Analysis of five near-complete genome assemblies of the tomato pathogen Cladosporium fulvum uncovers additional accessory chromosomes and structural variations induced by transposable elements effecting the loss of avirulence genes.

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.

First Draft Genome Resource for the Tomato Black Leaf Mold Pathogen Pseudocercospora fuligena.

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 N50 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.

A chromosome-scale genome assembly of the grape powdery mildew pathogen Erysiphe necator reveals its genomic architecture and previously unknown features of its biology.

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 graminis. 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.

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.

A chromosome-scale genome assembly of the tomato pathogen Cladosporium fulvum reveals a compartmentalized genome architecture and the presence of a dispensable chromosome.

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.

Characterization of the mitochondrial genomes of three powdery mildew pathogens reveals remarkable variation in size and nucleotide composition.

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.

The mitochondrial genome of the grape powdery mildew pathogen Erysiphe necator is intron rich and exhibits a distinct gene organization.

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.

Draft genome sequence of Xylaria sp., the causal agent of taproot decline of soybean in the southern United States.

The draft genome of Xylaria sp. isolate MSU_SB201401, causal agent of taproot decline of soybean in the southern U.S., is presented here. The genome assembly was 56.7 Mb in size with an L50 of 246. A total of 10,880 putative protein-encoding genes were predicted, including 647 genes encoding carbohydrate-active enzymes and 1053 genes encoding secreted proteins. This is the first draft genome of a plant-pathogenic Xylaria sp. associated with soybean. The draft genome of Xylaria sp. isolate MSU_SB201401 will provide an important resource for future experiments to determine the molecular basis of pathogenesis.

The genome sequence of Bipolaris cookei reveals mechanisms of pathogenesis underlying target leaf spot of sorghum.

Bipolaris cookei (=Bipolaris sorghicola) causes target leaf spot, one of the most prevalent foliar diseases of sorghum. Little is known about the molecular basis of pathogenesis in B. cookei, in large part due to a paucity of resources for molecular genetics, such as a reference genome. Here, a draft genome sequence of B. cookei was obtained and analyzed. A hybrid assembly strategy utilizing Illumina and Pacific Biosciences sequencing technologies produced a draft nuclear genome of 36.1 Mb, organized into 321 scaffolds with L50 of 31 and N50 of 378 kb, from which 11,189 genes were predicted. Additionally, a finished mitochondrial genome sequence of 135,790 bp was obtained, which contained 75 predicted genes. Comparative genomics revealed that B. cookei possessed substantially fewer carbohydrate-active enzymes and secreted proteins than closely related Bipolaris species. Novel genes involved in secondary metabolism, including genes implicated in ophiobolin biosynthesis, were identified. Among 37 B. cookei genes induced during sorghum infection, one encodes a putative effector with a limited taxonomic distribution among plant pathogenic fungi. The draft genome sequence of B. cookei provided novel insights into target leaf spot of sorghum and is an important resource for future investigation.

Comparative genomics of maize ear rot pathogens reveals expansion of carbohydrate-active enzymes and secondary metabolism backbone genes in Stenocarpella maydis.

Stenocarpella maydis is a plant pathogenic fungus that causes Diplodia ear rot, one of the most destructive diseases of maize. To date, little information is available regarding the molecular basis of pathogenesis in this organism, in part due to limited genomic resources. In this study, a 54.8 Mb draft genome assembly of S. maydis was obtained with Illumina and PacBio sequencing technologies, and analyzed. Comparative genomic analyses with the predominant maize ear rot pathogens Aspergillus flavus, Fusarium verticillioides, and Fusarium graminearum revealed an expanded set of carbohydrate-active enzymes for cellulose and hemicellulose degradation in S. maydis. Analyses of predicted genes involved in starch degradation revealed six putative α-amylases, four extracellular and two intracellular, and two putative γ-amylases, one of which appears to have been acquired from bacteria via horizontal transfer. Additionally, 87 backbone genes involved in secondary metabolism were identified, which represents one of the largest known assemblages among Pezizomycotina species. Numerous secondary metabolite gene clusters were identified, including two clusters likely involved in the biosynthesis of diplodiatoxin and chaetoglobosins. The draft genome of S. maydis presented here will serve as a useful resource for molecular genetics, functional genomics, and analyses of population diversity in this organism.

Taxonomic Resolution of the Nematophagous Fungal Isolate ARF18 via Genome Sequencing.

The taxonomically uncharacterized nematophagous fungus ARF18, which parasitizes cysts, juveniles, and adults of the soybean cyst nematode (Heterodera glycines), was proposed as a nematode biological control agent in 1991. A 46.3-Mb draft genome sequence of this fungus is presented, and a tentative taxonomic identification as a novel species of Brachyphoris is proposed.