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Fungi are a diverse kingdom characterized by remarkable genomic plasticity that facilitates pathogenicity and adaptation to adverse environmental conditions. In this review, we delve into the dynamic organization of fungal genomes and its implications for host adaptation and antifungal resistance. We examine key features and the heterogeneity of genomes across different fungal species, including but not limited to their chromosome content, DNA composition, distribution and arrangement of their content across chromosomes, and other major traits. We further highlight how this variability in genomic traits influences their virulence and adaptation to adverse conditions. Fungal genomes exhibit large variations in size, gene content, and structural features, such as abundance of transposable elements (TEs), compartmentalization into gene-rich and TE-rich regions, and the presence or absence of dispensable chromosomes. Genomic structural variations are equally diverse in fungi, ranging from whole-chromosome duplications that may enhance tolerance to antifungal compounds, to targeted deletion of effector encoding genes that may promote virulence. Finally, the often-overlooked fungal mitochondrial genomes can also affect virulence and resistance to fungicides. Such and other features of fungal genome organization are reviewed and discussed in the context of host-microbe interactions and antifungal resistance., Competing Interests: Conflict of interest Authors declare that they have no competing interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Fungal plant pathogens have dynamic genomes that allow them to rapidly adapt to adverse conditions and overcome host resistance. One way by which this dynamic genome plasticity is expressed is through effector gene loss, which enables plant pathogens to overcome recognition by cognate resistance genes in the host. However, the exact nature of these loses remains elusive in many fungi. This includes the tomato pathogen Cladosporium fulvum, which is the first fungal plant pathogen from which avirulence (Avr) genes were ever cloned and in which loss of Avr genes is often reported as a means of overcoming recognition by cognate tomato Cf resistance genes. A recent near-complete reference genome assembly of C. fulvum isolate Race 5 revealed a compartmentalized genome architecture and the presence of an accessory chromosome, thereby creating a basis for studying genome plasticity in fungal plant pathogens and its impact on avirulence genes., Results: Here, we obtained near-complete genome assemblies of four additional C. fulvum isolates. The genome assemblies had similar sizes (66.96 to 67.78 Mb), number of predicted genes (14,895 to 14,981), and estimated completeness (98.8 to 98.9%). Comparative analysis that included the genome of isolate Race 5 revealed high levels of synteny and colinearity, which extended to the density and distribution of repetitive elements and of repeat-induced point (RIP) mutations across homologous chromosomes. Nonetheless, structural variations, likely mediated by transposable elements and effecting the deletion of the avirulence genes Avr4E, Avr5, and Avr9, were also identified. The isolates further shared a core set of 13 chromosomes, but two accessory chromosomes were identified as well. Accessory chromosomes were significantly smaller in size, and one carried pseudogenized copies of two effector genes. Whole-genome alignments further revealed genomic islands of near-zero nucleotide diversity interspersed with islands of high nucleotide diversity that co-localized with repeat-rich regions. These regions were likely generated by RIP, which generally asymmetrically affected the genome of C. fulvum., Conclusions: Our results reveal new evolutionary aspects of the C. fulvum genome and provide new insights on the importance of genomic structural variations in overcoming host resistance in fungal plant pathogens., (© 2024. The Author(s).)

Information on the presence and severity of grape powdery mildew (GPM), caused by Erysiphe necator , has long been used to guide management decisions. While recent advances in the available molecular diagnostic assays and particle samplers have made monitoring easier, there is still a need for more efficient field collection of E. necator . The use of vineyard worker gloves worn during canopy manipulation as a sampler (glove swab) of E. necator was compared with samples identified by visual assessment with subsequent molecular confirmation (leaf swabs) and airborne spore samples collected by rotating-arm impaction traps (impaction traps). Samples from United States commercial vineyards in Oregon, Washington, and California were analyzed using two TaqMan qPCR assays targeting the internal transcribed spacer regions or cytochrome b gene of E. necator . Based on qPCR assays, visual disease assessments misidentified GPM up to 59% of the time with a higher frequency of misidentification occurring earlier in the growing season. Comparison of the aggregated leaf swab results for a row ( n = 915) to the row's corresponding glove swab had 60% agreement. The latent class analysis (LCA) indicated that glove swabs were more sensitive than leaf swabs in detecting E. necator presence. The impaction trap results had 77% agreement to glove swabs ( n = 206) taken from the same blocks. The LCAs estimated that the glove swabs and impaction trap samplers varied each year in which was more sensitive for detection. This likely indicates that these methods have similar levels of uncertainty and provide equivalent information. Additionally, all samplers, once E. necator was detected, were similarly sensitive and specific for detection of the A-143 resistance allele. Together, these results suggest that glove swabs are an effective sampling method for monitoring the presence of E. necator and, subsequently, the G143A amino acid substitution associated with resistance to quinone outside inhibitor fungicides in vineyards. Glove swabs could reduce sampling costs due to the lack of need for specialized equipment and time required for swab collection and processing ., Competing Interests: The author(s) declare no conflict of interest.

Erysiphe necator is an obligate fungal pathogen that causes grape powdery mildew, globally the most important disease on grapevines. Previous attempts to obtain a quality genome assembly for this pathogen were hindered by its high repetitive DNA content. Here, chromatin conformation capture (Hi-C) with long-read PacBio sequencing was combined to obtain a chromosome-scale assembly and a high-quality annotation for E. necator isolate EnFRAME01. The resulting 81.1 Mb genome assembly is 98% complete and consists of 34 scaffolds, 11 of which represent complete chromosomes. All chromosomes contain large centromeric-like regions and lack synteny to the 11 chromosomes of the cereal PM pathogen Blumeria gramini s. Further analysis of their composition showed that repeats and transposable elements (TEs) occupy 62.7% of their content. TEs were almost evenly interspersed outside centromeric and telomeric regions and massively overlapped with regions of annotated genes, suggesting that they could have a significant functional impact. Abundant gene duplicates were observed as well, particularly in genes encoding candidate secreted effector proteins. Moreover, younger in age gene duplicates exhibited more relaxed selection pressure and were more likely to be located physically close in the genome than older duplicates. A total of 122 genes with copy number variations among six isolates of E. necator were also identified and were enriched in genes that were duplicated in EnFRAME01, indicating they may reflect an adaptive variation. Taken together, our study illuminates higher-order genomic architectural features of E. necator and provides a valuable resource for studying genomic structural variations in this pathogen. IMPORTANCE Grape powdery mildew caused by the ascomycete fungus Erysiphe necator is economically the most important and recurrent disease in vineyards across the world. The obligate biotrophic nature of E. necator hinders the use of typical genetic methods to elucidate its pathogenicity and adaptation to adverse conditions, and thus comparative genomics has been a major method to study its genome biology. However, the current reference genome of E. necator isolate C-strain is highly fragmented with many non-coding regions left unassembled. This incompleteness prohibits in-depth comparative genomic analyses and the study of genomic structural variations (SVs) that are known to affect several aspects of microbial life, including fitness, virulence, and host adaptation. By obtaining a chromosome-scale genome assembly and a high-quality gene annotation for E. necator , we reveal the organization of its chromosomal content, unearth previously unknown features of its biology, and provide a reference for studying genomic SVs in this pathogen., Competing Interests: The authors declare no conflict of interest.

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., (© 2023 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Succinate dehydrogenase inhibitors (SDHIs) are fungicides used in control of numerous fungal plant pathogens, including Erysiphe necator , the causal agent of grapevine powdery mildew (GPM). Here, the sdhb , sdhc , and sdhd genes of E. necator were screened for mutations that may be associated with SDHI resistance. GPM samples were collected from 2017 to 2020 from the U.S. states of California, Oregon, Washington, and Michigan, and the Canadian province of British Columbia. Forty-five polymorphisms were identified in the three sdh genes, 17 of which caused missense mutations. Of these, the SDHC-p.I244V substitution was shown in this study to reduce sensitivity of E. necator to boscalid and fluopyram, whereas the SDHC-p.G25R substitution did not affect SDHI sensitivity. Of the other 15 missense mutations, the SDHC-p.H242R substitution was shown in previous studies to reduce sensitivity of E. necator toward boscalid, whereas the equivalents of the SDHB-p.H242L, SDHC-p.A83V, and SDHD-p.I71F substitutions were shown to reduce sensitivity to SDHIs in other fungi. Generally, only a single amino acid substitution was present in the SDHB, SDHC, or SDHD subunit of E. necator isolates, but missense mutations putatively associated with SDHI resistance were widely distributed in the sampled areas and increased in frequency over time. Finally, isolates that had decreased sensitivity to boscalid or fluopyram were identified but with no or only the SDHC-p.G25R amino acid substitution present in SDHB, SDHC, and SDHD subunits. This suggests that target site mutations probably are not the only mechanism conferring resistance to SDHIs in E. necator.

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

Powdery mildews comprise a large group of economically important phytopathogenic fungi. However, limited information exists on their mitochondrial genomes. Here, we assembled and compared the mitochondrial genomes of the powdery mildew pathogens Blumeria graminis f. sp. tritici , Erysiphe pisi , and Golovinomyces cichoracearum . Included in the comparative analysis was also the mitochondrial genome of Erysiphe necator that was previously analysed. The mitochondrial genomes of the four Erysiphales exhibit a similar gene content and organization but a large variation in size, with sizes ranging from 109800 bp in B. graminis f. sp. tritici to 332165 bp in G. cichoracearum , which is the largest mitochondrial genome of a fungal pathogen reported to date. Further comparative analysis revealed an unusual bimodal GC distribution in the mitochondrial genomes of B. graminis f. sp. tritici and G. cichoracearum that was not previously observed in fungi. The cytochrome b ( cob ) genes of E. necator, E. pisi , and G. cichoracearum were also exceptionally rich in introns, which in turn harboured rare open reading frames encoding reverse transcriptases that were likely acquired horizontally. Golovinomyces cichoracearum had also the longest cob gene (45 kb) among 703 fungal cob genes analysed. Collectively, these results provide novel insights into the organization of mitochondrial genomes of powdery mildew pathogens and represent valuable resources for population genetics and evolutionary studies.

Powdery mildews are notorious fungal plant pathogens but only limited information exists on their genomes. Here we present the mitochondrial genome of the grape powdery mildew fungus Erysiphe necator and a high-quality mitochondrial gene annotation generated through cloning and Sanger sequencing of full-length cDNA clones. The E. necator mitochondrial genome consists of a circular DNA sequence of 188,577 bp that harbors a core set of 14 protein-coding genes that are typically present in fungal mitochondrial genomes, along with genes encoding the small and large ribosomal subunits, a ribosomal protein S3, and 25 mitochondrial-encoded transfer RNAs (mt-tRNAs). Interestingly, it also exhibits a distinct gene organization with atypical bicistronic-like expression of the nad4L/nad5 and atp6/nad3 gene pairs, and contains a large number of 70 introns, making it one of the richest in introns mitochondrial genomes among fungi. Sixty-four intronic ORFs were also found, most of which encoded homing endonucleases of the LAGLIDADG or GIY-YIG families. Further comparative analysis of five E. necator isolates revealed 203 polymorphic sites, but only five were located within exons of the core mitochondrial genes. These results provide insights into the organization of mitochondrial genomes of powdery mildews and represent valuable resources for population genetic and evolutionary studies.

Background: Pseudocercospora fijiensis is the causal agent of the black leaf streak disease (BLSD) of banana. Bananas are important global export commodities and a major staple food. Their susceptibility to BLSD pushes disease management towards excessive fungicide use, largely relying on multisite inhibitors and sterol demethylation inhibitors (DMIs). These fungicides are ubiquitous in plant disease control, targeting the CYP51 enzyme. We examined sensitivity to DMIs in P. fijiensis field isolates collected from various major banana production zones in Colombia, Costa Rica, Dominican Republic, Ecuador, the Philippines, Guadalupe, Martinique and Cameroon and determined the underlying genetic reasons for the observed phenotypes., Results: We observed a continuous range of sensitivity towards the DMI fungicides difenoconazole, epoxiconazole and propiconazole with clear cross-sensitivity. Sequence analyses of PfCYP51 in 266 isolates showed 28 independent amino acid substitutions, nine of which correlated with reduced sensitivity to DMIs. In addition to the mutations, we observed up to six insertions in the Pfcyp51 promoter. Such promoter insertions contain repeated elements with a palindromic core and correlate with the enhanced expression of Pfcyp51 and hence with reduced DMI sensitivity. Wild-type isolates from unsprayed bananas fields did not contain any promoter insertions., Conclusion: The presented data significantly contribute to understanding of the evolution and global distribution of DMI resistance mechanisms in P. fijiensis field populations and facilitate the prediction of different DMI efficacy. The overall reduced DMI sensitivity calls for the deployment of a wider range of solutions for sustainable control of this major banana disease. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

The bacterial strain Collimonas fungivorans Ter331 (C f Ter331) inhibits mycelial growth and spore germination in Aspergillus niger N402 ( An N402). The mechanisms underlying this antagonistic bacterial-fungal interaction have been extensively studied, but knowledge on the long-term outcome of this interaction is currently lacking. Here, we used experimental evolution to explore the dynamics of fungal adaptation to recurrent exposure to Cf Ter331. Specifically, five single-spore isolates (SSIs) of An N402 were evolved under three selection scenarios in liquid culture, i.e., (i) in the presence of Cf Ter331 for 80 growth cycles, (ii) in the absence of the bacterium for 80 cycles, and (iii) in the presence of Cf Ter331 for 40 cycles and then in its absence for 40 cycles. The evolved SSI lineages were then evaluated for phenotypic changes from the founder fungal strain, such as germinability with or without Cf Ter331. The analysis showed that recurrent exposure to Cf Ter331 selected for fungal lineages with reduced germinability and slower germination, even in the absence of Cf Ter331. In contrast, when An N402 evolved in the absence of the bacteria, lineages with increased germinability and faster germination were favored. SSIs that were first evolved in the presence of Cf Ter331 and then in its absence showed intermediate phenotypes but overall were more similar to SSIs that evolved in the absence of Cf Ter331 for 80 cycles. This suggests that traits acquired from exposure to Cf Ter331 were reversible upon removal of the selection pressure. Overall, our study provides insights into the effects on fungi from the long-term coculture with bacteria. IMPORTANCE The use of antagonistic bacteria for managing fungal diseases is becoming increasingly popular, and thus there is a need to understand the implications of their long-term use against fungi. Most efforts have so far focused on characterizing the antifungal properties and mode of action of the bacterial antagonists, but the possible outcomes of the persisting interaction between antagonistic bacteria and fungi are not well understood. In this study, we used experimental evolution in order to explore the evolutionary aspects of an antagonistic bacterial-fungal interaction, using the antifungal bacterium Collimonas fungivorans and the fungus Aspergillus niger as a model system. We show that evolution in the presence or absence of the bacteria selects for fungal lineages with opposing and conditionally beneficial traits, such as slow and fast spore germination, respectively. Overall, our studies reveal that fungal responses to biotic factors related to antagonism could be to some extent predictable and reversible.

Effectors are small, secreted proteins that promote pathogen virulence. Although key to microbial infections, unlocking the intrinsic function of effectors remains a challenge. We have previously shown that members of the fungal Avr4 effector family use a carbohydrate-binding module of family 14 (CBM14) to bind chitin in fungal cell walls and protect them from host chitinases during infection. Here, we show that gene duplication in the Avr4 family produced an Avr4-2 paralog with a previously unknown effector function. Specifically, we functionally characterize Pf Avr4-2, a paralog of Pf Avr4 in the tomato pathogen Pseudocercospora fuligena , and show that although it contains a CBM14 domain, it does not bind chitin or protect fungi against chitinases. Instead, Pf Avr4-2 interacts with highly de-esterified pectin in the plant's middle lamellae or primary cell walls and interferes with Ca 2+ -mediated cross-linking at cell-cell junction zones, thus loosening the plant cell wall structure and synergizing the activity of pathogen secreted endo-polygalacturonases., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Exploiting RNA interference (RNAi) in disease control through non-transformative methods that overcome the hurdle of producing transgenic plants has attracted much attention over the last years. Here, we explored such a method and used non-pathogenic bacteria as a versatile system for delivering RNAi to fungi. Specifically, the RNaseIII-null mutant strain of Escherichia coli HT115(DE3) was transformed with two plasmid vectors that enabled the constitutive or IPTG-inducible production of double-stranded RNAs (dsRNAs) against genes involved in aflatoxins production in Aspergillus flavus ( AflC ) or virulence of Botrytis cinerea ( BcSAS1 ). To facilitate the release of the dsRNAs, the bacterial cells were further genetically engineered to undergo a bacteriophage endolysin R-mediated autolysis, following a freeze-thaw cycle. Exposure under in vitro conditions of A. flavus or B. cinerea to living bacteria or their whole-cell autolysates induced silencing of AflC and BcSAS1 in a bacteria concentration-dependent manner, and instigated a reduction in aflatoxins production and mycelial growth, respectively. In planta applications of the living bacteria or their crude whole-cell autolysates produced similar results, thus creating a basis for translational research. These results demonstrate that bacteria can produce biologically active dsRNA against target genes in fungi and that bacteria-mediated RNAi can be used to control fungal pathogens.

Grapevine powdery mildew (GPM), caused by the fungus Erysiphe necator , is a constant threat to worldwide production of grape berries, requiring repeated use of fungicides for management. The frequent fungicide applications have resulted in resistance to commonly used quinone outside inhibitor (QoI) fungicides and the resistance is associated with single-nucleotide polymorphisms (SNPs) in the mitochondrial cytochrome b ). In this study, we attempted to detect the most common SNP causing a glycine to alanine substitution at amino acid position 143 (i.e., G143A) in the cytb ). In this study, we attempted to detect the most common SNP causing a glycine to alanine substitution at amino acid position 143 (i.e., G143A) in the cytb protein, to track this resistance using allele-specific TaqMan probe and digital-droplet PCR-based assays. Specificity and sensitivity of these assays showed that these two assays could discriminate SNPs and were effective on mixed samples. These diagnostic assays were implemented to survey E. necator samples collected from leaf and air samples from California and Oregon grape-growing regions. Sequencing of PCR amplicons and phenotyping of isolates also revealed that these assays accurately detected each allele (100% agreement), and there was an absolute agreement between the presence or absence of the G143A mutation and resistance to QoIs in the E. necator sampled. These results indicate that the developed diagnostic tools will help growers make informed decisions about fungicide selections and applications which, in turn, will facilitate GPM disease management and improve grape production systems.

Pseudocercospora fuligena is a fungus that causes black leaf mold, an important disease of tomato in tropical and subtropical regions of the world. Despite its economic importance, genomic resources for this pathogen are scarce and no reference genome was available thus far. Here, we report a 50.6-Mb genome assembly for P. fuligena, consisting of 348 contigs with an N 50 value of 0.407 Mb. In total, 13,764 protein-coding genes were predicted with an estimated BUSCO completeness of 98%. Among the predicted genes there were 179 candidate effectors, 445 carbohydrate-active enzymes, and 30 secondary metabolite gene clusters. The resources presented in this study will allow genome-wide comparative analyses and population genomic studies of this pathogen, ultimately improving management strategies for black leaf mold of tomato.

Collimonas fungivorans Ter331 (CfTer331) is a soil bacterium that produces collimomycin, a secondary metabolite that inhibits the vegetative growth of fungi. Here we show that CfTer331 can also interfere with fungal spore germination and that collimomycin biosynthesis is required for this activity. More specifically, in co-cultures of Aspergillus niger N402 (AnN402) co-nidiospores with CfTer331, the rate of transition from the isotropic to polarized stage of the germination process was reduced and the relatively few AnN402 conidiospores that completed the germination process were less likely to survive than those that were arrested in the isotropic phase. By contrast, a collimomycin-deficient mutant of CfTer331 had no effect on germination: in its presence, as in the absence or delayed presence of CfTer331, unhindered germination of conidiospores allowed rapid establishment of AnN402 mycelium and the subsequent acidification of the culture medium to the detriment of any bacteria present. However, when challenged early enough with CfTer331, the collimomycin-dependent arrest of the AnN402 germination process enabled CfTer331 to prevent AnN402 from forming mycelia and to gain dominance in the culture. We propose that the collimomycin-dependent arrest of spore germination represents an early intervention strategy used by CfTer331 to mitigate niche construction by fungi in nature., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Leveillula taurica is a major pathogen of tomato and several other crops that can cause substantial yield losses in favorable conditions for the fungus. Quinone outside inhibitor fungicides (Q o Is) are routinely used for the control of the pathogen in tomato fields across California, but their recurrent use could lead to the emergence of resistance against these compounds. Here, we partially cloned the cytochrome b gene from L. taurica ( Lt cytb ) and searched within populations of the fungus collected from tomato fields across California for mutations that confer resistance to Q o Is. A total of 21 single nucleotide polymorphisms (SNPs) were identified within a 704 bp fragment of the Lt cytb gene analyzed, of which five were non-synonymous substitutions. Among the most frequent SNPs encountered within field populations of the pathogen was the G143A substitution that confers high levels of resistance against Q o Is in several fungi. The other four amino acid substitutions were novel mutations, whose effect on Q o I resistance is currently unknown. Sequencing of the Lt cytb gene from individual single-cell conidia of the fungus further revealed that most SNPs, including the one leading to the G143A substitution, were present in a heteroplasmic state, indicating the co-existence of multiple mitotypes in single cells. Analysis of the field samples showed that the G143A substitution is predominantly heteroplasmic also within field populations of L. taurica in California, suggesting that Q o I resistance in this fungus is likely to be quantitative rather than qualitative.

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

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., (© 2017 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Pseudocercospora fijiensis , causal agent of the black Sigatoka disease (BSD) of Musa spp., has spread globally since its discovery in Fiji 1963 to all the banana and plantain growing areas across the globe. It is becoming the most damaging and economically important disease of this crop. The identification and characterization of genes that regulate infection processes and pathogenicity in P. fijiensis will provide important knowledge for the development of disease-resistant cultivars. In many fungal plant pathogens, the Fus3 and Slt2 are reported to be essential for pathogenicity. Fus3 regulates filamentous-invasion pathways including the formation of infection structures, sporulation, virulence, and invasive and filamentous growth, whereas Slt2 is involved in the cell-wall integrity pathway, virulence, invasive growth, and colonization in host tissues. Here, we used RNAi-mediated gene silencing to investigate the role of the Slt2 and Fus3 homologs in P. fijiensis in pathogen invasiveness, growth and pathogenicity. The PfSlt2 and PfFus3 silenced P. fijiensis transformants showed significantly lower gene expression and reduced virulence, invasive growth, and lower biomass in infected leaf tissues of East African Highland Banana (EAHB). This study suggests that Slt2 and Fus3 MAPK signaling pathways play important roles in plant infection and pathogenic growth of fungal pathogens. The silencing of these vital fungal genes through host-induced gene silencing (HIG) could be an alternative strategy for developing transgenic banana and plantain resistant to BSD.

Black Sigatoka disease, caused by Pseudocercospora fijiensis is a serious constraint to banana production worldwide. The disease continues to spread in new ecological niches and there is an urgent need to develop strategies for its control. The high osmolarity glycerol (HOG) pathway in Saccharomyces cerevisiae is well known to respond to changes in external osmolarity. HOG pathway activation leads to phosphorylation, activation and nuclear transduction of the HOG1 mitogen-activated protein kinases (MAPKs). The activated HOG1 triggers several responses to osmotic stress, including up or down regulation of different genes, regulation of protein translation, adjustments to cell cycle progression and synthesis of osmolyte glycerol. This study investigated the role of the MAPK-encoding PfHog1 gene on osmotic stress adaptation and virulence of P. fijie nsis. RNA interference-mediated gene silencing of PfHog1 significantly suppressed growth of P. fijiensis on potato dextrose agar media supplemented with 1 M NaCl, indicating that PfHog1 regulates osmotic stress. In addition, virulence of the PfHog1 -silenced mutants of P. fijiensis on banana was significantly reduced, as observed from the low rates of necrosis and disease development on the infected leaves. Staining with lacto phenol cotton blue further confirmed the impaired mycelial growth of the PfHog1 in the infected leaf tissues, which was further confirmed with quantification of the fungal biomass using absolute- quantitative PCR. Collectively, these findings demonstrate that PfHog1 plays a critical role in osmotic stress regulation and virulence of P. fijiensis on its host banana. Thus, PfHog1 could be an interesting target for the control of black Sigatoka disease in banana.

The Sigatoka disease complex, caused by the closely-related Dothideomycete fungi Pseudocercospora musae (yellow sigatoka), Pseudocercospora eumusae (eumusae leaf spot), and Pseudocercospora fijiensis (black sigatoka), is currently the most devastating disease on banana worldwide. The three species emerged on bananas from a recent common ancestor and show clear differences in virulence, with P. eumusae and P. fijiensis considered the most aggressive. In order to understand the genomic modifications associated with shifts in the species virulence spectra after speciation, and to identify their pathogenic core that can be exploited in disease management programs, we have sequenced and analyzed the genomes of P. eumusae and P. musae and compared them with the available genome sequence of P. fijiensis. Comparative analysis of genome architectures revealed significant differences in genome size, mainly due to different rates of LTR retrotransposon proliferation. Still, gene counts remained relatively equal and in the range of other Dothideomycetes. Phylogenetic reconstruction based on a set of 46 conserved single-copy genes strongly supported an earlier evolutionary radiation of P. fijiensis from P. musae and P. eumusae. However, pairwise analyses of gene content indicated that the more virulent P. eumusae and P. fijiensis share complementary patterns of expansions and contractions in core gene families related to metabolism and enzymatic degradation of plant cell walls, suggesting that the evolution of virulence in these two pathogens has, to some extent, been facilitated by convergent changes in metabolic pathways associated with nutrient acquisition and assimilation. In spite of their common ancestry and shared host-specificity, the three species retain fairly dissimilar repertoires of effector proteins, suggesting that they likely evolved different strategies for manipulating the host immune system. Finally, 234 gene families, including seven putative effectors, were exclusively present in the three Sigatoka species, and could thus be related to adaptation to the banana host.

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., Competing Interests: The authors have declared that no competing interests exist.