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April 21, 2020  |  

Transcriptome analysis reveals multiple signal network contributing to the Verticillium wilt resistance in eggplant

Verticillium wilt is a devastating disease in eggplants. In order to understand the molecular mechanism of disease resistance in eggplants, transcriptomes of Verticillium wilt infected eggplants were detected. A total of 480, 518, 887 and 1 046 Verticillium wilt related differentially expressed genes were identified at 6 (V6), 12 (V12), 24 (V24) and 48?h (V48), respectively. COG function classification revealed that most of DEGs functioned in “Amino acid transport and metabolism”, “Cytoskeleton” and “Cell motility”. In addition, compared the control plants (V0) to infected eggplants (V6-V48), a total of 111 common DEGs were identified. Except for “General function prediction only”, most of the DEGs enriched in “Signal transduction”. DEGs associated to different hormone signals, including GID1B, ROPGAP1, OPT3 and CDPK, were identified throughout the whole infection process. Cross-talk among defense signal pathways plays major roles in the Verticillium wilt disease resistance in eggplants.


April 21, 2020  |  

Global distribution of mating types shows limited opportunities for mating across populations of fungi causing boxwood blight disease.

Boxwood blight is a disease threat to natural and managed landscapes worldwide. To determine mating potential of the fungi responsible for the disease, Calonectria pseudonaviculata and C. henricotiae, we characterized their mating-type (MAT) loci. Genomes of C. henricotiae, C. pseudonaviculata and two other Calonectria species (C. leucothoes, C. naviculata) were sequenced and used to design PCR tests for mating-type from 268 isolates collected from four continents. All four Calonectria species have a MAT locus that is structurally consistent with the organization found in heterothallic ascomycetes, with just one idiomorph per individual isolate. Mating type was subdivided by species: all C. henricotiae isolates possessed the MAT1-1 idiomorph, whereas all C. pseudonaviculata isolates possessed the MAT1-2 idiomorph. To determine the potential for divergence at the MAT1 locus to present a barrier to interspecific hybridization, evolutionary analysis was conducted. Phylogenomic estimates showed that C. henricotiae and C. pseudonaviculata diverged approximately 2.1 Mya. However, syntenic comparisons, phylogenetic analyses, and estimates of nucleotide divergence across the MAT1 locus and proximal genes identified minimal divergence in this region of the genome. These results show that in North America and parts of Europe, where only C. pseudonaviculata resides, mating is constrained by the absence of MAT1-1. In regions of Europe where C. henricotiae and C. pseudonaviculata currently share the same host and geographic range, it remains to be determined whether or not these two recently diverged species are able to overcome species barriers to mate.Copyright © 2019 Elsevier Inc. All rights reserved.


April 21, 2020  |  

The complexity of the Sclerotinia sclerotiorum pathosystem in soybean: virulence factors, resistance mechanisms, and their exploitation to control Sclerotinia stem rot

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is a globally important, yield limiting disease of soybean. Progress has been made in our understanding of this pathosystem at the plant level, such as the key role of oxalic acid in disease development and the importance of cell wall-degrading enzymes and other secreted proteins. Unfortunately, advances have largely focused on the fungal side of this interaction and only provide glimpses into the plant mechanisms governing resistance to this pathogen. With the absence of commercially available resistant soybeans, chemical and cultural solutions are being used by farmers to manage SSR with limited success. Additional research is needed to identify S. sclerotiorum resistance mechanisms that can be exploited to improve genetic resistance in soybean and decrease reliance on spray regimes. Technologies such as transgenics and RNAi could be exploited to improve the level of resistance to S. sclerotiorum in soybean. This review offers insight into the hurdles of managing SSR at the plant level and potential solutions that might be adopted in the future.


April 21, 2020  |  

Interspecies conservation of organisation and function between nonhomologous regional centromeres.

Despite the conserved essential function of centromeres, centromeric DNA itself is not conserved. The histone-H3 variant, CENP-A, is the epigenetic mark that specifies centromere identity. Paradoxically, CENP-A normally assembles on particular sequences at specific genomic locations. To gain insight into the specification of complex centromeres, here we take an evolutionary approach, fully assembling genomes and centromeres of related fission yeasts. Centromere domain organization, but not sequence, is conserved between Schizosaccharomyces pombe, S. octosporus and S. cryophilus with a central CENP-ACnp1 domain flanked by heterochromatic outer-repeat regions. Conserved syntenic clusters of tRNA genes and 5S rRNA genes occur across the centromeres of S. octosporus and S. cryophilus, suggesting conserved function. Interestingly, nonhomologous centromere central-core sequences from S. octosporus and S. cryophilus are recognized in S. pombe, resulting in cross-species establishment of CENP-ACnp1 chromatin and functional kinetochores. Therefore, despite the lack of sequence conservation, Schizosaccharomyces centromere DNA possesses intrinsic conserved properties that promote assembly of CENP-A chromatin.


April 21, 2020  |  

Three phylogenetic groups have driven the recent population expansion of Cryptococcus neoformans.

Cryptococcus neoformans (C. neoformans var. grubii) is an environmentally acquired pathogen causing 181,000 HIV-associated deaths each year. We sequenced 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. The phylogeny of C. neoformans reveals a recent exponential population expansion, consistent with the increase in the number of susceptible hosts. In our study population, this expansion has been driven by three sub-clades of the C. neoformans VNIa lineage; VNIa-4, VNIa-5 and VNIa-93. These three sub-clades account for 91% of clinical isolates sequenced in our study. Combining the genome data with clinical information, we find that the VNIa-93 sub-clade, the most common sub-clade in Uganda and Malawi, was associated with better outcomes than VNIa-4 and VNIa-5, which predominate in Southeast Asia. This study lays the foundation for further work investigating the dominance of VNIa-4, VNIa-5 and VNIa-93 and the association between lineage and clinical phenotype.


April 21, 2020  |  

Genomic Analyses Reveal Evidence of Independent Evolution, Demographic History, and Extreme Environment Adaptation of Tibetan Plateau Agaricus bisporus.

Agaricus bisporus distributed in the Tibetan Plateau of China has high-stress resistance that is valuable for breeding improvements. However, its evolutionary history, specialization, and adaptation to the extreme Tibetan Plateau environment are largely unknown. Here, we performed de novo genome sequencing of a representative Tibetan Plateau wild strain ABM and comparative genomic analysis with the reported European strain H97 and H39. The assembled ABM genome was 30.4 Mb in size, and comprised 8,562 protein-coding genes. The ABM genome shared highly conserved syntenic blocks and a few inversions with H97 and H39. The phylogenetic tree constructed by 1,276 single-copy orthologous genes in nine fungal species showed that the Tibetan Plateau and European A. bisporus diverged ~5.5 million years ago. Population genomic analysis using genome resequencing of 29 strains revealed that the Tibetan Plateau population underwent significant differentiation from the European and American populations and evolved independently, and the global climate changes critically shaped the demographic history of the Tibetan Plateau population. Moreover, we identified key genes that are related to the cell wall and membrane system, and the development and defense systems regulated A. bisporus adapting to the harsh Tibetan Plateau environment. These findings highlight the value of genomic data in assessing the evolution and adaptation of mushrooms and will enhance future genetic improvements of A. bisporus.


April 21, 2020  |  

Characterization of the Castanopsis carlesii Deadwood Mycobiome by Pacbio Sequencing of the Full-Length Fungal Nuclear Ribosomal Internal Transcribed Spacer (ITS)

Short-read Next Generation Sequencing (NGS) platforms can easily and quickly generate thousands to hundreds of thousands of sequences per sample. However, the limited length of these sequences can cause problems during fungal taxonomic identification. Here we validate the use of Pacbio sequencing, a long-read NGS method, for characterizing the fungal community (mycobiome) of Castanopsis carlesii deadwood. We report the successful use of Pacbio sequencing to generate long-read sequences of the full-length (500 – 780 bp) fungal ITS regions of the Castanopsis carlesii mycobiome. Our results show that the studied deadwood mycobiome is taxonomically and functionally diverse, with an average of 85 fungal OTUs representing five functional groups (animal endosymbionts, endophytes, mycoparasites, plant pathogens, and saprotrophs). Based on relative abundance data, Basidiomycota were the most frequently detected phyla (50% of total sequences), followed by unidentified phyla and Ascomycota. However, based on presence/absence data, the most OTU-rich phyla were Ascomycota (58% of total OTUs, 72 OTUs) followed by Basidiomycota and unidentified phyla. The majority of fungal OTUs were identified as saprotrophs (70% of successfully function-assigned OTUs) followed by plant pathogens. Finally, we used phylogenetic analysis based on the full-length ITS sequences to confirm the species identification of 14/36 OTUs with high bootstrap support (99 – 100%). Based on the numbers of sequence reads obtained per sample, which ranged from 3,047 to 13,463, we conclude that Pacbio sequencing can be a powerful tool for characterizing moderate- and possibly high-complexity fungal communities.


April 21, 2020  |  

Hybridization is a recurrent evolutionary stimulus in wild yeast speciation.

Hybridization can result in reproductively isolated and phenotypically distinct lineages that evolve as independent hybrid species. How frequently hybridization leads to speciation remains largely unknown. Here we examine the potential recurrence of hybrid speciation in the wild yeast Saccharomyces paradoxus in North America, which comprises two endemic lineages SpB and SpC, and an incipient hybrid species, SpC*. Using whole-genome sequences from more than 300 strains, we uncover the hybrid origin of another group, SpD, that emerged from hybridization between SpC* and one of its parental species, the widespread SpB. We show that SpD has the potential to evolve as a novel hybrid species, because it displays phenotypic novelties that include an intermediate transcriptome profile, and partial reproductive isolation with its most abundant sympatric parental species, SpB. Our findings show that repetitive cycles of divergence and hybridization quickly generate diversity and reproductive isolation, providing the raw material for speciation by hybridization.


April 21, 2020  |  

Study of the whole genome, methylome and transcriptome of Cordyceps militaris.

The complete genome of Cordyceps militaris was sequenced using single-molecule real-time (SMRT) sequencing technology at a coverage over 300×. The genome size was 32.57?Mb, and 14 contigs ranging from 0.35 to 4.58?Mb with an N50 of 2.86?Mb were assembled, including 4 contigs with telomeric sequences on both ends and an additional 8 contigs with telomeric sequences on either the 5′ or 3′ end. A methylome database of the genome was constructed using SMRT and m4C and m6A methylated nucleotides, and many unknown modification types were identified. The major m6A methylation motif is GA and GGAG, and the major m4C methylation motif is GC or CG/GC. In the C. militaris genome DNA, there were four types of methylated nucleotides that we confirmed using high-resolution LCMS-IT-TOF. Using PacBio Iso-Seq, a total of 31,133 complete cDNA sequences were obtained in the fruiting body. The conserved domains of the nontranscribed regions of the genome include TATA boxes, which are the initial regions of genome replication. There were 406 structural variants between the HN and CM01 strains, and there were 1,114 structural variants between the HN and ATCC strains.


April 21, 2020  |  

Genomics-driven discovery of a biosynthetic gene cluster required for the synthesis of BII-Rafflesfungin from the fungus Phoma sp. F3723.

Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis.Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined.We describe the NRPS-t1PKS cluster ‘BIIRfg’ compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.


April 21, 2020  |  

De novo genome sequencing and secretome analysis of Tilletia indica inciting Karnal bunt of wheat provides pathogenesis-related genes.

Tilletia indica is an internationally quarantined fungal pathogen causing Karnal bunt of wheat. The present study carried out that the whole genome of T. indica was sequenced and identified transposable elements, pathogenicity-related genes using a comparative genomics approach. The T. indica genome assembly size of 33.7 MB was generated using Illumina and Pac Bio platforms with GC content of 55.0%. A total of 1737 scaffolds were obtained with N50 of 58,667 bp. The ab initio gene prediction was performed using Ustilago maydis as the reference species. A total number of 10,113 genes were predicted with an average gene size of 1945 bp out of which functionally annotated genes were 7262. A total number of 3216 protein-coding genes were assigned in different categories. Out of a total number of 1877 transposable elements, gypsy had the highest count (573). Total 5772 simple sequence repeats were identified in the genome assembly, and the most abundant simple sequence repeat type was trinucleotide having 42% of total SSRs. The comparative genome analysis suggested 3751 proteins of T. indica had orthologs in five fungi, whereas 126 proteins were unique to T. indica. Secretome analysis revealed the presence of 1014 secretory proteins and few carbohydrate-active enzymes in the genome. Some putative candidate pathogenicity-related genes were identified in the genome. The whole genome of T. indica will provide a window to understand the pathogenesis mechanism, fungal life cycle, survival of teliospores, and novel strategies for management of Karnal bunt disease of wheat.


April 21, 2020  |  

Genome of lethal Lepiota venenata and insights into the evolution of toxin-biosynthetic genes.

Genomes of lethal Amanita and Galerina mushrooms have gradually become available in the past ten years; in contrast the other known amanitin-producing genus, Lepiota, is still vacant in this aspect. A fatal mushroom poisoning case in China has led to acquisition of fresh L. venenata fruiting bodies, based on which a draft genome was obtained through PacBio and Illumina sequencing platforms. Toxin-biosynthetic MSDIN family and Porlyl oligopeptidase B (POPB) genes were mined from the genome and used for phylogenetic and statistical studies to gain insights into the evolution of the biosynthetic pathway.The analysis of the genome data illustrated that only one MSDIN, named LvAMA1, exits in the genome, along with a POPB gene. No POPA homolog was identified by direct homology searching, however, one additional POP gene, named LvPOPC, was cloned and the gene structure determined. Similar to ApAMA1 in A. phalloides and GmAMA1 in G. marginata, LvAMA1 directly encodes a-amanitin. The two toxin genes were mapped to the draft genome, and the structures analyzed. Furthermore, phylogenetic and statistical analyses were conducted to study the evolution history of the POPB genes. Compared to our previous report, the phylogenetic trees unambiguously showed that a monophyletic POPB lineage clearly conflicted with the species phylogeny. In contrast, phylogeny of POPA genes resembled the species phylogeny. Topology and divergence tests showed that the POPB lineage was robust and these genes exhibited significantly shorter genetic distances than those of the house-keeping rbp2, a characteristic feature of genes with horizontal gene transfer (HGT) background. Consistently, same scenario applied to the only MSDIN, LvAMA1, in the genome.To the best of our knowledge, this is the first reported genome of Lepiota. The analyses of the toxin genes indicate that the cyclic peptides are synthesized through a ribosomal mechanism. The toxin genes, LvAMA1 and LvPOPB, are not in the vicinity of each other. Phylogenetic and evolutionary studies suggest that HGT is the underlining cause for the occurrence of POPB and MSDIN in Amanita, Galerina and Lepiota, which are allocated in three distantly-related families.


April 21, 2020  |  

FGMP: assessing fungal genome completeness

Background: Inexpensive high-throughput DNA sequencing has democratized access to genetic information for most organisms so that research utilizing a genome or transcriptome of an organism is not limited to model systems. However, the quality of the assemblies of sampled genomes can vary greatly which hampers utility for comparisons and meaningful interpretation. The uncertainty of the completeness of a given genome sequence can limit feasibility of asserting patterns of high rates of gene loss reported in many lineages. Results: We propose a computational framework and sequence resource for assessing completeness of fungal genomes called FGMP (Fungal Genome Mapping Project). Our approach is based on evolutionary conserved sets of proteins and DNA elements and is applicable to various types of genomic data. We present a comparison of FGMP and state-of-the-art methods for genome completeness assessment utilizing 246 genome assemblies of fungi. We discuss genome assembly improvements/degradations in 57 cases where assemblies have been updated, as recorded by NCBI assembly archive. Conclusion: FGMP is an accurate tool for quantifying level of completion from fungal genomic data. It is particularly useful for non-model organisms without reference genomes and can be used directly on unassembled reads, which can help reducing genome sequencing costs.


April 21, 2020  |  

Comparative transcriptome analysis identified candidate genes involved in mycelium browning in Lentinula edodes.

Lentinula edodes is one of the most popular edible mushroom species in the world and contains useful medicinal components, such as lentinan. The light-induced formation of brown film on the vegetative mycelial tissues of L. edodes is an important process for ensuring the quantity and quality of this edible mushroom. To understand the molecular mechanisms underlying this critical developmental process in L. edodes, we characterized the morphological phenotypic changes in a strain, Chamaram, associated with abnormal brown film formation and compared its genome-wide transcriptional features.In the present study, we performed genome-wide transcriptome analyses of different vegetative mycelium growth phenotypes, namely, early white, normal brown, and defective dark yellow partial brown films phenotypes which were exposed to different light conditions. The analysis revealed the identification of clusters of genes specific to the light-induced brown film phenotypes. These genes were significantly associated with light sensing via photoreceptors such as FMN- and FAD-bindings, signal transduction by kinases and GPCRs, melanogenesis via activation of tyrosinases, and cell wall degradation by glucanases, chitinases, and laccases, which suggests these processes are involved in the formation of mycelial browning in L. edodes. Interestingly, hydrophobin genes such as SC1 and SC3 exhibited divergent expression levels in the normal and abnormal brown mycelial films, indicating the ability of these genes to act in fruiting body initiation and formation of dikaryotic mycelia. Furthermore, we identified the up-regulation of glycoside hydrolase domain-containing genes in the normal brown film but not in the abnormal film phenotype, suggesting that cell wall degradation in the normal brown film phenotype is crucial in the developmental processes related to the initiation and formation of fruiting bodies.This study systematically analysed the expression patterns of light-induced browning-related genes in L. edodes. Our findings provide information for further investigations of browning formation mechanisms in L. edodes and a foundation for future L. edodes breeding.


April 21, 2020  |  

Chromosome rearrangements shape the diversification of secondary metabolism in the cyclosporin producing fungus Tolypocladium inflatum.

Genes involved in production of secondary metabolites (SMs) in fungi are exceptionally diverse. Even strains of the same species may exhibit differences in metabolite production, a finding that has important implications for drug discovery. Unlike in other eukaryotes, genes producing SMs are often clustered and co-expressed in fungal genomes, but the genetic mechanisms involved in the creation and maintenance of these secondary metabolite biosynthetic gene clusters (SMBGCs) remains poorly understood.In order to address the role of genome architecture and chromosome scale structural variation in generating diversity of SMBGCs, we generated chromosome scale assemblies of six geographically diverse isolates of the insect pathogenic fungus Tolypocladium inflatum, producer of the multi-billion dollar lifesaving immunosuppressant drug cyclosporin, and utilized a Hi-C chromosome conformation capture approach to address the role of genome architecture and structural variation in generating intraspecific diversity in SMBGCs. Our results demonstrate that the exchange of DNA between heterologous chromosomes plays an important role in generating novelty in SMBGCs in fungi. In particular, we demonstrate movement of a polyketide synthase (PKS) and several adjacent genes by translocation to a new chromosome and genomic context, potentially generating a novel PKS cluster. We also provide evidence for inter-chromosomal recombination between nonribosomal peptide synthetases located within subtelomeres and uncover a polymorphic cluster present in only two strains that is closely related to the cluster responsible for biosynthesis of the mycotoxin aflatoxin (AF), a highly carcinogenic compound that is a major public health concern worldwide. In contrast, the cyclosporin cluster, located internally on chromosomes, was conserved across strains, suggesting selective maintenance of this important virulence factor for infection of insects.This research places the evolution of SMBGCs within the context of whole genome evolution and suggests a role for recombination between chromosomes in generating novel SMBGCs in the medicinal fungus Tolypocladium inflatum.


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