Abstract
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.
Highlights
Botrytis cinerea is considered as one of the most important plant pathogenic fungi, causing severe pre- and postharvest losses on a large variety of fruits, vegetables and other crops worldwide [1]
Botrytis cinerea is one of the economically most important plant pathogens worldwide, causing pre- and postharvest rot on numerous fruit and vegetable crops
By using an optimized transformation protocol based on the powerful CRISPR/Cas technology, we have been able to eliminate most of the currently known phytotoxic compounds in individual B. cinerea strains
Summary
Botrytis cinerea is considered as one of the most important plant pathogenic fungi, causing severe pre- and postharvest losses on a large variety of fruits, vegetables and other crops worldwide [1]. Several mechanisms have been reported to promote necrotrophic infection of B. cinerea, such as the secretion of plant cell death inducing proteins (CDIPs) and cell wall degrading enzymes, the release of phytotoxic metabolites and organic acids, and the acidification of the host tissue [2–4]. B. cinerea releases two major phytotoxic families of metabolites, the sesquiterpenoid botrydial and its relatives, and the polyketides botcinic acid and its derivatives, botcinins. Mutants deficient in the biosynthesis of either botrydial or botcinins were unaffected in virulence, a double mutant lacking both toxins showed reduced infection [9, 10]. Plant cell wall degrading enzymes (CWDE) are essential for tissue mazeration by necrotrophic pathogens, but because of their redundancy, the contributions of individual members for pathogenesis are difficult to determine. An endo-arabinanase (BcAra1) was found to be required for full
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