transgenic plants expressing EFR‐Cf‐9 were more resistant to the (hemi)biotrophic bacterial pathogens Pseudomonas amygdali pv. tabaci (Pta) 11528 and Pseudomonas syringae pv. tomato DC3000, and mounted an HR in response to high doses of Pta 11528 and P. carotovorum. Taken together, these data indicate that the EFR‐Cf‐9 chimera is a valuable tool for both investigating the molecular mechanisms responsible for the activation of defence responses by PRRs, and for potential biotechnological use to improve crop disease resistance.

A mutational analysis of the cytosolic domain of the tomato Cf-9 disease-resistance protein shows that membrane-proximal residues are important for Avr9-dependent necrosis.

The tomato Cf-9 gene encodes a membrane-anchored glycoprotein that imparts race-specific resistance against the tomato leaf mould fungus Cladosporium fulvum in response to the avirulence protein Avr9. Although the N-terminal half of the extracellular leucine-rich repeat (eLRR) domain of the Cf-9 protein determines its specificity for Avr9, the C-terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf-9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in Avr9-dependent necrosis triggered by Cf-9. Our results indicate that the membrane-proximal region of the cytosolic domain of Cf-9 plays an important role in Cf-9-mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf-9 function. An alanine mutation of T835 had no effect on Cf-9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf-9 function. We therefore postulate that phosphorylation/de-phosphorylation of T835 could act as a molecular switch to determine whether Cf-9 is in a primed or inactive state. Yeast two-hybrid analysis was used to show that the cytosolic domain of Cf-9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline-induced kink in the membrane-proximal region of the cytosolic domain of Cf-9 may be important for interaction with VAP27, which may, in turn, be important for Cf-9 function.

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.

Positive selection and intragenic recombination contribute to high allelic diversity in effector genes of Mycosphaerella fijiensis, causal agent of the black leaf streak disease of banana

Previously, we have determined the nonhost-mediated recognition of the MfAvr4 and MfEcp2 effector proteins from the banana pathogen Mycosphaerella fijiensis in tomato, by the cognate Cf-4 and Cf-Ecp2 resistance proteins, respectively. These two resistance proteins could thus mediate resistance against M. fijiensis if genetically transformed into banana (Musa spp.). However, disease resistance controlled by single dominant genes can be overcome by mutated effector alleles, whose products are not recognized by the cognate resistance proteins. Here, we surveyed the allelic variation within the MfAvr4, MfEcp2, MfEcp2-2 and MfEcp2-3 effector genes of M. fijiensis in a global population of the pathogen, and assayed its impact on recognition by the tomato Cf-4 and Cf-Ecp2 resistance proteins, respectively. We identified a large number of polymorphisms that could reflect a co-evolutionary arms race between host and pathogen. The analysis of nucleotide substitution patterns suggests that both positive selection and intragenic recombination have shaped the evolution of M. fijiensis effectors. Clear differences in allelic diversity were observed between strains originating from South-East Asia relative to strains from other banana-producing continents, consistent with the hypothesis that M. fijiensis originated in the Asian-Pacific region. Furthermore, transient co-expression of the MfAvr4 effector alleles and the tomato Cf-4 resistance gene, as well as of MfEcp2, MfEcp2-2 and MfEcp2-3 and the putative Cf-Ecp2 resistance gene, indicated that effector alleles able to overcome these resistance genes are already present in natural populations of the pathogen, thus questioning the durability of resistance that can be provided by these genes in the field.

Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on dicots and monocots.

Most fungal effectors characterized so far are species-specific and facilitate virulence on a particular host plant. During infection of its host tomato, Cladosporium fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce effector-triggered immunity in their presence. Here we show that homologs of the C. fulvum Avr4 and Ecp2 effectors are present in other pathogenic fungi of the Dothideomycete class, including Mycosphaerella fijiensis, the causal agent of black Sigatoka disease of banana. We demonstrate that the Avr4 homolog of M. fijiensis is a functional ortholog of C. fulvum Avr4 that protects fungal cell walls against hydrolysis by plant chitinases through binding to chitin and, despite the low overall sequence homology, triggers a Cf-4-mediated hypersensitive response (HR) in tomato. Furthermore, three homologs of C. fulvum Ecp2 are found in M. fijiensis, one of which induces different levels of necrosis or HR in tomato lines that lack or contain a putative cognate Cf-Ecp2 protein, respectively. In contrast to Avr4, which acts as a defensive virulence factor, M. fijiensis Ecp2 likely promotes virulence by interacting with a putative host target causing host cell necrosis, whereas Cf-Ecp2 could possibly guard the virulence target of Ecp2 and trigger a Cf-Ecp2-mediated HR. Overall our data suggest that Avr4 and Ecp2 represent core effectors that are collectively recognized by single cognate Cf-proteins. Transfer of these Cf genes to plant species that are attacked by fungi containing these cognate core effectors provides unique ways for breeding disease-resistant crops.

Regions of the Cf-9B Disease Resistance Protein Able to Cause Spontaneous Necrosis in Nicotiana benthamiana Lie Within the Region Controlling Pathogen Recognition in Tomato

The tomato Cf-9 and Cf-9B genes both confer resistance to the leaf mold fungus Cladosporium fulvum but only Cf-9 confers seedling resistance and recognizes the avirulence (Avr) protein Avr9 produced by C. fulvum. Using domain swaps, leucine-rich repeats (LRR) 5 to 15 of Cf-9 were shown to be required for Cf-9-specific resistance to C. fulvum in tomato, and the entire N-terminus up to LRR15 of Cf-9B was shown to be required for Cf-9B-specific resistance. Finer domain swaps showed that nine amino-acid differences in LRR 13 to 15 provided sufficient Cf-9-specific residues in a Cf-9B context for recognition of Avr9 in Nicotiana tabacum or sufficient Cf-9B residues in a Cf-9 context for a novel necrotic response caused by the expression of Cf-9B in N. benthamiana. The responses conferred by LRR 13 to 15 were enhanced by addition of LRR 10 to 12, and either region of Cf-9B was found to cause necrosis in N. benthamiana when the other was replaced by Cf-9 sequence in a Cf-9B context. As a consequence, the domain swap with LRR 13 to 15 of Cf-9 in a Cf-9B context gained the dual ability to recognize Avr9 and cause necrosis in N. benthamiana. Intriguingly, two Cf-9B-specific domain swaps gave differing results for necrosis assays in N. benthamiana compared with disease resistance assays in transgenic tomato. The different domain requirements in these two cases suggest that the two assays detect unrelated ligands or detect related ligands in slightly different ways. A heat-sensitive necrosis-inducing factor present in N. benthamiana intercellular washing fluids was found to cause a necrotic response in N. tabacum plants carrying Hcr9-9A, Cf-9B, and Cf-9 but not in plants carrying only Cf-9. We postulate that this necrosis-inducing factor is recognized by Cf-9B either directly as a ligand or indirectly as a regulator of Cf-9B autoactivity.

The Major Specificity-Determining Amino Acids of the Tomato Cf-9 Disease Resistance Protein Are at Hypervariable Solvent-Exposed Positions in the Central Leucine-Rich Repeats

The interaction between tomato and the leaf mold pathogen Cladosporium fulvum is controlled in a gene-for-gene manner by plant Cf genes that encode membrane-anchored extracytoplasmic leucine-rich repeat (LRR) glycoproteins, which confer recognition of their cognate fungal avirulence (Avr) proteins. Cf-9 and Cf-4 are two such proteins that are 91% identical yet recognize the sequence-unrelated fungal avirulence determinants Avr9 and Avr4, respectively. As shown previously, Cf-4 specificity is determined by three putative solvent-exposed residues in the central LRR and a deletion of two LRR relative to Cf-9. In this study, we focused on identifying the specificity determinants of Cf-9. We generated chimeras between Cf-9 and its close homologue Cf-9B and identified five amino acid residues that constitute major specificity determinants of Cf-9. Introduction of these residues into Cf-9B allowed recognition of Avr9. Consistent with a role in recognition specificity, the identified residues are putatively solvent exposed in the central LRR and occupy hypervariable positions in the global Cf alignment. One of the specificity residues is not found in any other known Cf protein, suggesting the importance of diversifying selection rather than sequence exchange between homologues. Interestingly, there is an overlap between the Cf-4 and Cf-9 specificity-determining residues, precluding a protein with dual specificity.

Recognitional Specificity and Evolution in the Tomato–Cladosporium fulvum Pathosystem

The interactions between plants and many biotrophic or hemibiotrophic pathogens are controlled by receptor proteins in the host and effector proteins delivered by the pathogen. Pathogen effectors facilitate pathogen growth through the suppression of host defenses and the manipulation of host metabolism, but recognition of a pathogen-effector protein by a host receptor enables the host to activate a suite of defense mechanisms that limit pathogen growth. In the tomato (Lycopersicon esculentum syn. Solanum lycopersicum)-Cladosporium fulvum (leaf mold fungus syn. Passalora fulva) pathosystem, the host receptors are plasma membrane-anchored, leucine-rich repeat, receptor-like proteins encoded by an array of Cf genes conferring resistance to C. fulvum. The pathogen effectors are mostly small, secreted, cysteine-rich, but otherwise largely dissimilar, extracellular proteins encoded by an array of avirulence (Avr) genes, so called because of their ability to trigger resistance and limit pathogen growth when the corresponding Cf gene is present in tomato. A number of Cf and Avr genes have been isolated, and details of the complex molecular interplay between tomato Cf proteins and C. fulvum effector proteins are beginning to emerge. Each effector appears to have a different role; probably most bind or modify different host proteins, but at least one has a passive role masking the pathogen. It is, therefore, not surprising that each effector is probably detected in a distinct and specific manner, some by direct binding, others as complexes with host proteins, and others via their modification of host proteins. The two papers accompanying this review contribute further to our understanding of the molecular specificity underlying effector perception by Cf proteins. This review, therefore, focuses on our current understanding of recognitional specificity in the tomato-C. fulvum pathosystem and highlights some of the critical questions that remain to be addressed. It also addresses the evolutionary causes and consequences of this specificity.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF VF-LIKE CANDIDATE GENES IN CULTIVATED APPLES AND SELECTIONS FROM MALUS SIEVERSII

The genetic locus for resistance to apple scab most frequently used in apple breeding is Vf, derived from Malus floribunda 821. For the Vf locus a cluster of four resistance gene paralogs encoding receptor-like proteins (RLP) with similarity to the tomato Cf resistance genes is known. Based on nucleotide alignments of published sequences of HcrVf1, HcrVf2, and HcrVf3, PCR primers were designed from the variable leucine-rich repeat (LRR) C1 subdomain. PCR products with high amino acid identity (85 to 100%) to HcrVf1 and HcrVf2 were obtained not only from M. floribunda 821 and Vf cultivars, but also from other apple scab resistance sources, such as ‘Russian Seedling’ R12740-7A or some ‘Antonovka’ accessions tested. A series of thirteen HcrVf candidate genes have been partly cloned from the PCR fragments spanning N-terminal LRRs 20 - 30. Expression analyses and mapping were focused on two specific Vf-homologous candidate genes, Vf1RSA and Vf2ARD, identified in resistant Malus genotypes known for carrying other scab resistance genes than Vf. RT-PCR experiments showed that Vf1RSA as well as the Vf2ARD are expressed under pathogen-free conditions. The results of a quantitative PCR-based transcription profiling performed for Vf2ARD suggest that this gene is scab-inducible in some resistant cultivars. Vf1RSA has been mapped, so far, without any recombination to the known Vf gene cluster on linkage group LG 1. The second candidate gene Vf2ARD is separated from the Vf gene cluster with a genetic distance of about 2 cM and might be a member of a second Vf-like locus on apple linkage group LG 1.

Identification and molecular analysis of candidate genes homologous toHcrVfgenes for scab resistance in apple

AbstractThe genetic locus for resistance to apple scab most frequently used in apple breeding isVf, derived fromMalus floribunda821. For theVflocus a cluster of four resistance gene paralogs (called asHcrVfgenes) encoding receptor‐like proteins (RLP) with similarity to the tomatoCfresistance genes is known. Based on published sequences forHcrVf1andHcrVf2PCR primers were designed from the domain B and the variable leucine‐rich repeat (LRR) C1 subdomain. PCR products with high amino acid identity (85–100%) toHcrVf1andHcrVf2were obtained not only fromM. floribunda821 andVfcultivars but also from other apple scab resistance sources, such as ‘Russian Seedling’ R12740‐7A (Vrresistance) or ‘Antonovka polutorafuntovaya’ (VAresistance). A series of 13HcrVfcandidate genes have been partly cloned from the PCR fragments spanning N‐terminal LRRs 20–30. A considerable number of amino acid exchanges within the solvent‐exposed xxLxLxx structural motives were detected among the homologous sequences. Expression analyses and mapping focused on a selectedVf‐homologous candidate gene (calledVf2ARD) identified in resistantMalusgenotypes known for carrying other scab resistance genes thanVf. RT‐PCR experiments showed thatVf2ARDis expressed under pathogen‐free conditions. The results of a quantitative PCR‐based transcription profiling suggest that this gene is scab‐inducible in some resistant cultivars.Vf2ARDhas been mapped on linkage group LG 1. It is separated from theVfgene cluster with a genetic distance of about 2 cM and might be a member of a secondVf ‐like locus on apple linkage group LG 1.

Gene silencing to investigate the roles of receptor-like proteins in Arabidopsis

Receptor-like proteins (RLPs) are cell surface receptors that play important roles in various processes. In several plant species RLPs have been found to play a role in disease resistance, including the tomato Cf and Ve proteins and the apple HcrVf proteins that mediate resistance against the fungal pathogens Cladosporium fulvum, Verticillium spp. and Venturia inaequalis, respectively. The Arabidopsis genome contains 57 AtRLP genes. Two of these, CLV2 (AtRLP10) and TMM (AtRLP17), have well-characterized functions in meristem and stomatal development, respectively, while AtRLP52 is required for defense against powdery mildew. We recently reported the assembly of a genome-wide collection of T-DNA insertion lines for the Arabidopsis AtRLP genes. This collection was functionally analyzed with respect to plant growth, development and sensitivity to various stress responses including pathogen susceptibility. Only few new phenotypes were discovered; while AtRLP41 was found to mediate abscisic acid sensitivity, AtRLP30 (and possibly AtRLP18) was found to be required for full non-host resistance to a bacterial pathogen. Possibly, identification of novel phenotypes is obscured by functional redundancy. Therefore, RNA interference (RNAi) to target the expression of multiple AtRLP genes simultaneously was employed followed by functional analysis of the RNAi lines.Addendum to: Wang G, Ellendorff U, Kemp B, Mansfield JW, Forsyth A, Mitchell K, Bastas K, Liu CM, Woods-Tör A, Zipfel C, de Wit PJGM, Jones JDG, Tör M, Thomma BPHJ. A genome-wide functional investigation into the roles of receptor-like proteins in Arabidopsis. Plant Physiol 2008; 147:503-17.

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.

Transcript profiling for Avr4/Cf-4- and Avr9/Cf-9-dependent defence gene expression

Tomato Cf genes confer resistance to the leaf mold pathogen Cladosporium fulvum. The Avr4/Cf-4- and Avr9/Cf-9-dependent hypersensitive responses (HRs) are distinct in cell death pattern, intensity, and sensitivity to environmental conditions. To understand the mechanism resulting in these differences, comparative transcript profiling for Avr4/Cf-4- and Avr9/Cf-9-dependent defence gene expression by cDNA-AFLP was performed previously. 367 ACE (Avr/Cf-elicited) transcript-derived fragments (TDFs) were identified, among which 189 were cloned and sequenced. In this study, we report another 89 ACE fragments. These ACE genes were associated with: defence, signal transduction, HR and cell death, transcriptional regulation, metabolism, protein synthesis, photosynthesis, membrane fusion, secretion and trafficking, miscellaneous biological processes, and genes with unknown function or with no significant similarity to known sequences. Among these sequences 43 (potentially encoding 36 types of proteins) were identified for the first time as genes differentially expressed during the development of Avr/Cf-dependent HR. Sequence and expression data from this study further support that transcription is reprogrammed to promote defence response and HR and repress photosynthesis in the Avr/Cf HR+ seedlings.

Involvement of cathepsin B in the plant disease resistance hypersensitive response

A diverse range of plant proteases are implicated in pathogen perception and in subsequent signalling and execution of disease resistance. We demonstrate, using protease inhibitors and virus-induced gene silencing (VIGS), that the plant papain cysteine protease cathepsin B is required for the disease resistance hypersensitive response (HR). VIGS of cathepsin B prevented programmed cell death (PCD) and compromised disease resistance induced by two distinct non-host bacterial pathogens. It also suppressed the HR triggered by transient co-expression of potato R3a and Phytophthora infestans Avr3a genes. However, VIGS of cathepsin B did not compromise HR following recognition of Cladosporium fulvum AVR4 by tomato Cf-4, indicating that plant PCD can be independent of cathepsin B. The non-host HR to Erwinia amylovora was accompanied by a transient increase in cathepsin B transcript level and enzymatic activity and induction of the HR marker gene Hsr203. VIGS of cathepsin B significantly reduced the induction of Hsr203 following E. amylovora challenge, further demonstrating a role for this protease in PCD. Whereas cathepsin B is often relocalized from the lysosome to the cytosol during animal PCD, plant cathepsin B is secreted into the apoplast, and is activated upon secretion in the absence of pathogen challenge.

Comparative transcript profiling by cDNA‐AFLP reveals similar patterns of Avr4/Cf‐4‐ and Avr9/Cf‐9‐dependent defence gene expression

Tomato Cf genes confer resistance to the fungal pathogen Cladosporium fulvum. Although the Cf-4 and Cf-9 proteins are very similar, the Cf-4- and Cf-9-dependent hypersensitive responses (HRs) are distinct in cell death pattern, intensity and sensitivity to environmental conditions. To investigate the mechanism leading to these differences, comparative transcript profiling of Avr4/Cf-4- and Avr9/Cf-9-dependent defence gene expression was performed. To do this, cDNA-AFLP analysis was conducted on Avr/Cf tomato seedlings undergoing early HR. Both Avr4/Cf-4 and Avr9/Cf-9 signalling elicited the same spectrum of genes, referred to here as Avr/Cf-elicited (ACE) genes. Of approximately 25 000 transcript-derived fragments (TDFs), 367 (1.5%) showed significant differential expression between HR(+) and HR(-) seedlings (either Avr4/Cf-4- or Avr9/Cf-9-dependent). However, 42.8% of the ACE TDFs (157/367 in total) showed quantitatively different expression in the two types of HR(+) seedlings. The majority of these (135/157, 86.0%) displayed significantly greater differential expression (either induced or repressed) in Avr4/Cf-4 HR(+) seedlings than in Avr9/Cf-9 HR(+) seedlings. Our results are consistent with the previous observation that Avr4/Cf-4-dependent HR is more severe than Avr9/Cf-9-dependent HR, and indicate that the distinction between Avr4/Cf-4- and Avr9/Cf-9-dependent HR is most probably a result of events upstream of the defence responses. Sequencing of 189 ACE fragments identified genes associated with: defence and resistance (33.3%), signal transduction (7.4%), HR and cell death (5.3%), transcriptional regulation and post-transcriptional modification (4.3%). Expression data revealed that defence response, respiration and biological oxidation are strongly induced while photosynthesis is severely repressed in the HR(+) seedlings.

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.

CITRX thioredoxin is a putative adaptor protein connecting Cf-9 and the ACIK1 protein kinase during the Cf-9/Avr9- induced defence response

Tomato Cf- 9, a receptor-like protein (RLP), confers resistance to races of the fungal pathogen Cladosporium fulvum that express the Avr9 avirulence gene. CITRX ( Cf-9- interacting thio redo xin) was previously identified in a yeast two-hybrid screen as a protein interacting with the cytoplasmic domain of Cf-9 and shown to be a negative regulator of the cell death induced after Cf-9/Avr9 interaction. ACIK1 is a Ser/Thr protein kinase that is specifically required for the Cf- 9 and Cf- 4 dependent defence response in tomato. In this paper we present data suggesting that CITRX may act as an adaptor recruiting the ACIK1 kinase to the cytoplasmic domain of Cf-9 upon elicitation with the Avr9 peptide. Interestingly, the catalytic activities of both CITRX and ACIK1 are not required for their interaction.