Improved Protoplast Production Protocol for Fungal Transformations Mediated by CRISPR/Cas9 in Botrytis cinerea Non-Sporulating Isolates.

Botrytis cinerea is a necrotrophic fungus that causes considerable economic losses in commercial crops. Fungi of the genus Botrytis exhibit great morphological and genetic variability, ranging from non-sporogenic and non-infective isolates to highly virulent sporogenic ones. There is growing interest in the different isolates in terms of their methodological applications aimed at gaining a deeper understanding of the biology of these fungal species for more efficient control of the infections they cause. This article describes an improvement in the protoplast production protocol from non-sporogenic isolates, resulting in viable protoplasts with regenerating capacity. The method improvements consist of a two-day incubation period with mycelium plugs and orbital shaking. Special mention is made of our preference for the VinoTaste Pro enzyme in the KC buffer as a replacement for Glucanex, as it enhances the efficacy of protoplast isolation in B459 and B371 isolates. The methodology described here has proven to be very useful for biotechnological applications such as genetic transformations mediated by the CRISPR/Cas9 tool.

Unravelling the Function of the Sesquiterpene Cyclase STC3 in the Lifecycle of Botrytis cinerea.

The genome sequencing of Botrytis cinerea supplies a general overview of the map of genes involved in secondary metabolite synthesis. B. cinerea genomic data reveals that this phytopathogenic fungus has seven sesquiterpene cyclase (Bcstc) genes that encode proteins involved in the farnesyl diphosphate cyclization. Three sesquiterpene cyclases (BcStc1, BcStc5 and BcStc7) are characterized, related to the biosynthesis of botrydial, abscisic acid and (+)-4-epi-eremophilenol, respectively. However, the role of the other four sesquiterpene cyclases (BcStc2, BcStc3, BcStc4 and BcStc6) remains unknown. BcStc3 is a well-conserved protein with homologues in many fungal species, and here, we undertake its functional characterization in the lifecycle of the fungus. A null mutant ΔBcstc3 and an overexpressed-Bcstc3 transformant (OvBcstc3) are generated, and both strains show the deregulation of those other sesquiterpene cyclase-encoding genes (Bcstc1, Bcstc5 and Bcstc7). These results suggest a co-regulation of the expression of the sesquiterpene cyclase gene family in B. cinerea. The phenotypic characterization of both transformants reveals that BcStc3 is involved in oxidative stress tolerance, the production of reactive oxygen species and virulence. The metabolomic analysis allows the isolation of characteristic polyketides and eremophilenols from the secondary metabolism of B. cinerea, although no sesquiterpenes different from those already described are identified.

Comparing Fungal Sensitivity to Isothiocyanate Products on Different Botrytis spp.

Glucosinolates, the main secondary metabolites accumulated in cruciferous flora, have a major impact on fortifying plant immunity against diverse pathogens. Although Botrytis cinerea exhibits varying sensitivity to these compounds, current research has yet to fully understand the intricate mechanisms governing its response to glucosinolates. Different species of the genus Botrytis were exposed to glucosinolate-derived isothiocyanates, revealing that B. fabae, B. deweyae, and B. convolute, species with the mfsG transporter gene (Bcin06g00026) not detected with PCR, were more sensitive to isothiocyanates than Botrytis species containing that gene, such as B. cinerea, B. pseudocinerea, and B. byssoidea. This finding was further corroborated by the inability of species with the mfsG gene not detected with PCR to infect plants with a high concentration of glucosinolate-derived isothiocyanates. These results challenge established correlations, revealing varying aggressiveness on different plant substrates. An expression analysis highlighted the gene's induction in the presence of isothiocyanate, and a bioinformatic investigation identified homologous genes in other Botrytis species. Our study underscored the importance of advanced biotechnology to help understand these proteins and thus offer innovative solutions for agriculture.

From Genes to Molecules, Secondary Metabolism in Botrytis cinerea: New Insights into Anamorphic and Teleomorphic Stages.

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.

Structures, Occurrences and Biosynthesis of 11,12,13-Tri-nor-Sesquiterpenes, an Intriguing Class of Bioactive Metabolites.

The compounds 11,12,13-tri-nor-sesquiterpenes are degraded sesquiterpenoids which have lost the C3 unit of isopropyl or isopropenyl at C-7 of the sesquiterpene skeleton. The irregular C-backbone originates from the oxidative removal of a C3 side chain from the C15 sesquiterpene, which arises from farnesyl diphosphate (FDP). The C12-framework is generated, generally, in all families of sesquiterpenes by oxidative cleavage of the C3 substituent, with the simultaneous introduction of a double bond. This article reviews the isolation, biosynthesis and biological activity of this special class of sesquiterpenes, the 11,12,13-tri-nor-sesquiterpenes.