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Staff Publications

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Assignment of a dubious gene cluster to melanin biosynthesis in the tomato fungal pathogen Cladosporium fulvum
Griffiths, Scott A. ; Cox, Russell J. ; Overdijk, Elysa J.R. ; Mesarich, Carl H. ; Saccomanno, Benedetta ; Lazarus, Colin M. ; Wit, Pierre J.G.M. de; Collemare, Jérôme - \ 2018
PLoS ONE 13 (2018)12. - ISSN 1932-6203

Pigments and phytotoxins are crucial for the survival and spread of plant pathogenic fungi. The genome of the tomato biotrophic fungal pathogen Cladosporium fulvum contains a predicted gene cluster (CfPKS1, CfPRF1, CfRDT1 and CfTSF1) that is syntenic with the characterized elsinochrome toxin gene cluster in the citrus pathogen Elsinoë fawcettii. However, a previous phylogenetic analysis suggested that CfPks1 might instead be involved in pigment production. Here, we report the characterization of the CfPKS1 gene cluster to resolve this ambiguity. Activation of the regulator CfTSF1 specifically induced the expression of CfPKS1 and CfRDT1, but not of CfPRF1. These co-regulated genes that define the CfPKS1 gene cluster are orthologous to genes involved in 1,3-dihydroxynaphthalene (DHN) melanin biosynthesis in other fungi. Heterologous expression of CfPKS1 in Aspergillus oryzae yielded 1,3,6,8-tetrahydroxynaphthalene, a typical precursor of DHN melanin. Δcfpks1 deletion mutants showed similar altered pigmentation to wild type treated with DHN melanin inhibitors. These mutants remained virulent on tomato, showing this gene cluster is not involved in pathogenicity. Altogether, our results showed that the CfPKS1 gene cluster is involved in the production of DHN melanin and suggests that elsinochrome production in E. fawcettii likely involves another gene cluster.

Specific Hypersensitive Response–Associated Recognition of New Apoplastic Effectors from Cladosporium fulvum in Wild Tomato
Mesarich, Carl H. ; Ӧkmen, Bilal ; Rovenich, Hanna ; Griffiths, Scott A. ; Wang, Changchun ; Karimi Jashni, Mansoor ; Mihajlovski, Aleksandar ; Collemare, Jérôme ; Hunziker, Lukas ; Deng, Cecilia H. ; Burgt, Ate Van Der; Beenen, Henriek G. ; Templeton, Matthew D. ; Bradshaw, Rosie E. ; Wit, Pierre J.G.M. De - \ 2018
Molecular Plant-Microbe Interactions 31 (2018)1. - ISSN 0894-0282 - p. 145 - 162.
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.
Down-regulation of cladofulvin biosynthesis is required for biotrophic growth of Cladosporium fulvum on tomato : A secondary metabolite prevents fungal biotrophy
Griffiths, Scott ; Mesarich, Carl H. ; Overdijk, Elysa J.R. ; Saccomanno, Benedetta ; Wit, Pierre J.G.M. De; Collemare, Jérôme - \ 2018
Molecular Plant Pathology 19 (2018)2. - ISSN 1464-6722 - p. 369 - 380.
Fungal biotrophy is associated with a reduced capacity to produce potentially toxic secondary metabolites (SMs). Yet, the genome of the biotrophic plant pathogen Cladosporium fulvum contains many SM biosynthetic gene clusters, with several related to toxin production. These gene clusters are, however, poorly expressed during colonisation of tomato. The sole detectable SM produced by C. fulvum during in vitro growth is the anthraquinone cladofulvin. Although this pigment is not detected in infected leaves, cladofulvin biosynthetic genes are expressed throughout the pre-penetration phase and during conidiation at the end of the infection cycle, but they are repressed during the biotrophic phase of tomato colonization. It was suggested that tight regulation of SM gene clusters is required for C. fulvum to behave as a biotrophic pathogen, while retaining potential fitness determinants for growth and survival outside its host. To address this hypothesis, we analysed the disease symptoms caused by mutant C. fulvum strains that do not produce or over-produce cladofulvin during the biotrophic growth phase. Non-producers infected tomato similar to wild type, suggesting that cladofulvin is not a virulence factor. In contrast, the cladofulvin over-producers caused strong necrosis and desiccation of tomato leaves, which in turn, arrested conidiation. Consistent with the role of pigments in survival against abiotic stresses, cladofulvin protects conidia against UV light and low temperature stress. Overall this study demonstrates that repression of cladofulvin production is required for C. fulvum to sustain its biotrophic lifestyle in tomato, while its production is important for survival outside its host.
Novel effectors identified in the apoplast of Cladosporium fulvum-infected tomato
Mesarich, C. ; Ökmen, B. ; Rövenich, H.J. ; Karimi Jashni, M. ; Wang, C. ; Griffiths, S.A. ; Collemare, J.A.R. ; Deng, C. ; Wit, P.J.G.M. de - \ 2016
Tomato leaf mold disease is caused by the biotrophic fungal pathogen Cladosporium fulvum. To colonize the leaf apoplast, C. fulvum secretes a collection of effector proteins that modulate host immune responses, as well as other proteins (e.g., carbohydrate-active enzymes or CAZys) that facilitate nutrient acquisition. In the presence of cognate Cf immune receptors, however, many of these proteins trigger immune responses that render the pathogen avirulent. Characterization of the C. fulvum apoplastic secretome is required to further understand the abovementioned processes, and to identify novel sources of resistance against this pathogen. We have used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to identify 141 secreted and surface-associated fungal proteins present in apoplastic fluid harvested from compatible C. fulvum–tomato interactions. In addition to the known effectors identified in previous studies, this collection contains >70 new C. fulvum candidate effector (CfCE) proteins. Using a Potato virus X (PVX)-based expression system, we show that nine of these CfCEs, including Ecp11-1, which has homology to AvrLm3 and AvrLmJ1 of Leptosphaeria maculans, trigger cell death in particular wild accessions of tomato. Thus, our study has likely uncovered novel avirulence effectors of C. fulvum, as well as Cf immune receptors in wild tomato with new specificities against this pathogen. An overview of the C. fulvum apoplastic secretome will be presented.
Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization
Griffiths, Scott ; Mesarich, Carl H. ; Saccomanno, Benedetta ; Vaisberg, Abraham ; Wit, Pierre J.G.M. de; Cox, Russell ; Collemare, Jérôme - \ 2016
Proceedings of the National Academy of Sciences of the United States of America 113 (2016)25. - ISSN 0027-8424 - p. 6851 - 6856.
Cytoxicity - Emodin - Gene cluster - Nataloe-emodin - Secondary metabolism

Anthraquinones are a large family of secondary metabolites (SMs) that are extensively studied for their diverse biological activities. These activities are determined by functional group decorations and the formation of dimers from anthraquinone monomers. Despite their numerous medicinal qualities, very few anthraquinone biosynthetic pathways have been elucidated so far, including the enzymatic dimerization steps. In this study, we report the elucidation of the biosynthesis of cladofulvin, an asymmetrical homodimer of nataloe-emodin produced by the fungus Cladosporium fulvum. A gene cluster of 10 genes controls cladofulvin biosynthesis, which begins with the production of atrochrysone carboxylic acid by the polyketide synthase ClaG and the β-lactamase ClaF. This compound is decarboxylated by ClaH to yield emodin, which is then converted to chrysophanol hydroquinone by the reductase ClaC and the dehydratase ClaB. We show that the predicted cytochrome P450 ClaM catalyzes the dimerization of nataloe-emodin to cladofulvin. Remarkably, such dimerization dramatically increases nataloe-emodin cytotoxicity against mammalian cell lines. These findings shed light on the enzymatic mechanisms involved in anthraquinone dimerization. Future characterization of the ClaM enzyme should facilitate engineering the biosynthesis of novel, potent, dimeric anthraquinones and structurally related compound families.

A conserved proline residue in Dothideomycete Avr4 effector proteins is required to trigger a Cf-4-dependent hypersensitive response
Mesarich, C.H. ; Stergiopoulos, I. ; Beenen, H.G. ; Cordovez da Cunha, V. ; Guo, Y. ; Karimi Jashni, M. ; Bradshaw, R.E. ; Wit, P.J.G.M. de - \ 2016
Molecular Plant Pathology 17 (2016)1. - ISSN 1464-6722 - p. 84 - 95.
CfAvr4, a chitin-binding effector protein produced by the Dothideomycete tomato pathogen Cladosporium fulvum, protects the cell wall of this fungus against hydrolysis by secreted host chitinases during infection. However, in the presence of the Cf-4 immune receptor of tomato, CfAvr4 triggers a hypersensitive response (HR), which renders the pathogen avirulent. Recently, several orthologues of CfAvr4 have been identified from phylogenetically closely related species of Dothideomycete fungi. Of these, DsAvr4 from Dothistroma septosporum also triggers a Cf-4-dependent HR, but CaAvr4 and CbAvr4 from Cercospora apii and Cercospora beticola, respectively, do not. All, however, bind chitin. To identify the region(s) and specific amino acid residue(s) of CfAvr4 and DsAvr4 required to trigger a Cf-4-dependent HR, chimeric and mutant proteins, in which specific protein regions or single amino acid residues, respectively, were exchanged between CfAvr4 and CaAvr4 or DsAvr4 and CbAvr4, were tested for their ability to trigger an HR in Nicotiana benthamiana plants transgenic for the Cf-4 immune receptor gene. Based on this approach, a single region common to CfAvr4 and DsAvr4 was determined to carry a conserved proline residue necessary for the elicitation of this HR. In support of this result, a Cf-4-dependent HR was triggered by mutant CaAvr4 and CbAvr4 proteins carrying an arginine-to-proline substitution at this position. This study provides the first step in deciphering how Avr4 orthologues from different Dothideomycete fungi trigger a Cf-4-dependent HR.
Understanding plant immunity as a surveillance system to detect invasion
Cook III, D.E. ; Mesarich, C.H. ; Thomma, B.P.H.J. - \ 2015
Annual Review of Phytopathology 53 (2015). - ISSN 0066-4286 - p. 541 - 563.
disease-resistance gene - bacterial elicitor flagellin - syringae effectors avrb - host-selective toxins - innate immunity - arabidopsis-thaliana - molecular-patterns - microbe interactions - durable resistance - necrotrophic pathogens
Various conceptual models to describe the plant immune system have been presented. The most recent paradigm to gain wide acceptance in the field is often referred to as the zigzag model, which reconciles the previously formulated gene-for-gene hypothesis with the recognition of general elicitors in a single model. This review focuses on the limitations of the current paradigm of molecular plant-microbe interactions and how it too narrowly defines the plant immune system. As such, we discuss an alternative view of plant innate immunity as a system that evolves to detect invasion. This view accommodates the range from mutualistic to parasitic symbioses that plants form with diverse organisms, as well as the spectrum of ligands that the plant immune system perceives. Finally, how this view can contribute to the current practice of resistance breeding is discussed.
Novel mutations detected in avirulence genes overcoming tomato Cf resistance genes in isolates of a Japanese population of Cladosporium fulvum
Iida, Y. ; Hof, P.M.J. van 't; Beenen, H.G. ; Mesarich, C.H. ; Kubota, M. ; Stergiopoulos, I. ; Mehrabi, A. ; Notsu, A. ; Fujiwara, K. ; Bahkali, A. ; Abd-Elsalam, K. ; Collemare, J. ; Wit, P.J.G.M. de - \ 2015
PLoS ONE 10 (2015)4. - ISSN 1932-6203 - 18 p.
fungal effector proteins - leaf mold - disease resistance - virulence factor - cf-4-mediated resistance - allelic variation - passalora-fulva - plant-pathogen - binding-site - avr4
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 battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant-pathogen interactions
Karimi Jashni, M. ; Mehrabi, R. ; Collemare, J. ; Mesarich, C.H. ; Wit, P.J.G.M. de - \ 2015
Frontiers in Plant Science 6 (2015). - ISSN 1664-462X - 7 p.
cf-2-dependent disease resistance - extracellular serine-protease - l. enhances resistance - class iv chitinases - phytophthora-infestans - cladosporium-fulvum - proteolytic-enzymes - antifungal activity - gene-expression - tomato
Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs) that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered immunity in host plants have been reported in the literature, but little is known about their role in compromising basal defense responses induced by microbe-associated molecular patterns. Recently, several reports appeared in literature on secreted fungal proteases that modify or degrade pathogenesis-related proteins, including plant chitinases or PIs that compromise their activities. This prompted us to review the recent advances on proteases and PIs involved in fungal virulence and plant defense. Proteases and PIs from plants and their fungal pathogens play an important role in the arms race between plants and pathogens, which has resulted in co-evolutionary diversification and adaptation shaping pathogen lifestyles.
Characterisation of Ave1 orthologs in Venturia scab pathogens
Wheeler, J. ; Kastner, P. ; Taranto, A. ; Shiller, J. ; Boshoven, J.C. ; Mesarich, C.H. ; Thomma, B.P.H.J. ; Deng, C. ; Bowen, J. ; Plummer, K.M. - \ 2015
In: Book of Abstracts 28th Fungal Genetics Conference. - - p. 211 - 211.
Most fungal effectors are genus, species or race-specific, however a few are more broadly conserved (e.g. ECP6), and some are discontinuously distributed within the Fungi (Ave1 & AvrLm6). Single orthologs of Ave1 from Verticillium dahliae, a virulence effector that activates Ve1-mediated resistance in tomato, have been identified in unrelated fungi (Colletotrichum higginsianum, Cercospora beticola, Fusarium oxysporum f. sp. lycopersici). A subset of these activate Ve1-mediated resistance in tomato (de Jonge et al. 2012). Ave1 also shares similarity to an ortholog in the phytopathogenic bacterium Xanthomonas axonopodis pv. citri, as well as to a common family of plant natriuretic peptides and expansins, involved in plant homeostasis and plant cell wall modifications (de Jonge et al. 2012). We have identified highly expanded Ave1-like gene families (with conserved predicted cysteine patterns & 37-57% overall aa identity) in the biotrophic scab fungi, Venturia inaequalis (5 genomes) & V. pirina. The orthologs are closely associated with repeats in Venturia genomes, however only a few appear to be impacted by RIP. Like VdAve1, Venturia orthologs have a conserved intron in the 5’UTR, which causes problems for automated gene calling, especially those packages informed by transcriptome data. Several of the Venturia orthologs are up-regulated during leaf infection (RNAseq data), and some are also highly expressed during in vitro growth on cellophane (RNAseq and proteomic data, Cook et al. 2014). Synthetic peptides (36 & 39 amino acids) from two V. inaequalis Ave1 orthologs, based on conserved homeostasis-regulating domains of plant proteins, affected Arabidopsis thaliana protoplasts (swelling) and guard cells (collapse) of Tradescantia leaves in epidermal peels exposed to the peptides (0.1µM) in solution. We hypothesize that Venturia Ave1 proteins may play a role during biotrophic infection in disturbing plant homeostasis and promoting nutrient release from plant cells.
The role of effectors, pamps, and secondary metabolites in adaptation of Cladosporium fulvum to tomato
Wit, P.J.G.M. de; Mesarich, C.H. ; Ökmen, B. ; Burgt, I.A. van der; Iida, Y. ; Battaglia, E. ; Beenen, H.G. ; Griffiths, S.A. ; Bradshaw, R.E. ; Collemare, J.A.R. - \ 2014
In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 41 - 41.
CS-28.1 - The biotrophic tomato pathogen Cladosporium fulvum is a Dothideomycete fungus that is most related to the hemi-biotrophic fungal pine pathogen Dothistroma septosporum. We are interested in understanding genomic adaptations in these and related fungi that can explain host plant-specificity. The C. fulvum genomic sequence allowed us to identify and study its complete effector catalogue of which many effectors had been identified already and of which three represent core effectors (Avr4, Ecp2 and Ecp6) occurring in several genera and species of fungal plant pathogens. The genome of D. septosporum harbors the highest number of homologs of C. fulvum effectors, but some are pseudogenized. Both C. fulvum and D. septosporum contain a large number of genes involved in the production of secondary metabolites. C. fulvum seems to adapt its host plant tomato by down-regulation or pseudogenization of genes involved in the production of toxic secondary metabolites. One clear example is dothiostromin, a toxin produced by D. septosporum during colonization of pine needle. C. fulvum contains all genes required for the production of this toxin, but a few crucial genes have been pseudogenized. Apart from new effectors (Avr5) we have also identified new pathogen-associated molecular patterns (PAMPs), as well as damage-associated molecular patterns (MAMPs) from C. fulvum. Both represent cell-wall degrading enzymes produced by C. fulvum during infection of tomato. We also found that C. fulvum has adapted to grow on tomato by secreting the enzyme a- tomatinase that hydrolyses the antifungal tomato saponin a-tomatine into the non-toxic compounds lycotetraose and tomatidine.
Dothideomycete Plant Pathogens Require Specific Virulence Factors for Colonization and Host Plants Have Developed Specific R Genes for Defence
Wit, P.J.G.M. de; Mesarich, C.H. ; Ökmen, B. ; Burgt, A. van der; Iida, Y. ; Battaglia, E. ; Beenen, H.G. ; Griffiths, S.A. ; Collemare, J.A.R. ; Bradshaw, R.E. - \ 2014
In: Book of Abstracts Joint Meeting American Phytopathological Society and Canadian Phytopathological Society. - - p. 44 - S.
The tomato pathogen Cladosporium fulvum is a Dothideomycete that is most related to the hemi-biotrophic fungal pine pathogen Dothistroma septosporum. We study genomic adaptations in these and related fungi that can explain host plant-specificity. The C. fulvum genomic sequence allowed us to identify and study its complete effector catalogue including three core effectors (Avr4, Ecp2 and Ecp6). The genome of D. septosporum harbors the highest number of homologs of C. fulvum effectors, but some are pseudogenized. Both C. fulvum and D. septosporum contain a large number of genes involved in the production of secondary metabolites. C. fulvum seems to adapt its host plant tomato by down-regulation or pseudogenization of genes involved in the production of toxic secondary metabolites. One example is dothiostromin, a toxin produced by D. septosporum during colonization of pine needle. C. fulvum contains all genes required for the production of this toxin, but a few of these genes are pseudogenized. Apart from new effectors (Avr5) we have also identified new pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) from C. fulvum. Both represent cell-wall degrading enzymes produced by C. fulvum during infection of tomato. We also found that C. fulvum has adapted to grow on tomato by secreting the enzyme a-tomatinase that hydrolyses the antifungal tomato saponin a-tomatine into the non-toxic compounds lycotetraose and tomatidine.
Role of effector proteins secreted by Cladosporium fulvum againist the mycoparasitic invasion
Iida, Y. ; Ökmen, B. ; Karimi Jashni, M. ; Mesarich, C.H. ; Ikeda, K. ; Collemare, J.A.R. ; Wit, P.J.G.M. de - \ 2014
In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 81 - 81.
P347 - Most fungal pathogens secrete effector molecules that function as virulence factors to facilitate disease on hosts, but they are also recognized by cognate plant resistance proteins to arrest fungal growth. Ten effector genes identified in leaf mold pathogen Cladosporium fulvum are strongly up-regulated during infection of the host plant tomato, but not or hardly in vitro. The fungal mycoparasite Dicyma puvinata is a well-known biocontrol agent that parasitizes several fungal leaf pathogens including C. fulvum. We found that in C. fulvum expression of most effector genes is up-regulated when it is co-cultured with D. puvinata. In addition, C. fulvum ¿ Avr4 and ¿Ecp6 deletion mutants are more susceptible to D. pulvinata than wild-type strain, suggesting that these effectors are not only important for defence of C. fulvum against tomato chitinases during infection but also to defend this fungus against mycoparasitic invasion. Most effectors are species-specific and a few are non-species-specific core effectors like Avr4 and Ecp6. Avr4 protects the C. fulvum cell wall from hydrolysis by tomato chitinase directly through chitin binding, whereas Ecp6 scavenges chitin fragments in the apoplastic region to prevent chitin-mediated elicitation of immune responses. These results suggest that chitinases from D. pulvinata are important for mycoparasitism, and C.fulvum deploys the same effectors to protect itself against both plants and mycoparasites.
Transcriptome sequencing: a powerful tool for the identification of avirulence effector genes from the tomato leaf mould pathogen Cladosporium fulvum
Mesarich, C.H. ; Ökmen, B. ; Burgt, I.A. van der; Griffiths, S.A. ; Wang, C. ; Rövenich, H. ; Beenen, H.G. ; Etalo, D.W. ; Joosten, M.H.A.J. ; Wit, P.J.G.M. de - \ 2014
In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 74 - 74.
P278 - Leaf mould disease of tomato (Solanum lycopersicum) is caused by the biotrophic fungal pathogen Cladosporium fulvum. Upon leaf entry, C. fulvum secretes a plethora of effector proteins to modulate host defence responses and promote successful infection. However, these effectors also trigger defence in tomato cultivars carrying cognate Cf disease resistance genes (avirulence effectors). Consequently, effector identification and functional characterization is required to further understand how C. fulvum facilitates infection, as well as the mechanisms involved in Cf-mediated effector perception and defence activation. Here, a combined bioinformatic and transcriptome sequencing approach was employed to identify avirulence effect or genes of C. fulvum. Firstly, we sought to clone the Avr5 avirulence effector gene, corresponding to the previously cloned Cf-5 resistance gene of tomato. RNA-Seq was performed on the sequenced race 0 strain (0WU; carries the Avr5 gene), as well as a race 5 strain (IPO 1979; lacks 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. The remaining CfCE proteins were then tested for their ability to elicit a hypersensitive response upon transient expression in wild tomato species containing unknown resistance traits. Using this approach, several potentially novel avirulence determinants of C. fulvum were identified.
Transcriptome sequencing uncovers the Avr5 avirulence gene of the tomato leaf mould pathogen Cladosporium fulvum
Mesarich, C.H. ; Griffiths, S.A. ; Burgt, A. van der; Okmen, B. ; Beenen, H. ; Etalo, D.W. ; Joosten, M.H.A.J. ; Wit, P.J.G.M. de - \ 2014
Molecular Plant-Microbe Interactions 27 (2014)8. - ISSN 0894-0282 - p. 846 - 857.
fungal effector proteins - cf-2-dependent disease resistance - aspergillus-nidulans - secreted proteins - candidate effectors - neurospora-crassa - allelic variation - virulence factor - signal peptides - plant-pathogens
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.
Ave1-like orthologs in Venturia: another expanded effector family emerges
Taranto, A. ; Jones, D. ; Shiller, J. ; Johnson, S. ; Hall, N. ; Cooke, I. ; Talbo, G. ; Mesarich, C.H. ; Thomma, B.P.H.J. ; Boshoven, J.C. ; Bowen, J. ; Deng, C. ; Templeton, M. ; Plummer, K.M. - \ 2013
In: Book of Abstracts 27th Fungal Genetics Conference, Asilomar, Pacific Grove, California, USA, 12-17 March 2013. - - p. 250 - 251.
Effectors are secreted by pathogens to modify plant physiology and establish disease. Plant immune receptors have evolved to recognise effectors and counter attack with defence responses. Most fungal effectors are lineage-specific, i.e. they are unique to a species, or to physiological races within a species. The availability of many whole genome sequences has revealed that some effectors are found in a discontinuous distribution within the fungal kingdom; a few phytopathogenic fungi (Colletotrichum higginsianum, Cercospora beticola, Fusarium oxysporum) possess an ortholog of Ave1 from Verticillium dahliae, an effector that activates Ve1-mediated resistance in tomato. A subset of these orthologs were shown to activate Ve1-mediated resistance in tomato. Unusually, Ave1 also shares similarity to an ortholog in the phytopathogenic bacterium Xanthomonas axonopodis, as well as to a widespread family of plant natriuretic peptides and expansins, involved in plant homeostasis and plant cell wall modification (de Jonge & van Esse et al. 2012). We have identified an expanded Ave1-like gene family in apple and pear scab fungi, Venturia inaequalis and V. pirina. These species also have expanded gene families with similarity to the Leptosphaeria maculans effector AvrLm6. V. pirina has 14 unique hits (best,1.43e-18) to VdAve1. V. inaequalis has 17 unique hits (best,1.07e-22) to VdAve1. The distribution of Ave1 orthologs is suggestive of one or more cross-kingdom gene transfer events. We are characterising Venturia Ave1-like genes to investigate the mode of gene multiplication; seek evidence of horizontal gene transfer; and determine the role of Ave1-like genes in pathogenicity. Ave1-like genes from non-Venturia fungi (and the bacterial gene) do not contain predicted introns, however, several (not all) V. inaequalis Ave1-like genes are predicted to contain introns. Codon usage bias among fungal, plant, and bacterial Ave1 orthologs, are being compared with the aim of inferring the kingdom of origin of the Venturia Ave1 orthologs. At least two ViAve1 orthologs are up-regulated during infection of apple. To determine whether the Venturia Ave1 proteins also activate a Ve1-mediated hypersensitive response, each has been co-expressed with tomato Ve1 in Nicotiana tabacum, using an Agrobacterium tumefaciens-mediated transient transformation assay.
Structure, dynamics and domain organization of the repeat protein Cin1 from the apple scab fungus
Mesarich, C.H. ; Schmitz, M. ; Tremouilhac, P. ; McGillivray, D.J. ; Templeton, M.D. ; Dingley, A.J. - \ 2012
Biochimica et Biophysica Acta. Proteins & Proteomics 1824 (2012)10. - ISSN 1570-9639 - p. 1118 - 1128.
model-free approach - magnetic-resonance relaxation - nmr structure determination - ray solution scattering - torsion angle dynamics - cell-wall protein - venturia-inaequalis - zinc-fingers - helicobacter-pylori - backbone dynamics
Venturia inaequalis is a hemi-biotrophic fungus that causes scab disease of apple. A recently-identified gene from this fungus, cin1 (cellophane-induced 1), is up-regulated over 1000-fold in planta and considerably on cellophane membranes, and encodes a cysteine-rich secreted protein of 523 residues with eight imperfect tandem repeats of ~ 60 amino acids. The Cin1 sequence has no homology to known proteins and appears to be genus-specific; however, Cin1 repeats and other repeat domains may be structurally similar. An NMR-derived structure of the first two repeat domains of Cin1 (Cin1-D1D2) and a low-resolution model of the full-length protein (Cin1-FL) using SAXS data were determined. The structure of Cin1-D1D2 reveals that each domain comprises a core helix–loop–helix (HLH) motif as part of a three-helix bundle, and is stabilized by two intra-domain disulfide bonds. Cin1-D1D2 adopts a unique protein fold as DALI and PDBeFOLD analysis identified no structural homology. A 15N backbone NMR dynamic analysis of Cin1-D1D2 showed that a short stretch of the inter-domain linker has large amplitude motions that give rise to reciprocal domain–domain mobility. This observation was supported by SAXS data modeling, where the scattering length density envelope remains thick at the domain–domain boundary, indicative of inter-domain dynamics. Cin1-FL SAXS data models a loosely-packed arrangement of domains, rather than the canonical parallel packing of adjacent HLH repeats observed in a-solenoid repeat proteins. Together, these data suggest that the repeat domains of Cin1 display a “beads-on-a-string” organization with inherent inter-domain flexibility that is likely to facilitate interactions with target ligands.
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