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

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.

Cytotoxic activity of Nep1‐like proteins on monocots

“…For B . cinerea , knockout mutants of multiple phytotoxic proteins, including Nep1 and Nep2, remained virulent on multiple host species, demonstrating the functional redundancy of virulence factors in Botrytis species (Leisen et al ., 2022). For these reasons, we did not make an effort to generate a Nep1 Nep2 double mutant in B .…”

Analysis of plant cell death-inducing proteins of the necrotrophic fungal pathogens Botrytis squamosa and Botrytis elliptica

“…This list still comprises a few dozen genes, and experiments are ongoing to purify CF samples through ion exchange chromatography and to test the activity of fractions for cell death-inducing activity. These efforts should result in reducing the number of candidate genes to a manageable number for functional analysis through CRISPR/Cas9-mediated deletion ( Leisen et al., 2020 ; Leisen et al., 2022 ).…”

Identification of candidate genes associated with resistance against race 0 of Colletotrichum lentis in Lens ervoides

“…Cell death in the form of a hypersensitive response (HR) or programmed cell death (PCD) is a highly effective defense strategy of plants against biotrophic pathogens as it deprives the pathogen of essential viable host plant cells 28 . However, thriving in the presence of cell death, this mechanism can also be manipulated by necrotrophic pathogens to effectively increase susceptibility 29 . In hemibiotrophs, HR could be fatal during the biotrophic phase, but may be promoted by the pathogen during the transition to the necrotrophic phase.…”