Population Genomics Reveals Molecular Determinants of Specialization to Tomato in the Polyphagous Fungal Pathogen Botrytis cinerea in France.

Many fungal plant pathogens encompass multiple populations specialized on different plant species. Understanding the factors underlying pathogen adaptation to their hosts is a major challenge of evolutionary microbiology, and it should help to prevent the emergence of new specialized pathogens on novel hosts. Previous studies have shown that French populations of the gray mold pathogen Botrytis cinerea parasitizing tomato and grapevine are differentiated from each other, and have higher aggressiveness on their host of origin than on other hosts, indicating some degree of host specialization in this polyphagous pathogen. Here, we aimed at identifying the genomic features underlying the specialization of B. cinerea populations to tomato and grapevine. Based on whole genome sequences of 32 isolates, we confirmed the subdivision of B. cinerea pathogens into two genetic clusters on grapevine and another, single cluster on tomato. Levels of genetic variation in the different clusters were similar, suggesting that the tomato-specific cluster has not recently emerged following a bottleneck. Using genome scans for selective sweeps and divergent selection, tests of positive selection based on polymorphism and divergence at synonymous and nonsynonymous sites, and analyses of presence and absence variation, we identified several candidate genes that represent possible determinants of host specialization in the tomato-associated population. This work deepens our understanding of the genomic changes underlying the specialization of fungal pathogen populations.

The polyphagous plant pathogenic fungus Botrytis cinerea encompasses host-specialized and generalist populations.

The host plant is often the main variable explaining population structure in fungal plant pathogens, because specialization contributes to reduce gene flow between populations associated with different hosts. Previous population genetic analysis revealed that French populations of the grey mould pathogen Botrytis cinerea were structured by hosts tomato and grapevine, suggesting host specialization in this highly polyphagous pathogen. However, these findings raised questions about the magnitude of this specialization and the possibility of specialization to other hosts. Here we report specialization of B. cinerea populations to tomato and grapevine hosts but not to other tested plants. Population genetic analysis revealed two pathogen clusters associated with tomato and grapevine, while the other clusters co-occurred on hydrangea, strawberry and bramble. Measurements of quantitative pathogenicity were consistent with host specialization of populations found on tomato, and to a lesser extent, populations found on grapevine. Pathogen populations from hydrangea and strawberry appeared to be generalist, while populations from bramble may be weakly specialized. Our results suggest that the polyphagous B. cinerea is more accurately described as a collection of generalist and specialist individuals in populations. This work opens new perspectives for grey mould management, while suggesting spatial optimization of crop organization within agricultural landscapes.

The formation of sesquiterpenoid presilphiperfolane and cameroonane metabolites in the Bcbot4 null mutant of Botrytis cinerea.

Botrytis cinerea is a polyphagous fungal parasite which causes serious damage to more than 200 plant species and consequent economic losses for commercial crops. This pathogen produces two families of phytotoxins, the botryanes and botcinins, which are involved in the infection mechanism. The B. cinerea genome has provided a complete picture of the genes involved in the biosynthesis of its secondary metabolites. The botrydial biosynthetic gene cluster has been identified. This cluster consists of seven genes, where the genes BcBOT1, BcBOT3 and BcBOT4 encode three mono-oxygenases. A study of the Bcbot4Δ null mutant revealed that this mono-oxygenase was involved in the hydroxylation at C-4 of the probotryane skeleton (C-11 of the presilphiperfolane skeleton). A detailed study of the Bcbot4Δ null mutant has been undertaken in order to study the metabolic fate of the presilphiperfolan-8-ol intermediate biosynthesized by this organism and in particular by this strain. As a result three new presilphiperfolanes and three new cameroonanes have been identified. The results suggest that the absence of the oxygen function at C-11 of the presilphiperfolane skeleton permits rearrangement to a cameroonane whilst hydroxylation at C-11 precludes this rearrangement. It is possible that the interactions of the C-11 hydroxylated derivatives perturb the stereo-electronic requirements for the migration of the C-11:C-7 sigma bond to C-8.

The botrydial biosynthetic gene cluster of Botrytis cinerea displays a bipartite genomic structure and is positively regulated by the putative Zn(II)2Cys6 transcription factor BcBot6.

Botrydial (BOT) is a non-host specific phytotoxin produced by the polyphagous phytopathogenic fungus Botrytis cinerea. The genomic region of the BOT biosynthetic gene cluster was investigated and revealed two additional genes named Bcbot6 and Bcbot7. Analysis revealed that the G+C/A+T-equilibrated regions that contain the Bcbot genes alternate with A+T-rich regions made of relics of transposable elements that have undergone repeat-induced point mutations (RIP). Furthermore, BcBot6, a Zn(II)2Cys6 putative transcription factor was identified as a nuclear protein and the major positive regulator of BOT biosynthesis. In addition, the phenotype of the ΔBcbot6 mutant indicated that BcBot6 and therefore BOT are dispensable for the development, pathogenicity and response to abiotic stresses in the B. cinerea strain B05.10. Finally, our data revealed that B. pseudocinerea, that is also polyphagous and lives in sympatry with B. cinerea, lacks the ability to produce BOT. Identification of BcBot6 as the major regulator of BOT synthesis is the first step towards a comprehensive understanding of the complete regulation network of BOT synthesis and of its ecological role in the B. cinerea life cycle.

A novel Zn2 Cys6 transcription factor BcGaaR regulates D-galacturonic acid utilization in Botrytis cinerea.

D-galacturonic acid (GalA) is the most abundant monosaccharide component of pectin. Previous transcriptome analysis in the plant pathogenic fungus Botrytis cinerea identified eight GalA-inducible genes involved in pectin decomposition, GalA transport and utilization. Co-expression of these genes indicates that a specific regulatory mechanism occurs in B. cinerea. In this study, promoter regions of these genes were analysed and eight conserved sequence motifs identified. The Bclga1 promoter, containing all these motifs, was functionally analysed and the motif designated GalA Responsive Element (GARE) was identified as the crucial cis-regulatory element in regulation of GalA utilization in B. cinerea. Yeast one-hybrid screening with the GARE motif led to identification of a novel Zn2 Cys6 transcription factor (TF), designated BcGaaR. Targeted knockout analysis revealed that BcGaaR is required for induction of GalA-inducible genes and growth of B. cinerea on GalA. A BcGaaR-GFP fusion protein was predominantly localized in nuclei in mycelium grown in GalA. Fluorescence in nuclei was much stronger in mycelium grown in GalA, as compared to fructose and glucose. This study provides the first report of a GalA-specific TF in filamentous fungi. Orthologs of BcGaaR are present in other ascomycete fungi that are able to utilize GalA, including Aspergillus spp., Trichoderma reesei and Neurospora crassa.

Botrytis cinerea virulence factors: new insights into a necrotrophic and polyphageous pathogen.

Botrytis cinerea is responsible for the gray mold disease on more than 200 host plants. This necrotrophic ascomycete displays the capacity to kill host cells through the production of toxins, reactive oxygen species and the induction of a plant-produced oxidative burst. Thanks to an arsenal of degrading enzymes, B. cinerea is then able to feed on different plant tissues. Recent molecular approaches, for example on characterizing components of signal transduction pathways, show that this fungus shares conserved virulence factors with other phytopathogens, but also highlight some Botrytis-specific features. The discovery of some first strain-specific virulence factors, together with population data, even suggests a possible host adaptation of the strains. The availability of the genome sequence now stimulates the development of high-throughput functional analysis to decipher the mechanisms involved in the large host range of this species.