Angela H. Williams

Genome Sequencing and Comparative Genomics of the Broad Host-Range Pathogen Rhizoctonia solani AG8

Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle which is classified into fourteen reproductively incompatible anastomosis groups (AGs). One of these, AG8, is a devastating pathogen causing bare patch of cereals, brassicas and legumes. R. solani is a multinucleate heterokaryon containing significant heterozygosity within a single cell. This complexity posed significant challenges for the assembly of its genome. We present a high quality genome assembly of R. solani AG8 and a manually curated set of 13,964 genes supported by RNA-seq. The AG8 genome assembly used novel methods to produce a haploid representation of its heterokaryotic state. The whole-genomes of AG8, the rice pathogen AG1-IA and the potato pathogen AG3 were observed to be syntenic and co-linear. Genes and functions putatively relevant to pathogenicity were highlighted by comparing AG8 to known pathogenicity genes, orthology databases spanning 197 phytopathogenic taxa and AG1-IA. We also observed SNP-level “hypermutation” of CpG dinucleotides to TpG between AG8 nuclei, with similarities to repeat-induced point mutation (RIP). Interestingly, gene-coding regions were widely affected along with repetitive DNA, which has not been previously observed for RIP in mononuclear fungi of the Pezizomycotina. The rate of heterozygous SNP mutations within this single isolate of AG8 was observed to be higher than SNP mutation rates observed across populations of most fungal species compared. Comparative analyses were combined to predict biological processes relevant to AG8 and 308 proteins with effector-like characteristics, forming a valuable resource for further study of this pathosystem. Predicted effector-like proteins had elevated levels of non-synonymous point mutations relative to synonymous mutations (dN/dS), suggesting that they may be under diversifying selection pressures. In addition, the distant relationship to sequenced necrotrophs of the Ascomycota suggests the R. solani genome sequence may prove to be a useful resource in future comparative analysis of plant pathogens.

The genome sequence of the biocontrol fungus Metarhizium anisopliae and comparative genomics of Metarhizium species

BackgroundMetarhizium anisopliae is an important fungal biocontrol agent of insect pests of agricultural crops. Genomics can aid the successful commercialization of biopesticides by identification of key genes differentiating closely related species, selection of virulent microbial isolates which are amenable to industrial scale production and formulation and through the reduction of phenotypic variability. The genome of Metarhizium isolate ARSEF23 was recently published as a model for M. anisopliae, however phylogenetic analysis has since re-classified this isolate as M. robertsii. We present a new annotated genome sequence of M. anisopliae (isolate Ma69) and whole genome comparison to M. robertsii (ARSEF23) and M. acridum (CQMa 102).ResultsWhole genome analysis of M. anisopliae indicates significant macrosynteny with M. robertsii but with some large genomic inversions. In comparison to M. acridum, the genome of M. anisopliae shares lower sequence homology. While alignments overall are co-linear, the genome of M. acridum is not contiguous enough to conclusively observe macrosynteny. Mating type gene analysis revealed both MAT1-1 and MAT1-2 genes present in M. anisopliae suggesting putative homothallism, despite having no known teleomorph, in contrast with the putatively heterothallic M. acridum isolate CQMa 102 (MAT1-2) and M. robertsii isolate ARSEF23 (altered MAT1-1). Repetitive DNA and RIP analysis revealed M. acridum to have twice the repetitive content of the other two species and M. anisopliae to be five times more RIP affected than M. robertsii. We also present an initial bioinformatic survey of candidate pathogenicity genes in M. anisopliae.ConclusionsThe annotated genome of M. anisopliae is an important resource for the identification of virulence genes specific to M. anisopliae and development of species- and strain- specific assays. New insight into the possibility of homothallism and RIP affectedness has important implications for the development of M. anisopliae as a biopesticide as it may indicate the potential for greater inherent diversity in this species than the other species. This could present opportunities to select isolates with unique combinations of pathogenicity factors, or it may point to instability in the species, a negative attribute in a biopesticide.

Comparative genomics and prediction of conditionally dispensable sequences in legume–infecting Fusarium oxysporum formae speciales facilitates identification of candidate effectors

BackgroundSoil-borne fungi of the Fusarium oxysporum species complex cause devastating wilt disease on many crops including legumes that supply human dietary protein needs across many parts of the globe. We present and compare draft genome assemblies for three legume-infecting formae speciales (ff. spp.): F. oxysporum f. sp. ciceris (Foc-38-1) and f. sp. pisi (Fop-37622), significant pathogens of chickpea and pea respectively, the world’s second and third most important grain legumes, and lastly f. sp. medicaginis (Fom-5190a) for which we developed a model legume pathosystem utilising Medicago truncatula.ResultsFocusing on the identification of pathogenicity gene content, we leveraged the reference genomes of Fusarium pathogens F. oxysporum f. sp. lycopersici (tomato-infecting) and F. solani (pea-infecting) and their well-characterised core and dispensable chromosomes to predict genomic organisation in the newly sequenced legume-infecting isolates. Dispensable chromosomes are not essential for growth and in Fusarium species are known to be enriched in host-specificity and pathogenicity-associated genes. Comparative genomics of the publicly available Fusarium species revealed differential patterns of sequence conservation across F. oxysporum formae speciales, with legume-pathogenic formae speciales not exhibiting greater sequence conservation between them relative to non-legume-infecting formae speciales, possibly indicating the lack of a common ancestral source for legume pathogenicity. Combining predicted dispensable gene content with in planta expression in the model legume-infecting isolate, we identified small conserved regions and candidate effectors, four of which shared greatest similarity to proteins from another legume-infecting ff. spp.ConclusionsWe demonstrate that distinction of core and potential dispensable genomic regions of novel F. oxysporum genomes is an effective tool to facilitate effector discovery and the identification of gene content possibly linked to host specificity. While the legume-infecting isolates didn’t share large genomic regions of pathogenicity-related content, smaller regions and candidate effector proteins were highly conserved, suggesting that they may play specific roles in inducing disease on legume hosts.

Evaluation of Secretion Prediction Highlights Differing Approaches Needed for Oomycete and Fungal Effectors

The steadily increasing number of sequenced fungal and oomycete genomes has enabled detailed studies of how these eukaryotic microbes infect plants and cause devastating losses in food crops. During infection, fungal and oomycete pathogens secrete effector molecules which manipulate host plant cell processes to the pathogens advantage. Proteinaceous effectors are synthesized intracellularly and must be externalized to interact with host cells. Computational prediction of secreted proteins from genomic sequences is an important technique to narrow down the candidate effector repertoire for subsequent experimental validation. In this study, we benchmark secretion prediction tools on experimentally validated fungal and oomycete effectors. We observe that for a set of fungal SwissProt protein sequences, SignalP 4 and the neural network predictors of SignalP 3 (D-score) and SignalP 2 perform best. For effector prediction in particular, the use of a sensitive method can be desirable to obtain the most complete candidate effector set. We show that the neural network predictors of SignalP 2 and 3, as well as TargetP were the most sensitive tools for fungal effector secretion prediction, whereas the hidden Markov model predictors of SignalP 2 and 3 were the most sensitive tools for oomycete effectors. Thus, previous versions of SignalP retain value for oomycete effector prediction, as the current version, SignalP 4, was unable to reliably predict the signal peptide of the oomycete Crinkler effectors in the test set. Our assessment of subcellular localization predictors shows that cytoplasmic effectors are often predicted as not extracellular. This limits the reliability of secretion predictions that depend on these tools. We present our assessment with a view to informing future pathogenomics studies and suggest revised pipelines for secretion prediction to obtain optimal effector predictions in fungi and oomycetes.

Phoma medicaginis stimulates the induction of the octadecanoid and phenylpropanoid pathways in Medicago truncatula: M. truncatula's defence response to P. medicaginis

Gene expression changes and metabolite abundances were measured during the interaction of Medicago truncatula with the fungal necrotrophic pathogen Phoma medicaginis in leaf tissue of susceptible and resistant accessions. Over 330 genes were differentially expressed in plants infected with P. medicaginis relative to mock-inoculated plants at 12 h post-inoculation. Of these, 191 were induced in either the resistant or the susceptible accession, with 143 genes repressed. Expression changes were observed in genes involved in the oxidative burst, cell wall strengthening and lipid metabolism, as well as several transcription factors. Genes related to salicylic acid, jasmonate and ethylene responses were up-regulated, as well as genes leading to the production of jasmonic acid. Significant induction of genes in the phenylpropanoid pathway leading to lignin and isoflavonoid biosynthesis occurred. High-pressure liquid chromatography with UV detection (HPLC-UV) identified several phenolic compounds induced by P. medicaginis, as well as constitutively higher levels of phenolic compounds, in the resistant M. truncatula accession. Differentially regulated genes induced in both the resistant and susceptible accessions, but with different kinetics, and constitutively more highly expressed and induced phenolic compounds provide candidates for functional analysis. Taken together, these results highlight the importance of the octadecanoid and phenylpropanoid pathways in defence against this necrotrophic pathogen.

Comprehensive Annotation of the Parastagonospora nodorum Reference Genome Using Next-Generation Genomics, Transcriptomics and Proteogenomics

Parastagonospora nodorum, the causal agent of Septoria nodorum blotch (SNB), is an economically important pathogen of wheat (Triticum spp.), and a model for the study of necrotrophic pathology and genome evolution. The reference P. nodorum strain SN15 was the first Dothideomycete with a published genome sequence, and has been used as the basis for comparison within and between species. Here we present an updated reference genome assembly with corrections of SNP and indel errors in the underlying genome assembly from deep resequencing data as well as extensive manual annotation of gene models using transcriptomic and proteomic sources of evidence (https://github.com/robsyme/Parastagonospora_nodorum_SN15). The updated assembly and annotation includes 8,366 genes with modified protein sequence and 866 new genes. This study shows the benefits of using a wide variety of experimental methods allied to expert curation to generate a reliable set of gene models.

Utilizing Gene Tree Variation to Identify Candidate Effector Genes in Zymoseptoria tritici

Zymoseptoria tritici is a host-specific, necrotrophic pathogen of wheat. Infection by Z. tritici is characterized by its extended latent period, which typically lasts 2 wks, and is followed by extensive host cell death, and rapid proliferation of fungal biomass. This work characterizes the level of genomic variation in 13 isolates, for which we have measured virulence on 11 wheat cultivars with differential resistance genes. Between the reference isolate, IPO323, and the 13 Australian isolates we identified over 800,000 single nucleotide polymorphisms, of which ∼10% had an effect on the coding regions of the genome. Furthermore, we identified over 1700 probable presence/absence polymorphisms in genes across the Australian isolates using de novo assembly. Finally, we developed a gene tree sorting method that quickly identifies groups of isolates within a single gene alignment whose sequence haplotypes correspond with virulence scores on a single wheat cultivar. Using this method, we have identified < 100 candidate effector genes whose gene sequence correlates with virulence toward a wheat cultivar carrying a major resistance gene.

Repeat-Induced Point Mutation: A Fungal-Specific, Endogenous Mutagenesis Process

Repeat-induced point mutation (RIP) is a form of genome mutation that is targeted towards repeated DNA sequences and which is observed only in certain fungal taxa; the Pezizomycotina (filamentous Ascomycota) and some species of the Basidiomycota. RIP is widely believed to have evolved to protect fungal genomes against transposon replication.

Draft genome sequence of Bacillus thuringiensis strain DAR 81934, which exhibits molluscicidal activity.

ABSTRACT Bacillus thuringiensis has been widely used as a biopesticide for a long time. Its molluscicidal activity, however, is rarely realized. Here, we report the genome sequence of B. thuringiensis strain DAR 81934, a strain with molluscicidal activity against the pest snail Cernuella virgata.

Genomic and comparative analysis of the Class Dothideomycetes

The class Dothideomycetes is a recently defined taxon within the phylum Ascomycota, with approximately 20 000 member species. Many species are important phytopathogens employing diverse pathogenicity strategies. This review compares the currently available genome sequences, including their mitochondrial sequences. The genomes of Dothideomycetes exhibit remarkable plasticity, characterised by extensive rearrangement of gene order and orientation. Dothideomycetes also display the ability to take advantage of repeat induced point mutation (RIP), a fungal specific mechanism to protect their genomes against transposon invasion. RIP also affects areas of the genome adjacent to transposon insertion sites conferring the adaptive advantage of rapid generation of diversity in fungal pathogenicity effector genes. In addition, Dothideomycetes have the propensity for lateral gene transfer which can involve the transmission of pathogenicity effectors. The combination of these mechanisms has led to the ascendance of many Dothideomycetes as the dominant fungal pathogens of their cultivated host plant species.