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Alternative splicing (AS) is a key element of eukaryotic gene expression that increases transcript and proteome diversity in cells, thereby altering their responses to external stimuli and stresses. While AS has been intensively researched in plants and animals, its frequency, conservation, and putative impact on virulence, are relatively still understudied in plant pathogenic fungi. Here, we profiled the AS events occurring in genes of Cladosporium fulvum isolates Race 5 and Race 4, during nearly a complete compatible infection cycle on their tomato host. Our studies revealed extensive heterogeneity in the transcript isoforms assembled from different isolates, infections, and infection timepoints, as over 80% of the transcript isoforms were singletons that were detected in only a single sample. Despite that, nearly 40% of the protein-coding genes in each isolate were predicted to be recurrently AS across the disparate infection timepoints, infections, and the two isolates. Of these, 37.5% were common to both isolates and 59% resulted in multiple protein isoforms, thereby putatively increasing proteome diversity in the pathogen by 31% during infections. An enrichment analysis showed that AS mostly affected genes likely to be involved in the transport of nutrients, regulation of gene expression, and monooxygenase activity, suggesting a role for AS in finetuning adaptation of C. fulvum on its tomato host during infections. Tracing the location of the AS genes on the fungal chromosomes showed that they were mostly located in repeat-rich regions of the core chromosomes, indicating a causal connection between gene location on the genome and propensity to AS. Finally, multiple cases of differential isoform usage in AS genes of C. fulvum were identified, suggesting that modulation of AS at different infection stages may be another way by which pathogens refine infections on their hosts., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zaccaron et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Fungal 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., Results: Here, 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., Conclusions: Our 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., (© 2024. The Author(s).)

High tunnels extend the growing season of high value crops, including tomatoes, but the environmental conditions within high tunnels favor the spread of the tomato leaf mold pathogen, Passalora fulva (syn. Cladosporium fulvum ). Tomato leaf mold results in defoliation, and if severe, losses in yield. Despite substantial research, little is known regarding the genetic structure and diversity of populations of P . fulva associated with high tunnel tomato production in the United States. From 2016 to 2019, a total of 50 P . fulva isolates were collected from tomato leaf samples in high tunnels in the Northeast and Minnesota. Other Cladosporium species were also isolated from the leaf surfaces. Koch's postulates were conducted to confirm that P . fulva was the cause of the disease symptoms observed. Race determination experiments revealed that the isolates belonged to either race 0 (six isolates) or race 2 (44 isolates). Polymorphisms were identified within four previously characterized effector genes: Avr2 , Avr4 , Avr4e , and Avr9 . The largest number of polymorphisms were observed for Avr2 . Both mating type genes, MAT1 - 1 - 1 and MAT1 - 2 - 1 , were present in the isolate collection. For further insights into the pathogen diversity, the 50 isolates were genotyped at 7,514 single-nucleotide polymorphism loci using genotyping-by-sequencing. Differentiation by region but not by year was observed. Within the collection of 50 isolates, there were 18 distinct genotypes. Information regarding P . fulva population diversity will enable better management recommendations for growers, as high tunnel production of tomatoes expands.

Tomato leaf mold disease caused by Cladosporium fulvum ( C. fulvum ) is one of the most common diseases affecting greenhouse tomato production. Cf proteins can recognize corresponding AVR proteins produced by C. fulvum , and Cf genes are associated with leaf mold resistance. Given that there are many physiological races of C. fulvum and that these races rapidly mutate, resistance to common Cf genes (such as Cf-2 , Cf-4 , Cf-5 , and Cf-9 ) has decreased. In the field, Ont7813 plants (carrying the Cf-13 gene) show effective resistance to C. fulvum ; thus, these plants could be used as new, disease-resistant materials. To explore the mechanism of the Cf-13 -mediated resistance response, transcriptome sequencing was performed on three replicates each of Ont7813 ( Cf-13 ) and Moneymaker (MM; carrying the Cf-0 gene) at 0, 9, and 15 days after inoculation (dai) for a total of 18 samples. In total, 943 genes were differentially expressed, specifically in the Ont7813 response process as compared to the Moneymaker response process. Gene ontology (GO) classification of these 943 differentially expressed genes (DEGs) showed that GO terms, including "hydrogen peroxide metabolic process (GO_Process)", "secondary active transmembrane transporter activity (GO_Function)", and "mismatch repair complex (GO_Component)", which were the same as 11 other GO terms, were significantly enriched. An analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that many key regulatory genes of the Cf-13 -mediated resistance response processes were involved in the "plant hormone signal transduction" pathway, the "plant-pathogen interaction" pathway, and the "MAPK signaling pathway-plant" pathway. Moreover, during C. fulvum infection, jasmonic acid (JA) and salicylic acid (SA) contents significantly increased in Ont7813 at the early stage. These results lay a vital foundation for further understanding the molecular mechanism of the Cf-13 gene in response to C. fulvum infection.

Cladosporium fulvum is a fungal pathogen that causes leaf mould of tomato. The reference genome of this pathogen was released in 2012 but its high repetitive DNA content prevented a contiguous assembly and further prohibited the analysis of its genome architecture. In this study, we combined third generation sequencing technology with the Hi-C chromatin conformation capture technique, to produce a high-quality and near complete genome assembly and gene annotation of a Race 5 isolate of C. fulvum . The resulting genome assembly contained 67.17 Mb organized into 14 chromosomes (Chr1-to-Chr14), all of which were assembled telomere-to-telomere. The smallest of the chromosomes, Chr14, is only 460 kb in size and contains 25 genes that all encode hypothetical proteins. Notably, PCR assays revealed that Chr14 was absent in 19 out of 24 isolates of a world-wide collection of C. fulvum , indicating that Chr14 is dispensable. Thus, C. fulvum is currently the second species of Capnodiales shown to harbour dispensable chromosomes. The genome of C. fulvum Race 5 is 49.7 % repetitive and contains 14 690 predicted genes with an estimated completeness of 98.9%, currently one of the highest among the Capnodiales. Genome structure analysis revealed a compartmentalized architecture composed of gene-dense and repeat-poor regions interspersed with gene-sparse and repeat-rich regions. Nearly 39.2 % of the C. fulvum Race 5 genome is affected by Repeat-Induced Point (RIP) mutations and evidence of RIP leakage toward non-repetitive regions was observed in all chromosomes, indicating the RIP plays an important role in the evolution of this pathogen. Finally, 345 genes encoding candidate effectors were identified in C. fulvum Race 5, with a significant enrichment of their location in gene-sparse regions, in accordance with the 'two-speed genome' model of evolution. Overall, the new reference genome of C. fulvum presents several notable features and is a valuable resource for studies in plant pathogens.

This study was conducted at the Agriculture College University of Karbala, Iraq to isolate and morphologically and molecularly diagnose thirteen Cladosporium isolates collected from tomato plant residues present in desert regions of Najaf and Karbala provinces, Iraq. We diagnosed the obtained isolates by PCR amplification using the ITS1 and ITS4 universal primer pair followed by sequencing. PCR amplification and analysis of nucleotide sequences using the BLAST program showed that all isolated fungi belong to Cladosporium sphaerospermum. Analysis of the nucleotide sequences of the identified C. sphaerospermum isolates 2, 6, 9, and 10 showed a genetic similarity reached 99%, 98%, 99%, and 99%, respectively, with those previously registered at the National Center for Biotechnology Information (NCBl). By comparing the nucleotide sequences of the identified C. sphaerospermum isolates with the sequences belong to the same fungi and available at NCBI, it was revealed that the identified C. sphaerospermum isolates 2, 6, 9, and 10 have a genetic variation with those previously recorded at the National Center for Biotechnology Information (NCBl); therefore, the identified sequences of C. sphaerospermum isolates have been registered in GenBank database (NCBI) under the accession numbers MN896004, MN896107, MN896963, and MN896971, respectively.

The molecular interactions between tomato and Cladosporium fulvum have been an important model for molecular plant pathology. Complex genetic loci on tomato chromosomes 1 and 6 harbor genes for resistance to Cladosporium fulvum , encoding receptor like-proteins that perceive distinct Cladosporium fulvum effectors and trigger plant defenses. Here, we report classical mapping strategies for loci in tomato accessions that respond to Cladosporium fulvum effector Ecp5, which is very sequence-monomorphic. We screened 139 wild tomato accessions for an Ecp5-induced hypersensitive response, and in five accessions, the Ecp5-induced hypersensitive response segregated as a monogenic trait, mapping to distinct loci in the tomato genome. We identified at least three loci on chromosomes 1, 7 and 12 that harbor distinct Cf-Ecp5 genes in four different accessions. Our mapping showed that the Cf-Ecp5 in Solanum pimpinellifolium G1.1161 is located at the Milky Way locus. The Cf-Ecp5 in Solanum pimpinellifolium LA0722 was mapped to the bottom arm of chromosome 7, while the Cf-Ecp5 genes in Solanum lycopersicum Ontario 7522 and Solanum pimpinellifolium LA2852 were mapped to the same locus on the top arm of chromosome 12. Bi-parental crosses between accessions carrying distinct Cf-Ecp5 genes revealed putative genetically unlinked suppressors of the Ecp5-induced hypersensitive response. Our mapping also showed that Cf-11 is located on chromosome 11, close to the Cf-3 locus. The Ecp5-induced hypersensitive response is widely distributed within tomato species and is variable in strength. This novel example of convergent evolution could be used for choosing different functional Cf-Ecp5 genes according to individual plant breeding needs., (Copyright © 2020 Iakovidis et al.)

Effectors are microbial-derived secreted proteins with an essential function in modulating host immunity during infections. CfAvr4, an effector protein from the tomato pathogen Cladosporium fulvum and the founding member of a fungal effector family, promotes parasitism through binding fungal chitin and protecting it from chitinases. Binding of Avr4 to chitin is mediated by a carbohydrate-binding module of family 14 (CBM14), an abundant CBM across all domains of life. To date, the structural basis of chitin-binding by Avr4 effector proteins and of recognition by the cognate Cf-4 plant immune receptor are still poorly understood. Using X-ray crystallography, we solved the crystal structure of CfAvr4 in complex with chitohexaose [(GlcNAc)6] at 1.95Å resolution. This is the first co-crystal structure of a CBM14 protein together with its ligand that further reveals the molecular mechanism of (GlcNAc)6 binding by Avr4 effector proteins and CBM14 family members in general. The structure showed that two molecules of CfAvr4 interact through the ligand and form a three-dimensional molecular sandwich that encapsulates two (GlcNAc)6 molecules within the dimeric assembly. Contrary to previous assumptions made with other CBM14 members, the chitohexaose-binding domain (ChBD) extends to the entire length of CfAvr4 with the reducing end of (GlcNAc)6 positioned near the N-terminus and the non-reducing end at the C-terminus. Site-directed mutagenesis of residues interacting with (GlcNAc)6 enabled the elucidation of the precise topography and amino acid composition of Avr4's ChBD and further showed that these residues do not individually mediate the recognition of CfAvr4 by the Cf-4 immune receptor. Instead, the studies highlighted the dependency of Cf-4-mediated recognition on CfAvr4's stability and resistance against proteolysis in the leaf apoplast, and provided the evidence for structurally separating intrinsic function from immune receptor recognition in this effector family., Competing Interests: The authors have declared that no competing interests exist.

Tomato leaf mold disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes that encode receptor-like proteins (RLPs). These RLPs recognize specific SSP effectors to initiate a hypersensitive response (HR) that renders the pathogen avirulent. C. fulvum strains capable of overcoming one or more of all cloned Cf genes have now emerged. To combat these strains, new Cf genes are required. An effectoromics approach was employed to identify wild tomato accessions carrying new Cf genes. Proteomics and transcriptome sequencing were first used to identify 70 apoplastic in planta-induced C. fulvum SSPs. Based on sequence homology, 61 of these SSPs were novel or lacked known functional domains. Seven, however, had predicted structural homology to antimicrobial proteins, suggesting a possible role in mediating antagonistic microbe-microbe interactions in planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs, using the Potato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry new Cf genes available for incorporation into cultivated tomato.

Cladosporium fulvum is a non-obligate biotrophic fungal tomato pathogen for which fifteen secondary metabolite (SM) gene clusters were previously identified in its genome. However, most of these SM biosynthetic pathways remain cryptic during growth in planta and in different in vitro conditions. The sole SM produced in vitro is the pigment cladofulvin. In this study, we attempted to activate cryptic pathways in order to identify new compounds produced by C. fulvum. For this purpose, we manipulated orthologues of the global regulators VeA, LaeA and HdaA known to regulate SM biosynthesis in other fungal species. In C. fulvum, deleting or over-expressing these regulators yielded no new detectable SMs. Yet, quantification of cladofulvin revealed that CfHdaA is an activator whilst CfVeA and CfLaeA seemed to act as repressors of cladofulvin production. In the wild type strain, cladofulvin biosynthesis was affected by the carbon source, with highest production under carbon limitation and traces only in presence of saccharose. Repression of cladofulvin production by saccharose was dependent on both CfVeA and CfLaeA. Deletion of CfVeA or CfLaeA caused production of sterile mycelia, whilst Δcfhdaa deletion mutants sporulated, suggesting that cladofulvin production is not linked to asexual reproduction. Profiling the transcription of these regulators showed that CfHdaA-mediated regulation of cladofulvin production is independent of both CfVeA and CfLaeA. Our data suggest CfLaeA directly affects cladofulvin production whilst the effect of CfVeA is indirect, suggesting a role for CfLaeA outside of the Velvet complex. In conclusion, our results showed that regulation of SM production in C. fulvum is different from other fungi and indicate that manipulation of global regulators is not a universal tool to discover new fungal natural products., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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.

The Cf-5 gene of tomato confers resistance to strains of the fungal pathogen Cladosporium fulvum carrying the avirulence gene Avr5. Although Cf-5 has been cloned, Avr5 has remained elusive. We report the cloning of Avr5 using a combined bioinformatic and transcriptome sequencing approach. RNA-Seq was performed on the sequenced race 0 strain (0WU; carrying Avr5), as well as a race 5 strain (IPO 1979; lacking a functional Avr5 gene) during infection of susceptible tomato. Forty-four in planta-induced C. fulvum candidate effector (CfCE) genes of 0WU were identified that putatively encode a secreted, small cysteine-rich protein. An expressed transcript sequence comparison between strains revealed two polymorphic CfCE genes in IPO 1979. One of these conferred avirulence to IPO 1979 on Cf-5 tomato following complementation with the corresponding 0WU allele, confirming identification of Avr5. Complementation also led to increased fungal biomass during infection of susceptible tomato, signifying a role for Avr5 in virulence. Seven of eight race 5 strains investigated escape Cf-5-mediated resistance through deletion of the Avr5 gene. Avr5 is heavily flanked by repetitive elements, suggesting that repeat instability, in combination with Cf-5-mediated selection pressure, has led to the emergence of race 5 strains deleted for the Avr5 gene.

Fungal Wor1-like proteins are conserved transcriptional regulators that are reported to regulate the virulence of several plant pathogenic fungi by affecting the expression of virulence genes. Here, we report the functional analysis of CfWor1, the homologue of Wor1 in Cladosporium fulvum. Δcfwor1 mutants produce sclerotium-like structures and rough hyphae, which are covered with a black extracellular matrix. These mutants do not sporulate and are no longer virulent on tomato. A CE.CfWor1 transformant that constitutively expresses CfWor1 produces fewer spores with altered morphology and is also reduced in virulence. RNA-seq and RT-qrtPCR analyses suggest that reduced virulence of Δcfwor1 mutants is due to global downregulation of transcription, translation and mitochondrial respiratory chain. The reduced virulence of the CE.CfWor1 transformant is likely due to downregulation of effector genes. Complementation of a non-virulent Δfosge1 (Wor1-homologue) mutant of Fusarium oxysporum f. sp. lycopersici with CfWor1 restored expression of the SIX effector genes in this fungus, but not its virulence. Chimeric proteins of CfWor1/FoSge1 also only partially restored defects of the Δfosge1 mutant, suggesting that these transcriptional regulators have functionally diverged. Altogether, our results suggest that CfWor1 primarily regulates development of C. fulvum, which indirectly affects the expression of a subset of virulence genes., (© 2014 John Wiley & Sons Ltd.)

Cladosporium fulvum is a biotrophic fungal pathogen that causes leaf mould of tomato. Analysis of its genome suggested a high potential for production of secondary metabolites (SM), which might be harmful to plants and animals. Here, we have analysed in detail the predicted SM gene clusters of C. fulvum employing phylogenetic and comparative genomic approaches. Expression of the SM core genes was measured by RT-qrtPCR and produced SMs were determined by LC-MS and NMR analyses. The genome of C. fulvum contains six gene clusters that are conserved in other fungal species, which have undergone rearrangements and gene losses associated with the presence of transposable elements. Although being a biotroph, C. fulvum has the potential to produce elsinochrome and cercosporin toxins. However, the corresponding core genes are not expressed during infection of tomato. Only two core genes, PKS6 and NPS9, show high expression in planta, but both are significantly down regulated during colonization of the mesophyll tissue. In vitro SM profiling detected only one major compound that was identified as cladofulvin. PKS6 is likely involved in the production of this pigment because it is the only core gene significantly expressed under these conditions. Cladofulvin does not cause necrosis on Solanaceae plants and does not show any antimicrobial activity. In contrast to other biotrophic fungi that have a reduced SM production capacity, our studies on C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent another mechanism associated with a biotrophic lifestyle.

Identification of hypersensitive cell death (HCD) regulators is essential to dissect the molecular mechanisms underlying plant disease resistance. In this study, combined proteomic and RNA interfering (RNAi) analyses were employed to identify genes required for the HCD conferred by the tomato resistance gene Cf-4 and the Cladosporium fulvum avirulence gene Avr4. Forty-nine proteins differentially expressed in the tomato seedlings mounting and those not mounting Cf-4/Avr4-dependent HCD were identified through proteomic analysis. Among them were a variety of defence-related proteins including a cysteine protease, Pip1, an operative target of another C. fulvum effector, Avr2. Additionally, glutathione-mediated antioxidation is a major response to Cf-4/Avr4-dependent HCD. Functional analysis through tobacco rattle virus-induced gene silencing and transient RNAi assays of the chosen 16 differentially expressed proteins revealed that seven genes, which encode Pip1 homologue NbPip1, a SIPK type MAP kinase Nbf4, an asparagine synthetase NbAsn, a trypsin inhibitor LeMir-like protein NbMir, a small GTP-binding protein, a late embryogenesis-like protein, and an ASR4-like protein, were required for Cf-4/Avr4-dependent HCD. Furthermore, the former four genes were essential for Cf-9/Avr9-dependent HCD; NbPip1, NbAsn, and NbMir, but not Nbf4, affected a nonadaptive bacterial pathogen Xanthomonas oryzae pv. oryzae-induced HCD in Nicotiana benthamiana. These data demonstrate that Pip1 and LeMir may play a general role in HCD and plant immunity and that the application of combined proteomic and RNA interfering analyses is an efficient strategy to identify genes required for HCD, disease resistance, and probably other biological processes in plants.

Coevolution between hosts and pathogens is thought to occur between interacting molecules of both species. This results in the maintenance of genetic diversity at pathogen antigens (or so-called effectors) and host resistance genes such as the major histocompatibility complex (MHC) in mammals or resistance (R) genes in plants. In plant-pathogen interactions, the current paradigm posits that a specific defense response is activated upon recognition of pathogen effectors via interaction with their corresponding R proteins. According to the "Guard-Hypothesis," R proteins (the "guards") can sense modification of target molecules in the host (the "guardees") by pathogen effectors and subsequently trigger the defense response. Multiple studies have reported high genetic diversity at R genes maintained by balancing selection. In contrast, little is known about the evolutionary mechanisms shaping the guardee, which may be subject to contrasting evolutionary forces. Here we show that the evolution of the guardee RCR3 is characterized by gene duplication, frequent gene conversion, and balancing selection in the wild tomato species Solanum peruvianum. Investigating the functional characteristics of 54 natural variants through in vitro and in planta assays, we detected differences in recognition of the pathogen effector through interaction with the guardee, as well as substantial variation in the strength of the defense response. This variation is maintained by balancing selection at each copy of the RCR3 gene. Our analyses pinpoint three amino acid polymorphisms with key functional consequences for the coevolution between the guardee (RCR3) and its guard (Cf-2). We conclude that, in addition to coevolution at the "guardee-effector" interface for pathogen recognition, natural selection acts on the "guard-guardee" interface. Guardee evolution may be governed by a counterbalance between improved activation in the presence and prevention of auto-immune responses in the absence of the corresponding pathogen., Competing Interests: The authors have declared that no competing interests exist.

Accurate evaluation of disease phenotypes is considered a key step to study plant-microbe interactions, as the rate of host colonization by the pathogenic microbe directly reflects whether the defense response of the plant is compromised. Although several techniques were developed to quantitate the amount of infection, only a few of them are inherently suitable for large disease screens. Here, I describe an unbiased method to quantitate disease phenotypes which manifest themselves by visible symptoms contrasting with the remaining unaffected parts of the host tissue. The method utilizes a macro plugin written for the image processing program "ImageJ" to calculate two values which determine the disease index for a specific treatment. In case the disease symptoms are not clear, a transgenic pathogenic fungus expressing the GUS gene is suitable for high-throughput disease screens, since staining for GUS activity facilitates an easy detection of the blue-stained pathogen. I illustrate the versatility of this method by analyzing a data set from a functional silencing screening experiment in resistant tomato that was inoculated with a GUS-expressing strain of the fungus Cladosporium fulvum. The method calculates a disease index for each silenced plant and thereby provides a basis for the unbiased identification of candidate host genes required for full resistance to this fungus.

The Cladosporium fulvum Avr2 effector is a novel type of cysteine protease inhibitor with eight cysteine residues that are all involved in disulphide bonds. We have produced wild-type Avr2 protein in Pichia pastoris and determined its disulphide bond pattern. By site-directed mutagenesis of all eight cysteine residues, we show that three of the four disulphide bonds are required for Avr2 stability. The six C-terminal amino acid residues of Avr2 contain one disulphide bond that is not embedded in its overall structure. Avr2 is not processed by the tomato cysteine protease Rcr3 and is an uncompetitive inhibitor of Rcr3. We also produced mutant Avr2 proteins in which selected amino acid residues were individually replaced by alanine, and, in one mutant, all six C-terminal amino acid residues were deleted. We determined the inhibitory constant (K(i) ) of these mutants for Rcr3 and their ability to trigger a Cf-2-mediated hypersensitive response (HR) in tomato. We found that the two C-terminal cysteine residues and the six amino acid C-terminal tail of Avr2 are required for both Rcr3 inhibitory activity and the ability to trigger a Cf-2-mediated HR. Individual replacement of the lysine-17, lysine-20 or tyrosine-21 residue by alanine did not affect significantly the biological activity of Avr2. Overall, our data suggest that the affinity of the Avr2 mutants for Rcr3 correlates with their ability to trigger a Cf-2-mediated HR., (© 2010 The Authors. Molecular Plant Pathology © 2010 BSPP and Blackwell Publishing Ltd.)

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.

Hydrophobins are amphipathic molecules which form part of fungal cell walls and extracellular matrices and perform a variety of roles in fungal growth and development. The tomato pathogen Cladosporium fulvum has six hydrophobin genes, HCf-1 to -6. We have devised an epitope tagging approach for establishing hydrophobin localization during growth in culture and in plants. In this paper we localize HCf-2, -3, -4 and -5 and compare the data to our previous observations for HCf-1 and -6. In culture, HCf-1, -2, -3 and 4 localize to conidia and also appear on aerial hyphae. HCf-4 is unique in that it appears on submerged hyphae. HCf-5 expression is tightly regulated and appears on aerial hyphae early on during growth. Only HCf-1, -3 and -6 were observed during infection; HCf-3 appears on both conidia and emerging germ tubes. We also show that HCf-6 is secreted and coats surfaces under and around growing hyphae and demonstrate the effect of deleting HCf-6 on the adhesion of germinating C. fulvum conidia to glass slides.

During tomato leaf colonization, the biotrophic fungus Cladosporium fulvum secretes several effector proteins into the apoplast. Eight effectors have previously been characterized and show no significant homology to each other or to other fungal genes. To discover novel C. fulvum effectors that might play a role in virulence, we utilized two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to visualize proteins secreted during C. fulvum-tomato interactions. Three novel C. fulvum proteins were identified: CfPhiA, Ecp6 and Ecp7. CfPhiA shows homology to proteins found on fungal sporogenous cells called phialides. Ecp6 contains lysin motifs (LysM domains) that are recognized as carbohydrate-binding modules. Ecp7 encodes a small, cysteine-rich protein with no homology to known proteins. Heterologous expression of Ecp6 significantly increased the virulence of the vascular pathogen Fusarium oxysporum on tomato. Furthermore, by RNA interference (RNAi)-mediated gene silencing we demonstrate that Ecp6 is instrumental for C. fulvum virulence on tomato. Hardly any allelic variation was observed in the Ecp6 coding region of a worldwide collection of C. fulvum strains. Although none of the C. fulvum effectors identified so far have obvious orthologues in other organisms, conserved Ecp6 orthologues were identified in various fungal species. Homology-based modelling suggests that the LysM domains of C. fulvum Ecp6 may be involved in chitin binding.

Aims: The aim of this study was to develop a sensitive real-time polymerase chain reaction (PCR) assay for the rapid detection of Cladosporium fulvum in tomato leaves., Methods and Results: Three PCR primer pairs were designed based on the nucleotide sequences of: (i) the internal transcribed spacer regions of ribosomal RNA; (ii) a microsatellite region amplified by the microsatellite primer M13; and (iii) the beta-tubulin gene of C. fulvum. Each primer pair amplified the expected target DNA fragment from geographically diverse isolates of C. fulvum. No PCR products were amplified with these primer pairs from DNA of other fungal species. Among the three pairs of primers, the primer pair CfF1/CfR1 developed based on the microsatellite region was the most sensitive. Using this sensitive primer pair, a real-time PCR assay was developed to detect early infection of C. fulvum in tomato leaves., Significance and Impact of the Study: DNA regions amplified by the microsatellite primer M13 have a high potential for developing highly sensitive species-specific PCR primers for the detection of phytopathogenic fungi. The real-time PCR assay developed in this study is useful in monitoring early infection of C. fulvum, and can help growers make timely decisions on fungicide application.

The allelic variation in four avirulence (Avr) and four extracellular protein (Ecp)-encoding genes of the tomato pathogen Cladosporium fulvum was analyzed for a worldwide collection of strains. The majority of polymorphisms observed in the Avr genes are deletions, point mutations, or insertions of transposon-like elements that are associated with transitions from avirulence to virulence, indicating adaptive evolution of the Avr genes to the cognate C. fulvum resistance genes that are deployed in commercial tomato lines. Large differences in types of polymorphisms between the Avr genes were observed, especially between Avr2 (indels) and Avr4 (amino-acid substitutions), indicating that selection pressure favors different types of adaptation. In contrast, only a limited number of polymorphisms were observed in the Ecp genes, which mostly involved synonymous modifications. A haplotype network based on the polymorphisms observed in the effector genes revealed a complex pattern of evolution marked by reticulations that suggests the occurrence of genetic recombination in this presumed asexual fungus. This, as well as the identification of strains with identical polymorphisms in Avr and Ecp genes but with opposite mating-type genes, suggests that development of complex races can be the combined result of positive selection and genetic recombination.

The biotrophic fungal pathogen Cladosporium fulvum (syn. Passalora fulva) is the causal agent of tomato leaf mold. The Avr4 protein belongs to a set of effectors that is secreted by C. fulvum during infection and is thought to play a role in pathogen virulence. Previous studies have shown that Avr4 binds to chitin present in fungal cell walls and that, through this binding, Avr4 can protect these cell walls against hydrolysis by plant chitinases. In this study, we demonstrate that Avr4 expression in Arabidopsis results in increased virulence of several fungal pathogens with exposed chitin in their cell walls, whereas the virulence of a bacterium and an oomycete remained unaltered. Heterologous expression of Avr4 in tomato increased the virulence of Fusarium oxysporum f. sp. lycopersici. Through tomato GeneChip analyses, we demonstrate that Avr4 expression in tomato results in the induced expression of only a few genes. Finally, we demonstrate that silencing of the Avr4 gene in C. fulvum decreases its virulence on tomato. This is the first report on the intrinsic function of a fungal avirulence protein that has a counter-defensive activity required for full virulence of the pathogen.

Two mating-type genes, designated MAT1-1-1 and MAT1-2-1, were cloned and sequenced from the presumed asexual ascomycete Cladosporium fulvum (syn. Passalora fulva). The encoded products are highly homologous to mating-type proteins from members of the Mycosphaerellaceae, such as Mycosphaerella graminicola and Cercospora beticola. In addition, the two MAT idiomorphs of C. fulvum showed regions of homology and each contained one additional putative ORF without significant similarity to known sequences. The distribution of the two mating-type genes in a world-wide collection of 86 C. fulvum strains showed a departure from a 1:1 ratio (chi(2)=4.81, df=1). AFLP analysis revealed a high level of genotypic diversity, while strains of the fungus were identified with similar virulence spectra but distinct AFLP patterns and opposite mating-types. These features could suggest the occurrence of recombination in C. fulvum.

Nonhost resistance as a durable and broad-spectrum defence strategy is of great potential for agricultural applications. We have previously isolated a cDNA showing homology with genes encoding bZIP transcription factors from tomato leaf mould pathogen Cladosporium fulvum. Upon expression, the cDNA results in necrosis in C. fulvum host tomato and nonhost tobacco plants and is thus named CfHNNI1 (for C . f ulvum host and nonhost plant necrosis inducer 1). In the present study we report the induction of necrosis in a variety of nonhost plant species belonging to three families by the transient in planta expression of CfHNNI1 using virus-based vectors. Additionally, transient expression of CfHNNI1 also induced expression of the HR marker gene LeHSR203 and greatly reduced the accumulation of recombinant Potato virus X. Stable CfHNNI1 transgenic tobacco plants were generated in which the expression of CfHNNI1 is under the control of the pathogen-inducible hsr203J promoter. When infected with the oomycetes pathogen Phytophthora parasitica var. nicotianae, these transgenic plants manifested enhanced expression of CfHNNI1 and subsequent accumulation of CfHNNI1 protein, resulting in high expression of the HSR203J and PR genes, and strong resistance to the pathogen. The CfHNNI1 transgenic plants also exhibited induced resistance to Pseudomonas syringae pv. tabaci and Tobacco mosaic virus. Furthermore, CfHNNI1 was highly expressed and the protein was translocated into plant cells during the incompatible interactions between C. fulvum and host and nonhost plants. Our results demonstrate that CfHNNI1 is a potential general elicitor of hypersensitive response and nonhost resistance.

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 thioredoxin) 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.

To identify genes required for the hypersensitive response (HR), we performed expression profiling of tomato plants mounting a synchronized HR, followed by functional analysis of differentially expressed genes. By cDNA-AFLP analysis, the expression profile of tomato plants containing both the Cf-4 resistance gene against Cladosporium fulvum and the matching Avr4 avirulence gene of this fungus was compared with that of control plants. About 1% of the transcript-derived fragments (442 out of 50,000) were derived from a differentially expressed gene. Based on their sequence and expression, 192 fragments, referred to as Avr4-responsive tomato (ART) fragments, were selected for VIGS (virus-induced gene silencing) in Cf-4-transgenic Nicotiana benthamiana. Inoculated plants were analyzed for compromised HR by agroinfiltration of either the C. fulvum Avr4 gene or the Inf1 gene of Phytophthora infestans, which invokes a HR in wild-type N. benthamiana. VIGS using 15 of the ART fragments resulted in a compromised HR, whereas VIGS with fragments of ART genes encoding HSP90, a nuclear GTPase, an L19 ribosomal protein, and most interestingly, a nucleotide binding-leucine rich repeat (NB-LRR)-type protein severely suppressed the HR induced both by Avr4 and Inf1. Requirement of an NB-LRR protein (designated NRC1, for NB-LRR protein required for HR-associated cell death 1) for Cf resistance protein function as well as Inf1-mediated HR suggests a convergence of signaling pathways and supports the recent observation that NB-LRR proteins play a role in signal transduction cascades downstream of resistance proteins.

Cladosporium fulvum (syn. Passalora fulva) is a biotrophic fungal pathogen that causes leaf mould on tomato (Solanum esculentum). The fungus grows exclusively in the tomato leaf apoplast where it secretes several small (<15 kDa) cysteine-rich proteins that are thought to play a role in disease establishment. To investigate the role of these proteins, and to identify their in planta targets, a targeted proteomics approach was undertaken. C. fulvum proteins were expressed as recombinant fusion proteins carrying various affinity-tags at either their C- or N-terminus. Although these fusion proteins were correctly expressed and secreted into the leaf apoplast, detection of affinity-tagged C. fulvum proteins failed, and affinity purification did not result in the recovery of these proteins. However, when using C. fulvum effector protein-specific antibodies, specific signals were obtained for the different proteins. It is concluded that the stability of the in planta expressed recombinant fusion proteins is insufficient, which results in removal of the affinity-tag from the fusion proteins, irrespective of the C- or N-terminal fusion or the nature of the affinity-tag. Similar phenomena were observed when the fusion proteins were expressed in other Solanaceous species, but not when expressed in Arabidopsis thaliana.

Gene-for-gene resistance is a well-known type of plant disease resistance. It is governed by plant resistance (R) genes and their matching pathogen avirulence (Avr) genes. This resistance is characterized by a hypersensitive response (HR) resulting from the interaction between products of a complementary R and Avr gene pair. The pathosystem of tomato (Lycopersicon esculentum Mill.) and its leaf mold fungal pathogen Cladosporium fulvum is a model system to study gene-for-gene resistance. HR occurs in tomato seedlings carrying a tomato Cf resistance gene and a matching C. fulvum Avr gene, including, for example, Cf-4/Avr4 and Cf-9/Avr9. Employing Cf/Avr tomato seedlings that both express a Cf gene and the matching Avr gene, here we report that both Cf-4/Avr4- and Cf-9/Avr9-dependent HR is delayed and reduced under high humidity (95%), and that Cf-9/Avr9-dependent HR is more sensitive to high humidity when compared to Cf-4/Avr4-dependent HR. Furthermore, high humidity acts synergistically with high temperature on HR suppression, resulting in complete blocking of the HR. The transcript profile of over 60 genes, related to HR, signaling and defense, in Cf/Avr seedlings grown under high humidity and thus showing no HR, significantly differed from that of the same seedlings grown under normal humidity and thus showing HR. These results demonstrate that high humidity probably acts at a very early point of the Cf downstream signaling pathway, or alternatively influences the interaction between the Cf and the Avr proteins.

The interaction between tomato and the leaf mold pathogen Cladosporium fulvum is controlled in a gene-for-gene manner. This interaction has provided useful insights to the molecular basis of recognition specificity in plant disease resistance (R) proteins, disease resistance (R) gene evolution, R-protein mediated signaling, and cellular responses to pathogen attack. Tomato Cf genes encode type I membrane-associated receptor-like proteins (RLPs) comprised predominantly of extracellular leucine-rich repeats (eLRRs) and which are anchored in the plasma membrane. Cf proteins recognize fungal avirulence (Avr) peptides secreted into the leaf apoplast during infection. A direct interaction of Cf proteins with their cognate Avr proteins has not been demonstrated and the molecular mechanism of Avr protein perception is not known. Following ligand perception Cf proteins trigger a hypersensitive response (HR) and the arrest of pathogen development. Cf proteins lack an obvious signaling domain, suggesting that defense response activation is mediated through interactions with other partners. Avr protein perception results in the rapid accumulation of active oxygen species (AOS), changes in cellular ion fluxes, activation of protein kinase cascades, changes in gene expression and, possibly, targeted protein degradation. Here we review our current understanding of Cf-mediated responses in resistance to C. fulvum.

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.

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.

The Cladosporium fulvum (Cf)-4 gene of tomato confers resistance to the fungus C. fulvum, expressing the corresponding avirulence (Avr)4 gene, which codes for an elicitor protein. Little is known about how such mechanisms work, but previous studies have shown that elicitor recognition activates Ca(2+) signalling and protein kinases, such as mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK). Here, we provide evidence that a new signalling component, the lipid second messenger phosphatidic acid (PA), is produced within a few minutes of AVR4/Cf-4 interaction. Using transgenic tobacco cells expressing the tomato Cf-4-resistance gene as a model system, phospholipid signalling pathways were studied by pre-labelling the cells with (32)P(i) and assaying for the formation of lipid signals after challenge with the fungal elicitor AVR4. A dramatic rapid response was an increase in (32)P-PA, together with its metabolic product diacylglycerol pyrophosphate (DGPP). AVR4 increased the levels of PA and DGPP in a Cf-4(+)-, time- and dose-dependent manner, while the non-matching elicitor AVR9 did not trigger any response. In general, PA signalling can be triggered by two different pathways: via phospholipase D (PLD), which generates PA directly by hydrolysing structural phospholipids like phosphatidylcholine (PC), or via PLC, which generates diacylglycerol (DAG) that is subsequently phosphorylated to PA by DAG kinase (DGK). To determine the origin of the AVR4-induced PA formation, a PLD-specific transphosphatidylation assay and a differential (32)P-labelling protocol were used. The results clearly demonstrated that most PA was produced via the phosphorylation of DAG. Neomycin and U73122, inhibitors of PLC activity, inhibited AVR4-induced PA accumulation, suggesting that the increase in DGK activity was because of increased PLC activity producing DAG. Lastly, evidence is provided that PLC signalling and, in particular, PA production could play a role in triggering responses, such as the AVR4-induced oxidative burst. For example, PLC inhibitors inhibited the oxidative burst, and when PA was added to cells, an oxidative burst was induced.

Hydrophobins are central to developmental processes of filamentous fungi. HCf-1 and HCf-6 are two of the six hydrophobins identified in the plant pathogenic fungus Cladosporium fulvum. We have fused the viral epitope V5 to HCf-1 and HCf-6, introduced the recombinant genes into C. fulvum strains that lack the two genes, and localised the tagged proteins by immunofluorescence microscopy. HCf-1(V5) is abundant on conidia and aerial structures formed in vitro and emerging from disease lesions on infected tomato plants. This is consistent with the proposed function of HCf-1 in aerial development and dissemination of conidia. HCf-6(V5) is secreted onto the growth substrate by the hyphae and during invasion of plant tissues, which suggests a function in adhesion and infection. This was not supported by the phenotypic analysis of DeltaHCf-6 strains. Hydrophobins may play distinct roles due to precisely regulated spatial localisation during infection-related development of C. fulvum.

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.

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.

Cladosporiumfulvum is a semi-biotrophic pathogen, which causes leaf mold of tomato (Lycopersicon spp.). In our laboratory this pathosystem serves as a model to study gene-for-gene interactions between plants and pathogenic fungi (Joosten & De Wit 1999). Many avirulence (Avr) genes and matching resistance (CQ) genes have been cloned and we are now beginning to understand how their products can induce an array of plant defense responses, including the classic hypersensitive response (HR). Here, we will discuss the latest results of our molecular studies on this interaction. These include the isolation of: (i) two new Avr genes, Avr2 and Avr4E, (ii) the determination of the specificity determinants within the Cf-4 and Cf-9 genes by artificial domain swaps and introduction of point mutations, (iii) the analysis of polymorphism occurring in AVR9-responsive Cf genes occurring in natural populations of L. pimpinellifolium, and finally (iv) the description of an efficient method to identify early HR-related genes.

The gene-for-gene model postulates that for every gene determining resistance in the host plant, there is a corresponding gene conditioning avirulence in the pathogen. On the basis of this relationship, products of resistance (R) genes and matching avirulence (Avr) genes are predicted to interact. Here, we report on binding studies between the R gene product Cf-9 of tomato and the Avr gene product AVR9 of the pathogenic fungus Cladosporium fulvum. Because a high-affinity binding site (HABS) for AVR9 is present in tomato lines, with or without the Cf-9 resistance gene, as well as in other solanaceous plants, the Cf-9 protein was produced in COS and insect cells in order to perform binding studies in the absence of the HABS. Binding studies with radio-labeled AVR9 were performed with Cf-9-producing COS and insect cells and with membrane preparations of such cells. Furthermore, the Cf-9 gene was introduced in tobacco, which is known to be able to produce a functional Cf-9 protein. Binding of AVR9 to Cf-9 protein produced in tobacco was studied employing surface plasmon resonance and surface-enhanced laser desorption and ionization. Specific binding between Cf-9 and AVR9 was not detected with any of the procedures. The implications of this observation are discussed.

Here we describe the role of the Cladosporium fulvum nitrogen response factor 1 (Nrf1) gene in regulation of the expression of avirulence gene Avr9 and virulence on tomato. The Nrf1 gene, which was isolated by a polymerase chain reaction-based strategy, is predicted to encode a protein of 918 amino acid residues. The protein contains a putative zinc finger DNA-binding domain that shares 98% amino acid identity with the zinc finger of the major nitrogen regulatory proteins AREA and NIT2 of Aspergillus nidulans and Neurospora crassa, respectively. Functional equivalence of Nrf1 to areA was demonstrated by complementation of an A. nidulans areA loss-of-function mutant with Nrf1. Nrf1-deficient transformants of C. fulvum obtained by homologous recombination were unable to utilize nitrate and nitrite as a nitrogen source. In contrast to what was observed in the C. fulvum wild-type, the Avr9 gene was no longer induced under nitrogen-starvation conditions in Nrf1-deficient strains. On susceptible tomato plants, the Nrf1-deficient strains were as virulent as wild-type strains of C. fulvum, although the expression of the Avr9 gene was strongly reduced. In addition, Nrf1-deficient strains were still avirulent on tomato plants containing the functional Cf-9 resistance gene, indicating that in planta, apparently sufficient quantities of stable AVR9 elicitor are produced. Our results suggest that the NRF1 protein is a major regulator of the Avr9 gene.

The tomato resistance genes Cf-4 and Cf-9 confer specific, hypersensitive response-associated recognition of Cladosporium carrying the avirulence genes Avr4 and Avr9, respectively. Cf-4 and Cf-9 encode type I transmembrane proteins with extracellular leucine-rich repeats (LRRs). Compared with Cf-9, Cf-4 lacks two LRRs and differs in 78 amino acid residues. To investigate the relevance of these differences for specificity, we exchanged domains between Cf-4 and Cf-9, and mutant constructs were tested for mediating the hypersensitive response by transient coexpression with either Avr4 or Avr9. We show that the number of LRRs is essential for both Cf-4 and Cf-9 function. In addition, Cf-9 specificity resides entirely in the LRR domain and appears to be distributed over several distant LRRs. In contrast, Cf-4 specificity determinants reside in the N-terminal LRR-flanking domain and three amino acid residues in LRRs 13, 14, and 16. These residues are present at putative solvent-exposed positions, and all are required for full Cf-4 function. Finally, we show that Cf-9 carrying the specificity determinants of Cf-4 has recognitional specificity for AVR4. The data indicate that diversifying selection of solvent-exposed residues has been a more important factor in the generation of Cf-4 specificity than has sequence exchange between Cf-4 progenitor genes. The fact that most variant residues in Cf-4 are not essential for Cf-4 specificity indicates that the diverse decoration of R proteins is not fully adapted to confer recognition of a certain avirulence determinant but likely provides a basis for a versatile, adaptive recognition system.

The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins.

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.

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.

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.

The avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is highly induced during infection of tomato plants. Expression of the Avr9 gene can also be induced in vitro when cells are grown on synthetic liquid medium containing little or no nitrogen. The Avr9 promoter contains six copies of the sequence TAGATA and six additional copies of the core sequence GATA within 0.4 kb upstream of the translation start site. In the filamentous fungi Aspergillus nidulans and Neurospora crassa, these promoter sequences have been identified as the binding sites for a wide-domain GATA-type regulator (AREA in A. nidulans and NIT2 in N. crassa) involved in nitrogen utilization. Quantification of GUS activity of A. nidulans transformants containing a single copy of the fully active Avr9 promoter-uidA (GUS) reporter gene fusion in different areA backgrounds, following starvation for nitrogen, showed that induction of the Avr9 promoter is regulated similarly in A. nidulans and C. fulvum. This suggests that AREA can regulate the Avr9 promoter and that C. fulvum contains an AREA-like regulator that can bind to these specific sequence motifs. Comparison of the induction profiles of Avr9 and niaD showed that Avr9 expression is independent of NIRA, as is niaD expression upon nitrogen starvation. Studies with Avr9 promoter-uidA fusions in which all or most of these sequences had been deleted, showed that Avr9 promoter activity is dependent on the presence of these specific cis-regulatory elements, suggesting that they do indeed function in transcriptional regulation of the Avr9 gene.

Five different hydrophobin-encoding cDNA clones from Cladosporium fulvum were isolated from cDNA libraries, made from nutrient-depleted mycelium. One cDNA clone was identical to the previously isolated hydrophobin HCf-1. The other clones were named HCf-2, -3, -4 and -5. HCf-1, -2, -3 and -4 show a high degree of identity, and are predicted to encode class I hydrophobins. HCf-5 encodes a class II hydrophobin. The expression patterns of these hydrophobins at various stages of development, and in liquid media lacking either carbon or nitrogen, or both, showed clear differences. All hydrophobins were more strongly expressed during sporulation than before, with HCf-4 and -5 showing the highest increase. Expression of HCf genes in infected plants was also higher at 16 days than at 10 days after infection. The expression of HCf-5 in sporulating mycelium was much lower in planta than in vitro. All HCf genes were upregulated under conditions of nutrient deprivation. HCf-1, -2, -3 and -4 showed highest levels of transcription in medium lacking both carbon and nitrogen. Expression of HCf-5 was highest in medium lacking nitrogen but containing carbon. HCf-1 was generally the most abundant hydrophobin. The introduction of multiple copies of HCf-1, which caused co-suppression of the endogenous HCf-1 gene, was shown to affect the expression of HCf-2, -3 and -4 also. Expression of HCf-4 was suppressed, but expression of HCf-2 and -3 was upregulated. Expression of HCf-5 was not changed.

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.

The pathogenicity of fungal pathogens is presumably dependent on genes that are expressed during infection. In order to isolate such genes from the tomato pathogen Cladosporium fulvum, and to test the hypothesis that starvation-induced genes are also plant induced, a cDNA library was prepared from mycelia grown in a defined medium and then transferred to a starvation medium. The library was then screened with cDNA prepared from starved and replete fungal mycelium. Five unique, differentially expressed cDNAs were isolated from 1,000 clones screened. Northern (RNA) hybridization confirmed that all five were starvation induced. Interestingly, all five were also found to be plant induced. The identity of two of the clones was indicated by partial DNA sequencing as alcohol and aldehyde dehydrogenase. The observed correlation between starvation induction and plant induction in discussed.

The avirulence gene Avr4 conditions avirulence of the biotrophic fungus Cladosporium fulvum on tomato genotypes carrying resistance gene Cf-4 (MM-Cf4). Strains of the fungus that circumvent Cf-4-specific resistance show various single point mutations in the coding region of the Avr4 gene. Similar to expression of the Avr4 gene, expression of the various virulent avr4 alleles is specifically induced during pathogenesis. Polyclonal antibodies raised against the AVR4 elicitor, however, did not detect AVR4 isoforms in MM-Cf4 plants infected by the different virulent strains, indicating that these isoforms are unstable. To analyze whether the AVR4 isoforms still possess specific elicitor activity, the avr4 alleles were expressed in MM-Cf4 plants by using the potato virus X (PVX)-based expression system. Inoculation with PVX::Avr4 resulted in the development of spreading lesions, eventually leading to plant death, whereas the various PVX::avr4 derivatives induced symptoms ranging from severe necrosis to no lesions at all. We conclude that instability of the AVR4 isoforms that are produced by virulent strains is a crucial factor in circumvention of Cf-4-mediated resistance.