Sequential breakdown of the Cf-9 leaf mould resistance locus in tomato by Fulvia fulva.

Leaf mould, caused by Fulvia fulva, is a devastating disease of tomato plants. In many commercial tomato cultivars, resistance to this disease is governed by the Cf-9 locus, which encodes five paralogous receptor-like proteins. Two of these proteins confer resistance: Cf-9C recognises the previously identified F. fulva effector Avr9 and provides resistance during all plant growth stages, while Cf-9B recognises the yet-unidentified F. fulva effector Avr9B and provides mature plant resistance only. In recent years, F. fulva strains have emerged that can overcome the Cf-9 locus, with Cf-9C circumvented through Avr9 deletion. To understand how Cf-9B is circumvented, we set out to identify Avr9B. Comparative genomics, transient expression assays and gene complementation experiments were used to identify Avr9B, while gene sequencing was used to assess Avr9B allelic variation across a world-wide strain collection. A strict correlation between Avr9 deletion and resistance-breaking mutations in Avr9B was observed in strains recently collected from Cf-9 cultivars, whereas Avr9 deletion but no mutations in Avr9B were observed in older strains. This research showcases how F. fulva has evolved to sequentially break down the Cf-9 locus and stresses the urgent need for commercial tomato cultivars that carry novel, stacked resistance genes active against this pathogen.

The Venturia inaequalis effector repertoire is dominated by expanded families with predicted structural similarity, but unrelated sequence, to avirulence proteins from other plant-pathogenic fungi.

BACKGROUND: Scab, caused by the biotrophic fungus Venturia inaequalis, is the most economically important disease of apples worldwide. During infection, V. inaequalis occupies the subcuticular environment, where it secretes virulence factors, termed effectors, to promote host colonization. Consistent with other plant-pathogenic fungi, many of these effectors are expected to be non-enzymatic proteins, some of which can be recognized by corresponding host resistance proteins to activate plant defences, thus acting as avirulence determinants. To develop durable control strategies against scab, a better understanding of the roles that these effector proteins play in promoting subcuticular growth by V. inaequalis, as well as in activating, suppressing, or circumventing resistance protein-mediated defences in apple, is required. RESULTS: We generated the first comprehensive RNA-seq transcriptome of V. inaequalis during colonization of apple. Analysis of this transcriptome revealed five temporal waves of gene expression that peaked during early, mid, or mid-late infection. While the number of genes encoding secreted, non-enzymatic proteinaceous effector candidates (ECs) varied in each wave, most belonged to waves that peaked in expression during mid-late infection. Spectral clustering based on sequence similarity determined that the majority of ECs belonged to expanded protein families. To gain insights into function, the tertiary structures of ECs were predicted using AlphaFold2. Strikingly, despite an absence of sequence similarity, many ECs were predicted to have structural similarity to avirulence proteins from other plant-pathogenic fungi, including members of the MAX, LARS, ToxA and FOLD effector families. In addition, several other ECs, including an EC family with sequence similarity to the AvrLm6 avirulence effector from Leptosphaeria maculans, were predicted to adopt a KP6-like fold. Thus, proteins with a KP6-like fold represent another structural family of effectors shared among plant-pathogenic fungi. CONCLUSIONS: Our study reveals the transcriptomic profile underpinning subcuticular growth by V. inaequalis and provides an enriched list of ECs that can be investigated for roles in virulence and avirulence. Furthermore, our study supports the idea that numerous sequence-unrelated effectors across plant-pathogenic fungi share common structural folds. In doing so, our study gives weight to the hypothesis that many fungal effectors evolved from ancestral genes through duplication, followed by sequence diversification, to produce sequence-unrelated but structurally similar proteins.

Signatures of Post-Glacial Genetic Isolation and Human-Driven Migration in the Dothistroma Needle Blight Pathogen in Western Canada.

Many current tree improvement programs are incorporating assisted gene flow strategies to match reforestation efforts with future climates. This is the case for the lodgepole pine (Pinus contorta var. latifolia), the most extensively planted tree in western Canada. Knowledge of the structure and origin of pathogen populations associated with this tree would help improve the breeding effort. Recent outbreaks of the Dothistroma needle blight (DNB) pathogen Dothistroma septosporum on lodgepole pine in British Columbia and its discovery in Alberta plantations raised questions about the diversity and population structure of this pathogen in western Canada. Using genotyping-by-sequencing on 119 D. septosporum isolates from 16 natural pine populations and plantations from this area, we identified four genetic lineages, all distinct from the other DNB lineages from outside of North America. Modeling of the population history indicated that these lineages diverged between 31.4 and 7.2 thousand years ago, coinciding with the last glacial maximum and the postglacial recolonization of lodgepole pine in western North America. The lineage found in the Kispiox Valley from British Columbia, where an unprecedented DNB epidemic occurred in the 1990s, was close to demographic equilibrium and displayed a high level of haplotypic diversity. Two lineages found in Alberta and Prince George (British Columbia) showed departure from random mating and contemporary gene flow, likely resulting from pine breeding activities and material exchanges in these areas. The increased movement of planting material could have some major consequences by facilitating secondary contact between genetically isolated DNB lineages, possibly resulting in new epidemics.

Camellia Plant Resistance and Susceptibility to Petal Blight Disease Are Defined by the Timing of Defense Responses.

The family Sclerotiniaceae includes important phytopathogens, such as Botrytis cinerea and Sclerotinia sclerotiorum, that activate plant immune responses to facilitate infection propagation. The mechanisms of plant resistance to these necrotrophic pathogens are still poorly understood. To discover mechanisms of resistance, we used the Ciborinia camelliae (Sclerotiniaceae)-Camellia spp. pathosystem. This fungus induces rapid infection of the blooms of susceptible cultivar Nicky Crisp (Camellia japonica × Camellia pitardii var. pitardii), while Camellia lutchuensis is highly resistant. Genome-wide analysis of gene expression in resistant plants revealed fast modulation of host transcriptional activity 6 h after ascospore inoculation. Ascospores induced the same defense pathways in the susceptible Camellia cultivar but much delayed and coinciding with disease development. We next tested the hypothesis that differences in defense timing influences disease outcome. We induced early defense in the susceptible cultivar using methyl jasmonate and this strongly reduced disease development. Conversely, delaying the response in the resistant species, by infecting it with actively growing fungal mycelium, increased susceptibility. The same plant defense pathways, therefore, contribute to both resistance and susceptibility, suggesting that defense timing is a critical factor in plant health, and resistance against necrotrophic pathogens may occur during the initial biotrophy-like stages.

DsEcp2-1 is a polymorphic effector that restricts growth of Dothistroma septosporum in pine.

The detrimental effect of fungal pathogens on forest trees is an increasingly important problem that has implications for the health of our planet. Despite this, the study of molecular plant-microbe interactions in forest trees is in its infancy, and very little is known about the roles of effector molecules from forest pathogens. Dothistroma septosporum causes a devastating needle blight disease of pines, and intriguingly, is closely related to Cladosporium fulvum, a tomato pathogen in which pioneering effector biology studies have been carried out. Here, we studied D. septosporum effectors that are shared with C. fulvum, by comparing gene sequences from global isolates of D. septosporum and assessing effector function in both host and non-host plants. Many of the effectors were predicted to be non-functional in D. septosporum due to their pseudogenization or low expression in planta, suggesting adaptation to lifestyle and host. Effector sequences were polymorphic among a global collection of D. septosporum isolates, but there was no evidence for positive selection. The DsEcp2-1 effector elicited cell death in the non-host plant Nicotiana tabacum, whilst D. septosporum DsEcp2-1 mutants showed increased colonization of pine needles. Together these results suggest that DsEcp2-1 might be recognized by an immune receptor in both angiosperm and gymnosperm plants. This work may lead to the identification of plant targets for DsEcp2-1 that will provide much needed information on the molecular basis of gymnosperm-pathogen interactions in forests, and may also lead to novel methods of disease control.

Chromatin-level regulation of the fragmented dothistromin gene cluster in the forest pathogen Dothistroma septosporum.

Genes required for fungal secondary metabolite production are usually clustered, co-regulated and expressed in stationary growth phase. Chromatin modification has an important role in co-regulation of secondary metabolite genes. The virulence factor dothistromin, a relative of aflatoxin, provided a unique opportunity to study chromatin level regulation in a highly fragmented gene cluster that is switched on during early exponential growth phase. We analysed three histone modification marks by ChIP-qPCR and gene deletion in the pine pathogen Dothistroma septosporum to determine their effects on dothistromin gene expression across a time course and at different loci of the dispersed gene cluster. Changes in gene expression and dothistromin production were associated with changes in histone marks, with higher acetylation (H3K9ac) and lower methylation (H3K9me3, H3K27me3) during early exponential phase at the onset of dothistromin production. But while H3K27me3 directly influenced dothistromin genes dispersed across chromosome 12, effects of H3K9 acetylation and methylation were orchestrated mainly through a centrally located pathway regulator gene DsAflR. These results revealed that secondary metabolite production can be controlled at the chromatin-level despite the genes being dispersed. They also suggest that patterns of chromatin modification are important in adaptation of a virulence factor for a specific role in planta.

Specific Hypersensitive Response-Associated Recognition of New Apoplastic Effectors from Cladosporium fulvum in Wild Tomato.

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.

Evolution of polyketide synthesis in a Dothideomycete forest pathogen.

Fungal secondary metabolites have many important biological roles and some, like the toxic polyketide aflatoxin, have been intensively studied at the genetic level. Complete sets of polyketide synthase (PKS) genes can now be identified in fungal pathogens by whole genome sequencing and studied in order to predict the biosynthetic potential of those fungi. The pine needle pathogen Dothistroma septosporum is predicted to have only three functional PKS genes, a small number for a hemibiotrophic fungus. One of these genes is required for production of dothistromin, a polyketide virulence factor related to aflatoxin, whose biosynthetic genes are dispersed across one chromosome rather than being clustered. Here we evaluated the evolution of the other two genes, and their predicted gene clusters, using phylogenetic and population analyses. DsPks1 and its gene cluster are quite conserved amongst related fungi, whilst DsPks2 appears to be novel. The DsPks1 protein was predicted to be required for dihydroxynaphthalene (DHN) melanin biosynthesis but functional analysis of DsPks1 mutants showed that D. septosporum produced mainly dihydroxyphenylalanine (DOPA) melanin, which is produced by a PKS-independent pathway. Although the secondary metabolites made by these two PKS genes are not known, comparisons between strains of D. septosporum from different regions of the world revealed that both PKS core genes are under negative selection and we suggest they may have important cryptic roles in planta.

LaeA negatively regulates dothistromin production in the pine needle pathogen Dothistroma septosporum.

In filamentous fungi both pathway-specific and global regulators regulate genes involved in the biosynthesis of secondary metabolites. LaeA is a global regulator that was named for its mutant phenotype, loss of aflR expression, due to its effect on the aflatoxin-pathway regulator AflR in Aspergillus spp. The pine needle pathogen Dothistroma septosporum produces a polyketide virulence factor, dothistromin, that is chemically related to aflatoxin and whose pathway genes are also regulated by an ortholog of AflR. However, dothistromin biosynthesis is distinctive because it is switched on during early (rather than late) exponential growth phase and the genes are dispersed in six loci across one chromosome instead of being clustered. It was therefore of interest to determine whether the function of the global regulator LaeA is conserved in D. septosporum. To address this question, a LaeA ortholog (DsLaeA) was identified and its function analyzed in D. septosporum. In contrast to aflatoxin production in Aspergillus spp., deletion of DsLaeA resulted in enhanced dothistromin production and increased expression of the pathway regulatory gene DsAflR. Although expression of other putative secondary metabolite genes in D. septosporum showed a range of different responses to loss of DsLaeA function, thin layer chromatography revealed increased levels of a previously unknown metabolite in DsLaeA mutants. In addition, these mutants exhibited reduced asexual sporulation, germination and hydrophobicity. Our data suggest that although the developmental regulatory role of DsLaeA is conserved, its role in the regulation of secondary metabolism differs from that of LaeA in A. nidulans and appears to be species specific. This study provides a step towards understanding fundamental differences in regulation of clustered and fragmented groups of secondary metabolite genes that may shed light on understanding functional adaptation in secondary metabolism.

The genomes of the fungal plant pathogens Cladosporium fulvum and Dothistroma septosporum reveal adaptation to different hosts and lifestyles but also signatures of common ancestry.

We sequenced and compared the genomes of the Dothideomycete fungal plant pathogens Cladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu >61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an α-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulation.

Diverse lifestyles and strategies of plant pathogenesis encoded in the genomes of eighteen Dothideomycetes fungi.

The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress.

Dothistromin genes at multiple separate loci are regulated by AflR.

In fungi, genes involved in the production of secondary metabolites are generally clustered at one location. There are some exceptions, such as genes required for synthesis of dothistromin, a toxin that is a chemical analog of the aflatoxin precursor versicolorin A and made by the pine needle pathogen Dothistroma septosporum. The availability of the D. septosporum genome sequence enabled identification of putative dothistromin genes, including an ortholog of the aflatoxin regulatory gene AflR, and revealed that most of the genes are spread over six separate regions (loci) on chromosome 12 (1.3 Mb). Here we show that levels of expression of the widely dispersed genes in D. septosporum are not correlated with gene location with respect to their distance from a telomere, but that AflR regulates them. The production of dothistromin by D. septosporum in which the AflR gene was knocked out (ΔDsAflR) was drastically reduced, but still detectable. This is in contrast to orthologous ΔAflR mutants in Aspergillus species that lack any aflatoxin production. Expression patterns in ΔDsAflR mutants helped to predict the complete set of genes involved in dothistromin production. This included a short-chain aryl alcohol dehydrogenase (NorB), which is located on chromosome 11 rather than chromosome 12, but was 24-fold down regulated in ΔDsAflR. An orthologous set of dothistromin genes, organized in a similar fragmented cluster arrangement to that seen in D. septosporum, was found in the closely related tomato pathogen Cladosporium fulvum even though this species does not produce dothistromin. In C. fulvum, pseudogenization of key biosynthetic genes explains the lack of dothistromin production. The fragmented arrangement of dothistromin genes provides an example of coordinated control of a dispersed set of secondary metabolite genes; it also provides an example where loss of dothistromin production might have allowed adaptation to a new pathogenic lifestyle.

Fragmentation of an aflatoxin-like gene cluster in a forest pathogen.

Plant pathogens use a complex arsenal of weapons, such as toxic secondary metabolites, to invade and destroy their hosts. Knowledge of how secondary metabolite pathways evolved is central to understanding the evolution of host specificity. The secondary metabolite dothistromin is structurally similar to aflatoxins and is produced by the fungal pine pathogen Dothistroma septosporum. Our study focused on dothistromin genes, which are widely dispersed across one chromosome, to determine whether this unusual distributed arrangement evolved from an ancestral cluster. We combined comparative genomics and population genetics approaches to elucidate the origins of the dispersed arrangement of dothistromin genes over a broad evolutionary time-scale at the phylum, class and species levels. Orthologs of dothistromin genes were found in two major classes of fungi. Their organization is consistent with clustering of core pathway genes in a common ancestor, but with intermediate cluster fragmentation states in the Dothideomycetes fungi. Recombination hotspots in a D. septosporum population matched sites of gene acquisition and cluster fragmentation at higher evolutionary levels. The results suggest that fragmentation of a larger ancestral cluster gave rise to the arrangement seen in D. septosporum. We propose that cluster fragmentation may facilitate metabolic retooling and subsequent host adaptation of plant pathogens.

Ciborinia camelliae (Sclerotiniaceae) induces variable plant resistance responses in selected species of Camellia.

Ciborinia camelliae is the causal agent of Camellia flower blight. This fungal pathogen is a significant pest of the Camellia floriculture industry because it specifically infects the floral tissue of ornamental camellia cultivars leading to the rapid development of necrotic lesions and blight. This study aims to characterize natural resistance to Ciborinia camelliae within a selection of Camellia spp. Based on macroscopic lesion development, Camellia 'Nicky Crisp' and Camellia lutchuensis were chosen as compatible and incompatible hosts, respectively. Microscopic analyses of the incompatible Camellia lutchuensis-Ciborinia camelliae interaction revealed several hallmarks of induced plant resistance, including papillae formation, H2O2 accumulation, and localized cell death. The compatible Camellia Nicky Crisp-Ciborinia camelliae interaction failed to trigger a similar resistance response. Ciborinia camelliae growth in compatible tissue demonstrated a switch from biotrophy to necrotrophy, evident from the simultaneous development of secondary hyphae and necrotic lesions. Extension of resistance analyses to 39 additional Camellia spp. identified variable levels of resistance within the Camellia genus. The evidence presented supports a resistance breeding strategy for controlling Ciborinia camelliae on ornamental Camellia hybrids.

Cell Wall Carbohydrate Dynamics during the Differentiation of Infection Structures by the Apple Scab Fungus, Venturia inaequalis.

Scab, caused by the biotrophic fungal pathogen Venturia inaequalis, is the most economically important disease of apples. During infection, V. inaequalis colonizes the subcuticular host environment, where it develops specialized infection structures called runner hyphae and stromata. These structures are thought to be involved in nutrient acquisition and effector (virulence factor) delivery, but also give rise to conidia that further the infection cycle. Despite their importance, very little is known about how these structures are differentiated. Likewise, nothing is known about how these structures are protected from host defenses or recognition by the host immune system. To better understand these processes, we first performed a glycosidic linkage analysis of sporulating tubular hyphae from V. inaequalis developed in culture. This analysis revealed that the V. inaequalis cell wall is mostly composed of glucans (44%) and mannans (37%), whereas chitin represents a much smaller proportion (4%). Next, we used transcriptomics and confocal laser scanning microscopy to provide insights into the cell wall carbohydrate composition of runner hyphae and stromata. These analyses revealed that, during subcuticular host colonization, genes of V. inaequalis putatively associated with the biosynthesis of immunogenic carbohydrates, such as chitin and ß-1,6-glucan, are downregulated relative to growth in culture, while on the surface of runner hyphae and stromata, chitin is deacetylated to the less-immunogenic carbohydrate chitosan. These changes are anticipated to enable the subcuticular differentiation of runner hyphae and stromata by V. inaequalis, as well as to protect these structures from host defenses and recognition by the host immune system. IMPORTANCE Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control. A key feature of these pathogens is their ability to differentiate specialized subcuticular infection structures that, to date, remain largely understudied. This is typified by Venturia inaequalis, which causes scab, the most economically important disease of apples. In this study, we show that, during subcuticular host colonization, V. inaequalis downregulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and ß-1,6-glucan, and coats its subcuticular infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable host colonization by V. inaequalis and provide a foundation for understanding subcuticular host colonization by other plant-pathogenic fungi. Such an understanding is important, as it may inform the development of novel control strategies against subcuticular plant-pathogenic fungi.

Beyond the genomes of Fulvia fulva (syn. Cladosporium fulvum) and Dothistroma septosporum: New insights into how these fungal pathogens interact with their host plants.

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.

The veA gene of the pine needle pathogen Dothistroma septosporum regulates sporulation and secondary metabolism.

Fungi possess genetic systems to regulate the expression of genes involved in complex processes such as development and secondary metabolite biosynthesis. The product of the velvet gene veA, first identified and characterized in Aspergillus nidulans, is a key player in the regulation of both of these processes. Since its discovery and characterization in many Aspergillus species, VeA has been found to have similar functions in other fungi, including the Dothideomycete Mycosphaerella graminicola. Another Dothideomycete, Dothistroma septosporum, is a pine needle pathogen that produces dothistromin, a polyketide toxin very closely related to aflatoxin (AF) and sterigmatocystin (ST) synthesized by Aspergillus spp. Dothistromin is unusual in that, unlike most other secondary metabolites, it is produced mainly during the early exponential growth phase in culture. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of AF/ST in Aspergillus spp. To begin to address this question, a veA ortholog was identified and its function analyzed in D. septosporum. Inactivation of the veA gene resulted in reduced dothistromin production and a corresponding decrease in expression of dothistromin biosynthetic genes. Expression of other putative secondary metabolite genes in D. septosporum such as polyketide synthases and non-ribosomal peptide synthases showed a range of different responses to loss of Ds-veA. Asexual sporulation was also significantly reduced in the mutants, accompanied by a reduction in the expression of a putative stuA regulatory gene. The mutants were, however, able to infect Pinus radiata seedlings and complete their life cycle under laboratory conditions. Overall this work suggests that D. septosporum has a veA ortholog that is involved in the control of both developmental and secondary metabolite biosynthetic pathways.

Targeted Gene Mutations in the Forest Pathogen Dothistroma septosporum Using CRISPR/Cas9.

Dothistroma needle blight, caused by Dothistroma septosporum, has increased in incidence and severity over the last few decades and is now one of the most important global diseases of pines. Disease resistance breeding could be accelerated by knowledge of pathogen virulence factors and their host targets. However, this is hindered due to inefficient targeted gene disruption in D. septosporum, which is required for virulence gene characterisation. Here we report the first successful application of CRISPR/Cas9 gene editing to a Dothideomycete forest pathogen, D. septosporum. Disruption of the dothistromin pathway regulator gene AflR, with a known phenotype, was performed using nonhomologous end-joining repair with an efficiency of > 90%. Transformants with a range of disruption mutations in AflR were produced. Disruption of Ds74283, a D. septosporum gene encoding a secreted cell death elicitor, was also achieved using CRISPR/Cas9, by using a specific donor DNA repair template to aid selection where the phenotype was unknown. In this case, 100% of screened transformants were identified as disruptants. In establishing CRISPR/Cas9 as a tool for gene editing in D. septosporum, our research could fast track the functional characterisation of candidate virulence factors in D. septosporum and helps set the foundation for development of this technology in other forest pathogens.

Characterization of two conserved cell death elicitor families from the Dothideomycete fungal pathogens Dothistroma septosporum and Fulvia fulva (syn. Cladosporium fulvum).

Dothistroma septosporum (Ds) and Fulvia fulva (Ff; previously called Cladosporium fulvum) are two closely related Dothideomycete fungal species that cause Dothistroma needle blight in pine and leaf mold in tomato, respectively. During host colonization, these pathogens secrete virulence factors termed effectors to promote infection. In the presence of corresponding host immune receptors, however, these effectors activate plant defenses, including a localized cell death response that halts pathogen growth. We identified two apoplastic effector protein families, Ecp20 and Ecp32, which are conserved between the two pathogens. The Ecp20 family has four paralogues in both species, while the Ecp32 family has four paralogues in D. septosporum and five in F. fulva. Both families have members that are highly expressed during host infection. Members of the Ecp20 family have predicted structural similarity to proteins with a ß-barrel fold, including the Alt a 1 allergen from Alternaria alternata, while members of the Ecp32 family have predicted structural similarity to proteins with a ß-trefoil fold, such as trypsin inhibitors and lectins. Using Agrobacterium tumefaciens-mediated transient transformation assays, each family member was assessed for its ability to trigger cell death in leaves of the non-host species Nicotiana benthamiana and N. tabacum. Using this approach, FfEcp20-2, DsEcp20-3, and FfEcp20-3 from the Ecp20 family, and all members from the Ecp32 family, except for the Ds/FfEcp32-4 pair, triggered cell death in both species. This cell death was dependent on secretion of the effectors to the apoplast. In line with recognition by an extracellular immune receptor, cell death triggered by Ds/FfEcp20-3 and FfEcp32-3 was compromised in N. benthamiana silenced for BAK1 or SOBIR1, which encode extracellular co-receptors involved in transducing defense response signals following apoplastic effector recognition. We then investigated whether DsEcp20-3 and DsEcp20-4 triggered cell death in the host species Pinus radiata by directly infiltrating purified protein into pine needles. Strikingly, as in the non-host species, DsEcp20-3 triggered cell death, while DsEcp20-4 did not. Collectively, our study describes two new candidate effector families with cell death-eliciting activity from D. septosporum and F. fulva and provides evidence that members of these families are recognized by plant immune receptors.