Universität zu Köln

Leisen, Thomas ORCID: 0000-0003-0552-0323, Werner, Janina ORCID: 0000-0002-6421-4783, Pattar, Patrick, Safari, Nassim, Ymeri, Edita, Sommer, Frederik ORCID: 0000-0003-0247-4907, Schroda, Michael ORCID: 0000-0001-6872-0483, Suarez, Ivonne, Collado, Isidro G., Scheuring, David ORCID: 0000-0001-9048-3330 and Hahn, Matthias (2022). Multiple knockout mutants reveal a high redundancy of phytotoxic compounds contributing to necrotrophic pathogenesis of Botrytis cinerea. PLoS Pathog., 18 (3). SAN FRANCISCO: PUBLIC LIBRARY SCIENCE. ISSN 1553-7374

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Abstract

Author summaryBotrytis cinerea is one of the economically most important plant pathogens worldwide, causing pre- and postharvest rot on numerous fruit and vegetable crops. The molecular basis for its ability to invade and kill such a wide range of host plants is poorly understood. The fungus secretes numerous phytotoxic proteins and metabolites into the infected tissue, but their roles for infection have not yet been clarified. 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. The mutants, containing up to 12 gene knockouts, showed normal growth and differentiation in vitro, but significantly delayed infection and reduced lesion formation on different plant tissues. Nevertheless, these mutants remained virulent and still induced plant necrosis, which indicated that a much larger number than the currently known phytotoxic proteins are required for infection and host cell killing. Our work has addressed for the first time the functional complexity of fungal virulence factors, and has prepared the route towards a comprehensive understanding of the necrotrophic lifestyle of B. cinerea 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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Leisen, Thomas UNSPECIFIED orcid.org/0000-0003-0552-0323 UNSPECIFIED Werner, Janina UNSPECIFIED orcid.org/0000-0002-6421-4783 UNSPECIFIED Pattar, Patrick UNSPECIFIED UNSPECIFIED UNSPECIFIED Safari, Nassim UNSPECIFIED UNSPECIFIED UNSPECIFIED Ymeri, Edita UNSPECIFIED UNSPECIFIED UNSPECIFIED Sommer, Frederik UNSPECIFIED orcid.org/0000-0003-0247-4907 UNSPECIFIED Schroda, Michael UNSPECIFIED orcid.org/0000-0001-6872-0483 UNSPECIFIED Suarez, Ivonne UNSPECIFIED UNSPECIFIED UNSPECIFIED Collado, Isidro G. UNSPECIFIED UNSPECIFIED UNSPECIFIED Scheuring, David UNSPECIFIED orcid.org/0000-0001-9048-3330 UNSPECIFIED Hahn, Matthias UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-671774
DOI:	10.1371/journal.ppat.1010367
Journal or Publication Title:	PLoS Pathog.
Volume:	18
Number:	3
Date:	2022
Publisher:	PUBLIC LIBRARY SCIENCE
Place of Publication:	SAN FRANCISCO
ISSN:	1553-7374
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language HYPERSENSITIVE RESPONSE; DEFENSE RESPONSE; VIRULENCE FACTOR; CELL-DEATH; PROTEIN; RECEPTOR; ELICITS; ENDOPOLYGALACTURONASES; RESISTANCE; EFFECTORS Multiple languages Microbiology; Parasitology; Virology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/67177