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.
Cultivation of the phytopathogenic fungus Botrytis cinerea using sublethal amounts of copper sulfate yielded a cryptic sesquiterpenoids family, which displayed the basic chemical structure of (+)-4-epi-eremophil-9-ene. The biosynthesis pathway was established, and the route involved the likely transformation of the diphosphate of farnesyl (FDP), to give a cis-fused eudesmane cation, through (S)-hedycaryol, finally yielding the (+)-4-epi-eremophil-9-enol derivatives. An expression study of genes that code for the sesquiterpene cyclases (STC), including the recently reported gene Bcstc7 present in the B. cinerea genome, was performed in order to establish the STC involved in this biosynthesis. The results showed a higher expression level for the Bcstc7 gene with respect to the other stc1–5 genes in both wild-type strains, B05.10 and Botrytis cinerea UCA992. Deletion of the Bcstc7 gene eliminated (+)-4-epi-eremophilenol biosynthesis, which could be re-established by complementing the null mutant with the Bcstc7 gene. Chemical analysis suggested that STC7 is the principal enzyme responsible for the key step of cyclization of FDP to eremophil-9-en-11-ols. Furthermore, a thorough study of the two wild-types and the complemented mutant revealed four new eremophilenol derivatives whose structures are reported here.
The ascomycete Botrytis cinerea Pers. Fr., classified within the family Sclerotiniaceae, is the agent that causes grey mould disease which infects at least 1400 plant species, including crops of economic importance such as grapes and strawberries. The life cycle of B. cinerea consists of two phases: asexual (anamorph, Botrytis cinerea Pers. Fr.) and sexual (teleomorph, Botryotinia fuckeliana (de Bary) Wetzel). During the XVI International Symposium dedicated to the Botrytis fungus, which was held in Bari in June 2013, the scientific community unanimously decided to assign the most widely used name of the asexual form, Botrytis, to this genus of fungi. However, in the literature, we continue to find articles referring to both morphic stages. In this review, we take stock of the genes and metabolites reported for both morphic forms of B. cinerea between January 2015 and October 2022.
Botrytis cinerea has high potential for the production of specialized metabolites. The recent resequencing of the genome of the B05.10 strain using PacBio technology and the resulting update of the Ensembl Fungi (2017) database in the genome sequence have been instrumental in identifying new genes that could be involved in secondary metabolism. Thus, a new sesquiterpene cyclase (STC) coding gene (Bcstc7) has been included in the gene list from this phytopathogenic fungus. We recently constructed the null and complement transformants in STC7 which enabled us to functionally characterize this STC. Deletion of the Bcstc7 gene abolished (+)-4-epieremophilenol biosynthesis, and could then be re-established by complementing the null mutant with the Bcstc7 gene. Chemical analysis of the complemented transformant suggests that STC7 is the principal enzyme responsible for the key cyclization step of farnesyl diphosphate (FDP) to (+)-4-epi-eremophil-9-en-11-ols.A thorough analysis of the metabolites produced by two wild-type strains, B05.10 and UCA992, and the complemented mutant compl ΔBcstc7 niaD , revealed the isolation and structural characterization of six 11,12,13-trinor-eremophilene derivatives, in addition to a large number of known eremophilen-11-ol derivatives. The structural characterization was carried out by extensive spectroscopic techniques. The biosynthesis of these compounds is explained by a retroaldol reaction or by dehydration and oxidative cleavage of C11-C13 carbons. This is the first time that this interesting family of degraded eremophilenols has been isolated from the phytopathogenous fungus B. cinerea.
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