Chemically Induced Cryptic Sesquiterpenoids and Expression of Sesquiterpene Cyclases in Botrytis cinerea Revealed New Sporogenic (+)-4-Epieremophil-9-en-11-ols

Abstract

The sequencing of the genomes of the B05.10 and T4 strains of the fungus Botrytis cinerea revealed an abundance of novel biosynthetic gene clusters, the majority of which were unexpected on the basis of the previous analyses of the fermentation of these and closely related species. By systematic alteration of easy accessible cultivation parameters, using chemical induction with copper sulfate, we have found a cryptic sesquiterpenoid family with new structures related to eremophil-9-ene, which had the basic structure of the sesquiterpene (+)-5-epiaristolochene ((+)-4-epieremophil-9-ene). An expression study of the sesquiterpene cyclase genes present in the Botrytis cinerea genome, under culture conditions, is reported. In general, a 3 day delay and a higher BcSTC genes expression were observed when copper (5 ppm) was fed to the fermentation broth. In addition, to the observed effect on the BcBOT2 (BcSTC1) gene, involved in the biosynthesis of the botrydial toxin, a higher expression level for BcSTC3 and BcSTC4 was observed with respect to the control in the strain B05.10. Interestingly, under copper conditions, the BcSTC4 gene was the most expressed gene in the Botrytis cinerea UCA992 strain. In vitro evaluation of the biological role of these metabolites indicates that they contributed to the conidial development in B. cinerea and appear to be involved in self-regulation of the production of asexual spores. Furthermore, they promoted the formation of complex appressoria or infection cushions.

Biosynthesis of abscisic acid in fungi: identification of a sesquiterpene cyclase as the key enzyme in Botrytis cinerea

“…Bcstc1 / Bcbot2 which encodes the key enzyme for the biosynthesis of botrydial, and the previously characterized Bcaba1, Bcaba2, Bcaba3 and Bcaba4 genes were also included in the experiment. The expression profiles of Bcstc and Bcaba genes were established using the specific primers and three housekeeping genes as in our previous study (Pinedo et al ., ; Supporting Information Table S1). Actin, β‐tubulin and elongation factor EF‐1α‐coding genes showed a stable expression level during fermentation assays and were used as references to quantify the relative expression of STC‐coding and Bcaba genes.…”

“…These differences in metabolite production could be correlated with the levels of expression of the STC‐coding genes. By utilizing identical culture conditions, we could identify Bcstc1/Bcbot2 as the most expressed Bcstc gene in the strain B05.10 (Pinedo et al ., ) and Bcstc5/Bcaba5 as the most highly expressed Bcstc gene in the strain ATCC58025 (this study). Moreover, Bcstc5/Bcaba5 appeared to be approximately sixfold more highly expressed in ATCC58025 than in B05.10.…”

“…While Bcstc1/Bcbot2 and Bcstc5/Bcaba5 are now identified as the key genes for the biosynthesis of botrydial and ABA respectively, the role of the other Bcstc genes remains unknown. Most B. cinerea strains produce botrydial in significant amounts in standard culture conditions, i.e., 7–10 µg ml −1 (Reino et al ., ; Pinedo et al ., ; Porquier et al ., ), but the other sesquiterpenes are often below the detection level. In comparison, there is an apparent redirection of FPP‐derived metabolism in the strain ATCC58025, resulting in a significant overproduction of ABA.…”

“…For quantitative RT‐qPCR expression analyses, 1 µg of RNA of each sample was reverse‐transcribed into cDNA using DyNAmo cDNA Synthesis Kit (Thermo‐Scientific). A total of thirteen specific pairs of primers were used in this study; five of them were for the STC‐coding genes, four for the Bcaba genes, one for the P450 monooxygenase coding gene Bcin01g03510 and three pairs for amplification of housekeeping genes actin , β‐tubulin and EF‐1α (Pinedo et al ., ; Supporting Information Table S1). Conventional PCR confirmed the correct specificity and selectivity of each pair of primers, using genomic DNA or cDNA from strain ATCC58025 as template.…”

“…Conventional PCR confirmed the correct specificity and selectivity of each pair of primers, using genomic DNA or cDNA from strain ATCC58025 as template. Real‐time PCR amplifications were carried out using the CFX96 Touch™ Real‐Time PCR Detection System (BioRad) and Maxima SYBRGreen qPCR MasterMix (Thermo‐Scientific) as previously described (Pinedo et al ., ) Optimal primer concentrations and annealing temperature were established and set at 200 nM and 60°C respectively, except for the EF‐1α gene, whose annealing temperature was set at 56°C. The construction of standard curves, the calculation of Mean Normalized Expression and the statistical analysis of the expression of the STC‐coding genes were performed using methods from Gil‐Salas and colleagues ().…”