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Fulvia fulva and Dothistroma septosporum are closely related apoplastic pathogens with similar lifestyles but different hosts: F.fulva is a pathogen of tomato, whilst D.septosporum is a pathogen of pine trees. In 2012, the first genome sequences of these pathogens were published, with F.fulva and D.septosporum having highly fragmented and near-complete assemblies, respectively. Since then, significant advances have been made in unravelling their genome architectures. For instance, the genome of F.fulva has now been assembled into 14 chromosomes, 13 of which have synteny with the 14 chromosomes of D.septosporum, suggesting these pathogens are even more closely related than originally thought. Considerable advances have also been made in the identification and functional characterization of virulence factors (e.g., effector proteins and secondary metabolites) from these pathogens, thereby providing new insights into how they promote host colonization or activate plant defence responses. For example, it has now been established that effector proteins from both F.fulva and D.septosporum interact with cell-surface immune receptors and co-receptors to activate the plant immune system. Progress has also been made in understanding how F.fulva and D.septosporum have evolved with their host plants, whilst intensive research into pandemics of Dothistroma needle blight in the Northern Hemisphere has shed light on the origins, migration, and genetic diversity of the global D.septosporum population. In this review, we specifically summarize advances made in our understanding of the F.fulva-tomato and D.septosporum-pine pathosystems over the last 10 years.

Leaf mold caused by the fungus Cladosporium fulvum (syn. Passalora fulva) has been a serious disease in greenhouse tomatoes in Cuba, but less damage is seen in outdoor grown plants. This study was conducted to identify races of the causal agent of tomato leaf mold. A total of 36 single conidial isolates obtained from different provinces in Cuba and the reference strain race 0 were inoculated on a differential set of tomato cultivars carrying different Cf resistance genes. Further, a polymerase chain reaction (PCR) was developed using Avr9 gene-specific primers. As result, all isolates could overcome the Cf-9 resistance gene only, but not resistance genes Cf-2, Cf-4 and Cf-5 and must therefore be coined race 9. Race identity was also confirmed by PCR analysis, which showed that the Avr9 gene was not amplified in any of 36 single conidial isolates studied proving that they all lack this gene. This is the first report of the occurrence of race 9 of C. fulvum in Cuba. Pyramiding other Cf genes in hybrid of tomato may provide a more durable level of resistance to C. fulvum.

Proteases are key components of the hydrolytic enzyme arsenal employed by fungal pathogens to invade their host plants. The recent advances in -omics era have facilitated identification of functional proteases involved in plant-fungus interactions. By comparison of the publically available sequences of fungal genomes we found that the number of protease genes present in the genome of Cladosporium fulvum, a biotrophic tomato pathogen, is comparable with that of hemibiotrophs. To identify host plant inducible protease genes and their products, we performed transcriptome and proteome analyses of C. fulvumin vitro and in planta by means of RNA-Seq/RT-qPCR and mass spectrometry. Transcriptome data showed that 14 out of the 59 predicted proteases are expressed during in vitro and in planta growth of C. fulvum, of which nine belong to serine proteases S8 and S10 and the rest belong to metallo- and aspartic proteases. Mass spectrometry confirmed the presence of six proteases at proteome level during plant infection. Expression of limited number of proteases by C. fulvum might sustain biotrophic growth and benefits its stealth pathogenesis.

The Dothideomycete Pseudocercospora fijiensis, previously Mycosphaerella fijiensis, is the causal agent of black Sigatoka, one of the most destructive diseases of bananas and plantains. Disease management depends on fungicide applications, with a major contribution from sterol demethylation-inhibitors (DMIs). The continued use of DMIs places considerable selection pressure on natural P. fijiensis populations, enabling the selection of novel genotypes with reduced sensitivity. The hitherto explanatory mechanism for this reduced sensitivity was the presence of non-synonymous point mutations in the target gene Pfcyp51, encoding the sterol 14α-demethylase enzyme. Here, we demonstrate a second mechanism involved in DMI sensitivity of P. fijiensis. We identified a 19-bp element in the wild-type (wt) Pfcyp51 promoter that concatenates in strains with reduced DMI sensitivity. A polymerase chain reaction (PCR) assay identified up to six Pfcyp51 promoter repeats in four field populations of P. fijiensis in Costa Rica. We used transformation experiments to swap the wt promoter of a sensitive field isolate with a promoter from a strain with reduced DMI sensitivity that comprised multiple insertions. Comparative in vivo phenotyping showed a functional and proportional up-regulation of Pfcyp51, which consequently decreased DMI sensitivity. Our data demonstrate that point mutations in the Pfcyp51 coding domain, as well as promoter inserts, contribute to the reduced DMI sensitivity of P. fijiensis. These results provide new insights into the importance of the appropriate use of DMIs and the need for the discovery of new molecules for black Sigatoka management.

S.G., J.C., C.H.M. and P.J.G.M.d.W. were financially supported by a grant from the Royal Netherlands Academy of Sciences.; 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 the colonization 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 are repressed during the biotrophic phase of tomato colonization. It has been suggested that the tight regulation of SM gene clusters is required for C. fulvum to behave as a biotrophic pathogen, whilst 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 in a similar manner to the 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 the repression of cladofulvin production is required for C. fulvum to sustain its biotrophic lifestyle in tomato, whereas its production is important for survival outside its host.

Summary To facilitate infection, pathogens deploy a plethora of effectors to suppress basal host immunity induced by exogenous microbe‐associated or endogenous damage‐associated molecular patterns (DAMPs). In this study, we have characterized family 17 glycosyl hydrolases of the tomato pathogen Cladosporium fulvum (CfGH17) and studied their role in infection. Heterologous expression of CfGH17‐1 to 5 by potato virus X in different tomato cultivars showed that CfGH17‐1 and CfGH17‐5 enzymes induce cell death in Cf‐0, Cf‐1 and Cf‐5 but not in Cf‐Ecp3 tomato cultivars or tobacco. Moreover, CfGH17‐1 orthologues from other phytopathogens, including Dothistroma septosporum and Mycosphaerella fijiensis, also trigger cell death in tomato. CfGH17‐1 and CfGH17‐5 are predicted to be β‐1,3‐glucanases and their enzymatic activity is required for the induction of cell death. CfGH17‐1 hydrolyses laminarin, a linear 1,3‐β‐glucan with 1,6‐β linkages. CfGH17‐1 expression is down‐regulated during the biotrophic phase of infection and up‐regulated during the necrotrophic phase. Deletion of CfGH17‐1 in C. fulvum did not reduce virulence on tomato, while constitutive expression of CfGH17‐1 decreased virulence, suggesting that abundant presence of CfGH17‐1 during biotrophic growth may release a DAMP that activates plant defence responses. Under natural conditions CfGH17‐1 is suggested to play a role during saprophytic growth when the fungus thrives on dead host tissue, which is in line with its high levels of expression at late stages of infection when host tissues have become necrotic. We suggest that CfGH17‐1 releases a DAMP from the host cell wall that is recognized by a yet unknown host plant receptor.

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.

SnTox1 induces programmed cell death and the up-regulation of pathogenesis-related genes including chitinases. Additionally, SnTox1 has structural homology to several plant chitin-binding proteins. Therefore, we evaluated SnTox1 for chitin binding and localization. We transformed an avirulent strain of Parastagonospora nodorum as well as three nonpathogens of wheat (Triticum aestivum), including a necrotrophic pathogen of barley, a hemibiotrophic pathogen of sugar beet and a saprotroph, to evaluate the role of SnTox1 in infection and in protection from wheat chitinases. SnTox1 bound chitin and an SnTox1-green fluorescent fusion protein localized to the mycelial cell wall. Purified SnTox1 induced necrosis in the absence of the pathogen when sprayed on the leaf surface and appeared to remain on the leaf surface while inducing both epidermal and mesophyll cell death. SnTox1 protected the different fungi from chitinase degradation. SnTox1 was sufficient to change the host range of a necrotrophic pathogen but not a hemibiotroph or saprotroph. Collectively, this work shows that SnTox1 probably interacts with a receptor on the outside of the cell to induce cell death to acquire nutrients, but SnTox1 accomplishes a second role in that it protects against one aspect of the defense response, namely the effects of wheat chitinases.

In this review, I recount my personal history. My drive to study host-pathogen interactions was to find alternatives for agrochemicals, which was triggered after reading the book “Silent Spring” by Rachel Carson. I reflect on my research at the Laboratory of Phytopathology at Wageningen University, where I have worked for my entire career on the interaction between Cladosporium fulvum and tomato, and related gene-for-gene pathosystems. I describe different methods used to identify and sequence avirulence (Avr) genes from the pathogen and resistance (R) genes from the host. The major genes involved in classical gene-for-gene interactions have now been identified, and breeders can produce plants with multiple R genes providing durable and environmentally safe protection against pathogens. In some cases, this might require the use of genetically modified plants when R genes cannot be introduced by classical breeding.

Tomato leaf mould disease is caused by the biotrophic fungusCladosporium fulvum. During infection,C. fulvumproduces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed byCfimmune 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. fulvumstrains capable of overcoming one or more of all clonedCfgenes have now emerged. To combat these strains, newCfgenes are required. An effectoromics approach was employed to identify wild tomato accessions carrying newCfgenes. Proteomics and transcriptome sequencing were first used to identify 70 apoplasticin planta-inducedC. fulvumSSPs. 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 interactionsin planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs using thePotato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry newCfgenes available for incorporation into cultivated tomato.

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.

Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z.?tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In?planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z.?tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z.?tritici–wheat pathosystem

Zymoseptoria tritici is an economically important pathogen of wheat. However, the molecular basis of pathogenicity on wheat is still poorly understood. Here, we present a global survey of the proteins secreted by this fungus in the apoplast of resistant (cv. Shafir) and susceptible (cv. Obelisk) wheat cultivars after inoculation with reference Z. tritici strain IPO323. The fungal proteins present in apoplastic fluids were analyzed by gel electrophoresis and by data-independent acquisition liquid chromatography/mass spectrometry (LC/MSE) combined with data-dependent acquisition LC–MS/MS. Subsequent mapping mass spectrometry-derived peptide sequence data against the genome sequence of strain IPO323 identified 665 peptides in the MSE and 93 in the LC–MS/MS mode that matched to 85 proteins. The identified fungal proteins, including cell-wall degrading enzymes and proteases, might function in pathogenicity, but the functions of many remain unknown. Most fungal proteins accumulated in cv. Obelisk at the onset of necrotrophy. This inventory provides an excellent basis for future detailed studies on the role of these genes and their encoded proteins during pathogenesis in wheat.

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. ?cfwor1 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 ?cfwor1 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 ?fosge1 (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 ?fosge1 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.

The interactions between fungi and plants encompass a spectrum of ecologies ranging from saprotrophy (growth on dead plant material) through pathogenesis (growth of the fungus accompanied by disease on the plant) to symbiosis (growth of the fungus with growth enhancement of the plant). We consider pathogenesis in this article and the key roles played by a range of pathogen-encoded molecules that have collectively become known as effectors.

Zymoseptoria tritici causes the major fungal wheat disease septoria tritici blotch, and is increasingly being used as a model for transmission and population genetics, as well as host–pathogen interactions. Here, we study the biological function of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans, as a representative of a superfamily of regulatory proteins involved in dimorphic switching. In Z.?tritici, this gene is pivotal for pathogenesis, as ZtWor1 mutants were nonpathogenic and complementation restored the wild-type phenotypes. In?planta expression analyses showed that ZtWor1 is up-regulated during the initiation of colonization and fructification, and regulates candidate effector genes, including one that was discovered after comparative proteome analysis of the Z.?tritici wild-type strain and the ZtWor1 mutant, which was particularly expressed in?planta. Cell fusion and anastomosis occur frequently in ZtWor1 mutants, reminiscent of mutants of MgGpb1, the s-subunit of the heterotrimeric G protein. Comparative expression of ZtWor1 in knock-out strains of MgGpb1 and MgTpk2, the catalytic subunit of protein kinase A, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenesis in many fungal plant pathogens

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

Pseudogenes are genes with significant homology to functional genes but contain disruptive mutations (DMs) leading to production of non- or partially functional proteins. Little is known about pseudogenization in pathogenic fungi with different lifestyles. Here we report on identification of DMs causing pseudogenes in the genomes of the fungal plant pathogens Botrytis cinerea, Cladosporium fulvum, Dothistroma septosporum, Mycosphaerella fijiensis, Verticillium dahliae and Zymoseptoria tritici. In these fungi we have identified 1740 gene models containing 2795 DMs obtained by an alignment-based gene prediction method. The contribution of sequencing errors to DMs was minimized by analyses of resequenced genomes to obtain a refined data set of 924 gene models containing 1666 true DMs. The frequency of pseudogenes varied from 1 to 5% in the gene catalogues of these fungi, being the highest in the asexually reproducing fungi C. fulvum (4.9%), followed by D. septosporum (2.4%) and V. dahliae (2.1%). The majority of pseudogenes does not represent recent gene duplications, but members of multi-gene families and unitary genes. In general there was no bias for pseudogenization of specific genes in the six fungi. Single exceptions are those encoding secreted proteins including proteases which appeared more frequently pseudogenized in C. fulvum than in D. septosporum. Most pseudogenes present in these two phylogenically closely related fungi are not shared suggesting that they are related to adaptation to a different host (tomato versus pine) and lifestyle (biotroph versus hemi-biotroph)

· α-Tomatine is an antifungal glycoalkaloid that provides basal defense to tomato (Solanum lycopersicum). However, tomato pathogens overcome this basal defense barrier by the secretion of tomatinases that degrade α-tomatine into the less fungitoxic compounds β-tomatine and tomatidine. Although pathogenic on tomato, it has been reported that the biotrophic fungus Cladosporium fulvum is unable to detoxify α-tomatine. · Here, we present a functional analysis of the glycosyl hydrolase (GH10), CfTom1, which is orthologous to fungal tomatinases. · We show that C. fulvum hydrolyzes α-tomatine into tomatidine in vitro and during the infection of tomato, which is fully attributed to the activity of CfTom1, as shown by the heterologous expression of this enzyme in tomato. Accordingly, ∆cftom1 mutants of C. fulvum are more sensitive to α-tomatine and are less virulent than the wild-type fungus on tomato. · Although α-tomatine is thought to be localized in the vacuole, we show that it is also present in the apoplast, where it is hydrolyzed by CfTom1 on infection. The accumulation of tomatidine during infection appears to be toxic to tomato cells and does not suppress defense responses, as suggested previously. Altogether, our results show that CfTom1 is responsible for the detoxification of α-tomatine by C. fulvum, and is required for full virulence of this fungus on tomato.

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs., Author Summary Black Sigatoka or black leaf streak disease, caused by the ascomycete fungus Pseudocercospora fijiensis, inflicts huge costs on banana producers, due to crop losses and expenses for disease control. The global banana export trade relies on Cavendish clones that are highly susceptible to P. fijiensis. Sustainable production of the world’s number one fruit requires a better understanding of host resistance and sophisticated management of fungicide resistance in the pathogen. In the P. fijiensis genome sequence we identified an effector that induced an HR-like necrosis on a resistant banana accession but not on a susceptible cultivar. If validated, this assay may be useful for identifying resistance in banana breeding programs. We also used the genomic sequence to develop highly polymorphic molecular markers for analyzing P. fijiensis field populations and identified a strong enrichment (nearly 100%) for fungicide resistance markers in fungicide-treated banana plantations compared to untreated wild-type populations. This rapid evolution of fungicide resistance poses an immediate threat to sustainable banana production.

International audience; 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 beta-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.

Most fungal plant pathogens secrete effector proteins during pathogenesis to manipulate their host's defense and promote disease. These are so highly diverse in sequence and distribution, they are essentially considered as species-specific. However, we have recently shown the presence of homologous effectors in fungal species of the Dothideomycetes class. One such example is Ecp2, an effector originally described in the tomato pathogen Cladosporium fulvum but later detected in the plant pathogenic fungi Mycosphaerella fijiensis and Mycosphaerella graminicola as well. Here, using in silico sequence-similarity searches against a database of 135 fungal genomes and GenBank, we extend our queries for homologs of Ecp2 to the fungal kingdom and beyond, and further study their history of diversification. Our analyses show that Ecp2 homologs are members of an ancient and widely distributed superfamily of putative fungal effectors, which we term Hce2 for Homologs of C. fulvum Ecp2. Molecular evolutionary analyses show that the superfamily originated and diversified within the fungal kingdom, experiencing multiple lineage-specific expansions and losses that are consistent with the birth-and-death model of gene family evolution. Newly formed paralogs appear to be subject to diversification early after gene duplication events, whereas at later stages purifying selection acts to preserve diversity and the newly evolved putative functions. Some members of the Hce2 superfamily are fused to fungal Glycoside Hydrolase family 18 chitinases that show high similarity to the Zymocin killer toxin from the dairy yeast Kluyveromyces lactis, suggesting an analogous role in antagonistic interactions. The observed high rates of gene duplication and loss in the Hce2 superfamily, combined with diversification in both sequence and possibly functions within and between species, suggest that Hce2s are involved in adaptation to stresses and new ecological niches. Such findings address the need to rationalize effector biology and evolution beyond the perspective of solely host-microbe interactions.

Spliceosomal introns are noncoding sequences that separate exons in eukaryotic genes and are removed from pre-messenger RNAs by the splicing machinery. Their origin has remained a mystery in biology since their discovery [ [1] and [2]] because intron gains seem to be infrequent in many eukaryotic lineages [ [3] and [4]]. Although a few recent intron gains have been reported [ [5] and [6]], none of the proposed gain mechanisms [7] can convincingly explain the high number of introns in present-day eukaryotic genomes. Here we report on particular spliceosomal introns that share high sequence similarity and are reminiscent of introner elements [8]. These elements multiplied in unrelated genes of six fungal genomes and account for the vast majority of intron gains in these fungal species. Such introner-like elements (ILEs) contain all typical characteristics of regular spliceosomal introns (RSIs) [ [9] and [10]] but are longer and predicted to harbor more stable secondary structures. However, dating of multiplication events showed that they degenerate in sequence and length within 100,000 years to eventually become indistinguishable from RSIs. We suggest that ILEs not only account for intron gains in six fungi but also in ancestral eukaryotes to give rise to most RSIs by a yet unknown multiplication mechanism