Multiple knockout mutants reveal a high redundancy of phytotoxic compounds contributing to necrotrophic pathogenesis of Botrytis cinerea

作者：Thomas Leisen, J.A. Werner, Patrick Pattar, Nassim Safari, Edita Ymeri, Frederik Sommer, Michael Schroda, Ivonne Suárez, Isidro G. Collado, David Scheuring, Matthias Hahn · 发表于：PLoS Pathogens · 年份：2022 · DOI：10.1371/journal.ppat.1010367 · 被引用次数：120 · 研究领域：Plant-Microbe Interactions and Immunity、Plant Virus Research Studies、Plant Disease Resistance and Genetics

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 t...