Phased chromosome-scale genome assembly of an asexual, allopolyploid root-knot nematode reveals complex subgenomic structure.

We present the chromosome-scale genome assembly of the allopolyploid root-knot nematode Meloidogyne javanica. We show that the M. javanica genome is predominantly allotetraploid, comprising two subgenomes, A and B, that most likely originated from hybridisation of two ancestral parental species. The assembly was annotated using full-length non-chimeric transcripts, comparison to reference databases, and ab initio prediction techniques, and the subgenomes were phased using ancestral k-mer spectral analysis. Subgenome B appears to show fission of chromosomal contigs, and while there is substantial synteny between subgenomes, we also identified regions lacking synteny that may have diverged in the ancestral genomes prior to or following hybridisation. This annotated and phased genome assembly forms a significant resource for understanding the origins and genetics of these globally important plant pathogens.

Transposon-Mediated Horizontal Transfer of the Host-Specific Virulence Protein ToxA between Three Fungal Wheat Pathogens.

Most known examples of horizontal gene transfer (HGT) between eukaryotes are ancient. These events are identified primarily using phylogenetic methods on coding regions alone. Only rarely are there examples of HGT where noncoding DNA is also reported. The gene encoding the wheat virulence protein ToxA and the surrounding 14 kb is one of these rare examples. ToxA has been horizontally transferred between three fungal wheat pathogens (Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana) as part of a conserved ∼14 kb element which contains coding and noncoding regions. Here we used long-read sequencing to define the extent of HGT between these three fungal species. Construction of near-chromosomal-level assemblies enabled identification of terminal inverted repeats on either end of the 14 kb region, typical of a type II DNA transposon. This is the first description of ToxA with complete transposon features, which we call ToxhAT. In all three species, ToxhAT resides in a large (140-to-250 kb) transposon-rich genomic island which is absent in isolates that do not carry the gene (annotated here as toxa- ). We demonstrate that the horizontal transfer of ToxhAT between P. tritici-repentis and P. nodorum occurred as part of a large (∼80 kb) HGT which is now undergoing extensive decay. In B. sorokiniana, in contrast, ToxhAT and its resident genomic island are mobile within the genome. Together, these data provide insight into the noncoding regions that facilitate HGT between eukaryotes and into the genomic processes which mask the extent of HGT between these species.IMPORTANCE This work dissects the tripartite horizontal transfer of ToxA, a gene that has a direct negative impact on global wheat yields. Defining the extent of horizontally transferred DNA is important because it can provide clues to the mechanisms that facilitate HGT. Our analysis of ToxA and its surrounding 14 kb suggests that this gene was horizontally transferred in two independent events, with one event likely facilitated by a type II DNA transposon. These horizontal transfer events are now in various processes of decay in each species due to the repeated insertion of new transposons and subsequent rounds of targeted mutation by a fungal genome defense mechanism known as repeat induced point mutation. This work highlights the role that HGT plays in the evolution of host adaptation in eukaryotic pathogens. It also increases the growing body of evidence indicating that transposons facilitate adaptive HGT events between fungi present in similar environments and hosts.

Comparative analysis of the predicted secretomes of Rosaceae scab pathogens Venturia inaequalis and V. pirina reveals expanded effector families and putative determinants of host range.

BACKGROUND: Fungal plant pathogens belonging to the genus Venturia cause damaging scab diseases of members of the Rosaceae. In terms of economic impact, the most important of these are V. inaequalis, which infects apple, and V. pirina, which is a pathogen of European pear. Given that Venturia fungi colonise the sub-cuticular space without penetrating plant cells, it is assumed that effectors that contribute to virulence and determination of host range will be secreted into this plant-pathogen interface. Thus the predicted secretomes of a range of isolates of Venturia with distinct host-ranges were interrogated to reveal putative proteins involved in virulence and pathogenicity. RESULTS: Genomes of Venturia pirina (one European pear scab isolate) and Venturia inaequalis (three apple scab, and one loquat scab, isolates) were sequenced and the predicted secretomes of each isolate identified. RNA-Seq was conducted on the apple-specific V. inaequalis isolate Vi1 (in vitro and infected apple leaves) to highlight virulence and pathogenicity components of the secretome. Genes encoding over 600 small secreted proteins (candidate effectors) were identified, most of which are novel to Venturia, with expansion of putative effector families a feature of the genus. Numerous genes with similarity to Leptosphaeria maculans AvrLm6 and the Verticillium spp. Ave1 were identified. Candidates for avirulence effectors with cognate resistance genes involved in race-cultivar specificity were identified, as were putative proteins involved in host-species determination. Candidate effectors were found, on average, to be in regions of relatively low gene-density and in closer proximity to repeats (e.g. transposable elements), compared with core eukaryotic genes. CONCLUSIONS: Comparative secretomics has revealed candidate effectors from Venturia fungal plant pathogens that attack pome fruit. Effectors that are putative determinants of host range were identified; both those that may be involved in race-cultivar and host-species specificity. Since many of the effector candidates are in close proximity to repetitive sequences this may point to a possible mechanism for the effector gene family expansion observed and a route to diversification via transposition and repeat-induced point mutation.

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides.

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1-like sequences were observed in most cases; however, RALF27-like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27-like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen-activated protein kinase activation). Gene expression analysis confirmed that a RALF-encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant-pathogen interactions.

A Large Family of AvrLm6-like Genes in the Apple and Pear Scab Pathogens, Venturia inaequalis and Venturia pirina.

Venturia inaequalis and V. pirina are Dothideomycete fungi that cause apple scab and pear scab disease, respectively. Whole genome sequencing of V. inaequalis and V. pirina isolates has revealed predicted proteins with sequence similarity to AvrLm6, a Leptosphaeria maculans effector that triggers a resistance response in Brassica napus and B. juncea carrying the resistance gene, Rlm6. AvrLm6-like genes are present as large families (>15 members) in all sequenced strains of V. inaequalis and V. pirina, while in L. maculans, only AvrLm6 and a single paralog have been identified. The Venturia AvrLm6-like genes are located in gene-poor regions of the genomes, and mostly in close proximity to transposable elements, which may explain the expansion of these gene families. An AvrLm6-like gene from V. inaequalis with the highest sequence identity to AvrLm6 was unable to trigger a resistance response in Rlm6-carrying B. juncea. RNA-seq and qRT-PCR gene expression analyses, of in planta- and in vitro-grown V. inaequalis, has revealed that many of the AvrLm6-like genes are expressed during infection. An AvrLm6 homolog from V. inaequalis that is up-regulated during infection was shown (using an eYFP-fusion protein construct) to be localized to the sub-cuticular stroma during biotrophic infection of apple hypocotyls.

Proteogenomic analysis of the Venturia pirina (Pear Scab Fungus) secretome reveals potential effectors.

A proteogenomic analysis is presented for Venturia pirina, a fungus that causes scab disease on European pear (Pyrus communis). V. pirina is host-specific, and the infection is thought to be mediated by secreted effector proteins. Currently, only 36 V. pirina proteins are catalogued in GenBank, and the genome sequence is not publicly available. To identify putative effectors, V. pirina was grown in vitro on and in cellophane sheets mimicking its growth in infected leaves. Secreted extracts were analyzed by tandem mass spectrometry, and the data (ProteomeXchange identifier PXD000710) was queried against a protein database generated by combining in silico predicted transcripts with six frame translations of a whole genome sequence of V. pirina (GenBank Accession JEMP00000000 ). We identified 1088 distinct V. pirina protein groups (FDR 1%) including 1085 detected for the first time. Thirty novel (not in silico predicted) proteins were found, of which 14 were identified as potential effectors based on characteristic features of fungal effector protein sequences. We also used evidence from semitryptic peptides at the protein N-terminus to corroborate in silico signal peptide predictions for 22 proteins, including several potential effectors. The analysis highlights the utility of proteogenomics in the study of secreted effectors.