Comparative Pathogenomics Reveals Horizontally Acquired Novel Virulence Genes in Fungi Infecting Cereal Hosts

Comparative analyses of pathogen genomes provide new insights into how pathogens have evolved common and divergent virulence strategies to invade related plant species. Fusarium crown and root rots are important diseases of wheat and barley world-wide. In Australia, these diseases are primarily caused by the fungal pathogen Fusarium pseudograminearum. Comparative genomic analyses showed that the F. pseudograminearum genome encodes proteins that are present in other fungal pathogens of cereals but absent in non-cereal pathogens. In some cases, these cereal pathogen specific genes were also found in bacteria associated with plants. Phylogenetic analysis of selected F. pseudograminearum genes supported the hypothesis of horizontal gene transfer into diverse cereal pathogens. Two horizontally acquired genes with no previously known role in fungal pathogenesis were studied functionally via gene knockout methods and shown to significantly affect virulence of F. pseudograminearum on the cereal hosts wheat and barley. Our results indicate using comparative genomics to identify genes specific to pathogens of related hosts reveals novel virulence genes and illustrates the importance of horizontal gene transfer in the evolution of plant infecting fungal pathogens

CgDN3: An essential pathogenicity gene of Colletotrichum gloeosporiodes necessary to avert a hypersensitive-like response in the host Stylosanthes guianensis

A gene of Colletotrichum gloeosporioides that is induced by nitrogen starvation in axenic culture and is expressed at the early stages of infection of the host Stylosanthes guianensis has been identified and its role in pathogenicity tested. The sequence of this gene, named CgDN3, indicated that it encodes a protein of 74 amino acids that contains a predicted 18 amino acid signal sequence for secretion of a basic 54 amino acid mature protein with weak homology to an internal region of plant wall-associated receptor kinases. Mutants of C. gloeosporioides were produced by homologous recombination in which part of the coding sequence and promoter region of the CgDN3 gene was replaced with a hygromycin-resistance gene cassette. Mutations in the CgDN3 gene were confirmed in two independent transformants and Northern (RNA) analysis demonstrated the disrupted CgDN3 gene was not expressed. The mutants had faster mycelial growth rates in vitro but produced spores that germinated to form appressoria normally on the leaf surface. However, the CgDN3 mutants were unable to infect and reproduce on intact host leaves. Microscopic analysis revealed small clusters of necrotic host cells at inoculation sites on leaves, suggesting that these mutants elicited a localized, host hypersensitive-like response. The mutants were able to grow necrotrophically and reproduce on leaves when conidia were inoculated directly onto wound sites. The putative promoter region of the CgDN3 gene was fused to a gene encoding a modified jellyfish green fluorescent protein and introduced into the fungus. Following inoculation, strong expression of green fluorescent protein was observed in primary infection vesicles in infected epidermal cells with weaker expression evident in hyphae growing within infected leaf tissue. These findings indicate that CgDN3 encodes a novel pathogenicity determinant associated with the biotrophic phase of primary infection and required to avert a hypersensitive-like response by a compatible host

Statistical parsimony haplotype network for <i>FpAH1</i>/<i>PnAH1</i> in <i>Fusarium pseudograminearum</i> and <i>Phaeosphaeria</i> spp. Haplotype bubbles are proportional to sample size shown in parentheses.

<p>Dots on line segments indicate single point mutations.</p

Examples of <i>Fusarium pseudograminearum</i> genes that may be of fungal origin and are candidates for roles in cereal pathogenicity.

<p>Numbers indicate BLASTp bit scores from the reciprocal best blast hits.</p>†<p>Cereal pathogen</p

Summary of the <i>F. pseudograminearum</i> genome sequence assembly in comparison to selected fungal genome sequences obtained by short-read sequencing and the reference genome for <i>F. graminearum</i>.

*<p>not provided.</p>†<p>85× coverage with Illumina and 10× with 454.</p

Phylogram of fungal amidohydrolases found in <i>F. pseudograminearum</i> that appear to be of bacterial origin and a number of bacterial amidohydrolases.

<p>Fungal sequences are highlighted in grey boxes. Numbers on branches indicate approximate likelihood ratio test branch support values.</p

Alignment between <i>F. graminearum</i> and <i>F. pseudograminearum</i>.

<p><i>F. graminearum</i> chromosomes are ordered in decreasing size. (A) Dot-plot representing a whole genome alignment between <i>F. graminearum</i> isolate Ph1 and <i>F. pseudograminearum</i> isolate CS3096. The alignment was generated with NUCmer, part of the MUMmer 3 comparative sequence analysis package. Sequences were pre-masked for known repetitive elements and simple repeats using RepeatMasker. The dot plot represents the best 1∶1 alignment between the two genomes. Dots closest to the diagonal represent co-linearity between the two genomes. Red represents matches in the forward direction and blue is indicative of inversions in part of the mapped contig relative to the <i>F. graminearum</i> genome. (B) Distribution of gaps in the alignment between <i>F. pseudograminearum</i> and <i>F. graminearum</i>, relative to the <i>F. graminearum</i> genome. <i>F. graminearum</i> chromosomes were divided into 100 Kbp non-overlapping windows and the unaligned nucleotides in each window summed and expressed as a percentage.</p

Virulence assay of the <i>Fusarium pseudograminearum dienelactone hydrolase 1</i> mutant (ΔFpDLH1) mutant towards wheat cultivar Kennedy in crown rot (A and B) and root rot (C and D) assays.

<p>For A and B N = 3 with each biological replicate consisting of 4 paper towel rolls consisting of 7–8 plants per roll. For C and D N = 15–16 plants. Error bars represent the standard error of the mean. Letters indicate statistically significant differences at <i>P</i><0.05. CS3096 and CS3427 are the parental strains for the FpDLH1 mutants, 96T926 and 27T892 respectively. Mock treatments are inoculations performed with agar plugs that have not been colonized by <i>Fusarium</i>.</p

Summary of BLASTp analysis of FpAH1 versus the non-redundant protein database (nr) at NCBI.

†<p>BLASTp performed 18-May-2012.</p

Fusarium root-rot virulence assay of the <i>Fusarium pseudograminearum</i> amidohydrolase 1 mutant (ΔFpAH1) towards wheat cultivar Kennedy (A and B) and barley cultivar Golden Promise (C and D).

<p>N = 16 individual plants. CS3096 is the parental strain for the FpAH1 mutant (96T492), which was complemented with <i>FpAH1</i> driven by the <i>TrpC</i> promoter (96T681). Error bars represent the standard error of the mean. Different letters on A and C indicate statistically significant differences (<i>P</i><0.05) in pair-wise t-tests. B and D are the plants used to score the shoot length shown in A and C.</p