Catherine Sirven

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea–specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

Fungi have three tetraspanin families with distinct functions.

BackgroundTetraspanins are small membrane proteins that belong to a superfamily encompassing 33 members in human and mouse. These proteins act as organizers of membrane-signalling complexes. So far only two tetraspanin families have been identified in fungi. These are Pls1, which is required for pathogenicity of the plant pathogenic ascomycetes, Magnaporthe grisea, Botrytis cinerea and Colletotrichum lindemuthianum, and Tsp2, whose function is unknown. In this report, we describe a third family of tetraspanins (Tsp3) and a new family of tetraspanin-like proteins (Tpl1) in fungi. We also describe expression of some of these genes in M. grisea and a basidiomycete, Laccaria bicolor, and also their functional analysis in M. grisea.ResultsThe exhaustive search for tetraspanins in fungal genomes reveals that higher fungi (basidiomycetes and ascomycetes) contain three families of tetraspanins (Pls1, Tsp2 and Tsp3) with different distribution amongst phyla. Pls1 is found in ascomycetes and basidiomycetes, whereas Tsp2 is restricted to basidiomycetes and Tsp3 to ascomycetes. A unique copy of each of PLS1 and TSP3 was found in ascomycetes in contrast to TSP2, which has several paralogs in the basidiomycetes, Coprinus cinereus and Laccaria bicolor. A tetraspanin-like family (Tpl1) was also identified in ascomycetes. Transcriptional analyses in various tissues of L. bicolor and M. grisea showed that PLS1 and TSP2 are expressed in all tissues in L. bicolor and that TSP3 and TPL1 are overexpressed in the sexual fruiting bodies (perithecia) and mycelia of M. grisea, suggesting that these genes are not pseudogenes. Phenotypic analysis of gene replacementmutants Δtsp3 and Δtpl1 of M. grisea revealed a reduction of the pathogenicity only on rice, in contrast to Δpls1 mutants, which are completely non-pathogenic on barley and rice.ConclusionA new tetraspanin family (Tsp3) and a tetraspanin-like protein family (Tpl1) have been identified in fungi. Functional analysis by gene replacement showed that these proteins, as well as Pls1, are involved in the infection process of the plant pathogenic fungus M. grisea. The next challenge will be to decipher the role(s) of tetraspanins in a range of symbiotic, saprophytic and human pathogenic fungi.

Genome-wide analyses of chitin synthases identify horizontal gene transfers towards bacteria and allow a robust and unifying classification into fungi.

BackgroundChitin, the second most abundant biopolymer on earth after cellulose, is found in probably all fungi, many animals (mainly invertebrates), several protists and a few algae, playing an essential role in the development of many of them. This polysaccharide is produced by type 2 glycosyltransferases, called chitin synthases (CHS). There are several contradictory classifications of CHS isoenzymes and, as regards their evolutionary history, their origin and diversity is still a matter of debate.ResultsA genome-wide analysis resulted in the detection of more than eight hundred putative chitin synthases in proteomes associated with about 130 genomes. Phylogenetic analyses were performed with special care to avoid any pitfalls associated with the peculiarities of these sequences (e.g. highly variable regions, truncated or recombined sequences, long-branch attraction). This allowed us to revise and unify the fungal CHS classification and to study the evolutionary history of the CHS multigenic family. This update has the advantage of being user-friendly due to the development of a dedicated website (http://wwwabi.snv.jussieu.fr/public/CHSdb), and it includes any correspondences with previously published classifications and mutants. Concerning the evolutionary history of CHS, this family has mainly evolved via duplications and losses. However, it is likely that several horizontal gene transfers (HGT) also occurred in eukaryotic microorganisms and, even more surprisingly, in bacteria.ConclusionsThis comprehensive multi-species analysis contributes to the classification of fungal CHS, in particular by optimizing its robustness, consensuality and accessibility. It also highlights the importance of HGT in the evolutionary history of CHS and describes bacterial chs genes for the first time. Many of the bacteria that have acquired a chitin synthase are plant pathogens (e.g. Dickeya spp; Pectobacterium spp; Brenneria spp; Agrobacterium vitis and Pseudomonas cichorii). Whether they are able to produce a chitin exopolysaccharide or secrete chitooligosaccharides requires further investigation.

Novel Tools to Identify the Mode of Action of Fungicides as Exemplified with Fluopicolide

The expanding field of fungal genomics stimulates the development of genome wide functional tools and comparative analyses in plant pathogenic fungi. As a consequence, transcriptomic, proteomic and metabolomic studies coupled with high throughput forward and reverse genetics are now available in a significant number of fungal plant pathogens (e.g. Ustilago maydis, Magnaporthe grisea, Fusarium graminearum, Botrytis cinerea). Genomics together with classical biochemical tools and microscopy offer the possibility to accelerate the identification of the biochemical mode of action of novel fungicides. This knowledge is also required to discover efficiently novel antifungal compounds and to characterize and follow efficiently the emergence of resistance. The available genomic tools for plant pathogenic fungi will be reviewed as exemplified with the mode of action of fluopicolide, a novel fungicide active against Oomycetes. Biological studies performed with Phytophthora infestans and Plasmopara viticola showed that fluopicolide affects the release and motility of zoospores and the germination of cysts, as well as mycelial growth and sporulation. Biochemical studies showed that its mode of action differs from that of known anti-oomycetes compounds. Fluopicolide does not show cross-resistance to commercial fungicide classes such as phenylamides, strobilurins (QoIs) and carboxylic acid amides (CAAs). Cytological studies in P. infestans showed that fluopicolide specifically modifies the spatial and cellular distribution of proteins labelled by antibodies specific for animal cytoskeleton associated proteins spectrin. Treatments with fluopicolide induced a fast redistribution of spectrin-like protein(s) from the membrane to the cytoplasm in both hyphae and zoospores. Whereas animal spectrin(s) play an important role in membrane stability, they are poorly characterized in fungi and oomycetes. Cytoskeletal proteins such as actins, tubulins, integrins and spectrins provide structural stability to cells as they form a network sustaining the plasma membrane. Fluopicolide may interfere and destabilize this network leading to cell disorganization. This hypothesis is supported by the observation that treatments of zoospores lead to the relocalization of spectrin-like protein(s) into the cytoplasm within a few minutes followed immediately by cell swelling and burst. Preliminary data of gene expression profiling in P. sojae treated cells showed a differential expression (up and down regulation) of genes involved in vesicular transport. The link between golgi function, vesicle transport and cellular relocation of spectrin like proteins will be discussed.