Investigations on VELVET regulatory mutants confirm the role of host tissue acidification and secretion of proteins in the pathogenesis of Botrytis cinerea.

The Botrytis cinerea VELVET complex regulates light-dependent development and virulence. The goal of this study was to identify common virulence defects of several VELVET mutants and to reveal their molecular basis. Growth, differentiation, physiology, gene expression and infection of fungal strains were analyzed, and quantitative comparisons of in planta transcriptomes and secretomes were performed. VELVET mutants showed reduced release of citric acid, the major acid secreted by the wild-type, whereas no significant role for oxalic acid was observed. Furthermore, a common set of infection-related and secreted proteins was strongly underexpressed in the mutants. Quantitative secretome analysis with 15 N metabolic labeling revealed a correlation of changes in protein and mRNA levels between wild-type and mutants, indicating that transcript levels determine the abundance of secreted proteins. Infection sites kept at low pH partially restored lesion expansion and expression of virulence genes by the mutants. Drastic downregulation of proteases in the mutants was correlated with incomplete degradation of cellular host proteins at the infection site, but no evidence was obtained that aspartyl proteases are required for lesion formation. The B. cinerea VELVET complex controls pathogenic differentiation by regulating organic acid secretion, host tissue acidification, gene expression and protein secretion.

Genome Comparisons between Botrytis fabae and the Closely Related Gray Mold Fungus Botrytis cinerea Reveal Possible Explanations for Their Contrasting Host Ranges.

While Botrytis cinerea causes gray mold on many plants, its close relative, Botrytis fabae, is host-specifically infecting predominantly faba bean plants. To explore the basis for its narrow host range, a gapless genome sequence of B. fabae strain G12 (BfabG12) was generated. The BfabG12 genome encompasses 45.0 Mb, with 16 chromosomal telomere-to-telomere contigs that show high synteny and sequence similarity to the corresponding B. cinerea B05.10 (BcB0510) chromosomes. Compared to BcB0510, it is 6% larger, due to many AT-rich regions containing remnants of transposable elements, but encodes fewer genes (11,420 vs. 11,707), due to losses of chromosomal segments with up to 20 genes. The coding capacity of BfabG12 is further reduced by nearly 400 genes that had been inactivated by mutations leading to truncations compared to their BcB0510 orthologues. Several species-specific gene clusters for secondary metabolite biosynthesis with stage-specific expression were identified. Comparison of the proteins secreted during infection revealed high similarities, including 17 phytotoxic proteins that were detected in both species. Our data indicate that evolution of the host-specific B. fabae occurred from an ancestral pathogen with wide host range similar to B. cinerea and was accompanied by losses and degeneration of genes, thereby reducing its pathogenic flexibility.

Genetic Diversity of Botrytis cinerea Revealed by Multilocus Sequencing, and Identification of B. cinerea Populations Showing Genetic Isolation and Distinct Host Adaptation.

Botrytis cinerea is a world-wide occurring plant pathogen, causing pre- and post-harvest gray mold rot on a large number of fruit, vegetable, and flower crops. B. cinerea is closely related to Botrytis pseudocinerea, another broad host range species which often occurs in sympatry with B. cinerea, and to several host-specific species including Botrytis fabae and Botrytis calthae. B. cinerea populations have been shown to be genetically heterogeneous, and attempts have been made to correlate genetic markers to virulence and host adaptation. Here, we present the development of a multilocus sequence typing (MLST) scheme, with 10 genes selected for high variability and phylogenetic congruence, to evaluate the genetic diversity of B. cinerea, B. fabae, and B. pseudocinerea. Using PacBio-assisted simultaneous mass sequencing of PCR products, MLST analysis of about 100 strains from diverse geographical origins and years of isolation was performed, which resulted in high-resolution strain differentiation and robust species separation. Several B. cinerea strains formed an as yet unknown population, referred to as group B, which was well separated from all other B. cinerea strains. Furthermore, the gene cluster for biosynthesis of the phytotoxin botcinic acid was missing in B. cinerea B strains. B. cinerea strains from the monocot Iris pseudacorus were found to form a genetically distinct population, and contained an intact gene cluster for production of the red pigment bikaverin, which is usually degenerated in B. cinerea. Remarkably, these strains were much more aggressive on Iris than other B. cinerea strains, which is the first unequivocal example for host specialization in B. cinerea. Our data reveal new insights into the genetic diversity of B. cinerea and provide evidence for intraspecific differentiation and different degrees of host adaptation of this polyphagous necrotrophic pathogen.