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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.
CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse
organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized
Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary
selection and convenient screening for marker-free editing events. We demonstrate that
this approach provides superior editing rates compared to existing CRISPR/Cas-based
methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae.
Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated
editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major
determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk
replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were
found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels
towards different SDHI fungicides. The increased efficiency and easy handling of RNPbased cotransformation is expected to accelerate molecular research in B. cinerea and
other fungi.
Botrytis cinerea is a major plant pathogen infecting more than 1400 plant species. During invasion, the fungus rapidly kills host cells, which is believed to be supported by induction of programmed plant cell death. To comprehensively evaluate the contributions of most of the currently known plant cell death inducing proteins (CDIPs) and metabolites for necrotrophic infection, an optimized CRISPR/Cas9 protocol was established which allowed to perform serial marker-free mutagenesis to generate multiple deletion mutants lacking up to 12 CDIPs. Whole genome sequencing of a 6x and 12x deletion mutant revealed a low number of off-target mutations which were unrelated to Cas9-mediated cleavage. Secretome analyses confirmed the loss of secreted proteins encoded by the deleted genes. Infection tests with the mutants revealed a successive decrease in virulence with increasing numbers of mutated genes, and varying effects of the knockouts on different host plants. Comparative analysis of mutants confirmed significant roles of two polygalacturonases (PG1, PG2) and the phytotoxic metabolites botrydial and botcinins for infection, but revealed no or only weak effects of deletion of the other CDIPs. Nicotiana benthamiana plants with mutated or silenced coreceptors of pattern recognition receptors, SOBIR1 and BAK1, showed similar susceptibility as control plants to infection by B. cinerea wild type and a 12x deletion mutant. These results raise doubts about a major role of manipulation of these plant defence regulators for B. cinerea infection. Despite the loss of most of the known phytotoxic compounds, the on planta secretomes of the multiple mutants retained substantial phytotoxic activity, proving that further, as yet unknown CDIPs contribute to necrosis and virulence. Our study has addressed for the first time systematically the functional redundancy of fungal virulence factors, and demonstrates that B. cinerea releases a highly redundant cocktail of proteins to achieve necrotrophic infection of a wide variety of host plants.