Rhynchosporium comparative genomics
In cooperation with the Leibniz Institute on Aging (Fritz Lipmann Institute) in Jena, the Institute of Bioinformatics and Systems Biology at the Helmholtz Center in Munich, the James Hutton Institute in Dundee (Scotland) and the Rothamsted Research Station in Harpenden (England) the genomes of isolates from the five Rhynchosporium species were sequenced (454, Illumina PE/MP), assembled and annotated ab initio. The genome of one of the R. commune isolates was developed into the reference genome. It has a total size of 55 Mb with about 30% protein-coding sequence. A function can be assigned to only about half of the c. 12.200 genes.
Based on multilocus sequence comparisons we could confirm the phylogenetic classification of the genus Rhynchosporium into the Leotiomycetes class of the Ascomycetes. To resolve the relationships within the genus Rhynchosporium single nucleotide polymorphisms (SNPs) were used. The resulting phylogenetic tree shows that speciation of R. agropyri occurred shortly before R. commune and R. secalis diverged, but long after the evolution of the CCG branch with R. orthosoprum and R. lolii.
2. Sexual Reproduction
Sexual reproduction has never been observed in Rhynchosporium. Nevertheless both mating type loci, MAT1-1 und MAT1-2, that are characteristic for heterothallic Ascomycetes, were found in most field populations in an equimolar ratio. Our analysis of the Rhynchosporium genome demonstrates now that not only the MAT genes but also the entire genetic machinery required for sexual reproduction are present.
3. Cell wall degradation
The Rhynchosporium genome was annotated using the CAZY (Carbohydrate Active enZYme) database. The number of enzymes involved in the degradation of host cell walls is characteristic for hemibiotrophic and necrotrophic monocot-infecting fungi. However, we could not find clues for a possible role in the expression of fungal host specificity.
4. Secondary metabolism
Despite their great chemical diversity most secondary metabolites of fungi are formed via four major metabolic routes, which are characterized by key enzymes such as polyketide synthases (PKS: polyketides), non-ribosomal peptide synthetases (NRPS: peptides), terpene cyclases (TC: terpenes) and dimethylallyl tryptophan synthases (DMATS: indole alkaloids). In addition to 1 TC gene and 3 DMATS genes we found 9 PKS, 4 NRPS and 3 PKS-NRPS hybrid genes in the genomes of the three BCG species of Rhynchosporium. They often occur in clusters along with genes coding for different modifying enzymes as well as transcription factors and transporters. Three additional genes may be of particular interest in the context of host specificity, a PKS gene specific for R. commune as well as a PKS and a NRPS gene, both occurring isolate-specifically. Comparative analyses of the metabolite profiles of wild-type and PKS deletion mutants were initiated to resolve identity and function of the respective polyketide products.
5. Effector proteins
About 8% of the 12.200 R. commune genes code for the fungal secretome, i.e., for the entirety of the secreted proteins (similar numbers apply to the other Rhynchosporium species). Among them are the genes NIP1, NIP2 und NIP3, which we previously described to contribute quantitatively to fungal virulence. NIP3 is present as a single gene in all BCG species, whereas NIP1 is only found in R. commune (albeit in only about half of the isolates tested worldwide) and in R. orthosporum. The products of both genes stimulate the activity of the H+-ATPase in the plasma membrane of host cells. NIP2, whose mode of action is not known yet, also occurs as a single gene in the CCG species, whereas the BCG species harbor NIP2 gene families of up to 10 members. These families evolved largely before the divergence of the BCG species. Therefore, they – as well as NIP1 und NIP3 – could have been important in the host jump of the common ancestor. To functionally analyze the NIP2 genes we are currently developing an RNAi-based gene silencing system.
Using in silico methods we identified seven effector genes specifically occurring in the genome of R. commune. Surprisingly, deletion mutants showed stronger growth and some also caused a more rapid development of disease symptoms than the wild-type. Therefore, the function of these effectors appears to lie in slowing down fungal development. This suggests that optimizing fungal development on the host plants aims at extending the biotrophic phase of pathogenesis at the expense of the necrotrophic phase.
Rhynchosporium appears to exemplify that adaptation of a microorganism does not intend to provoke maximum damage to its plant host. Stabilization by secreted effectors of the generally very slow biotrophic development of the fungus may suggest that already the ancestors of the fungus lived as endophytes and not as pathogens on their original host plant.