Abstract
The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain.
Highlights
Botrytis cinerea is a notorious phytopathogenic ascomycete that causes gray mold disease in more than 200 host plant species and significantly damages large amounts of pre- and postharvest crops around the world every year [1,2]
The novel Agrobacterium vector pCBh1 for gene overexpression was constructed based on the pBHt2 backbone and contained two cassettes in its tumor-inducing plasmid DNA (T-DNA) region: a hygromycin-resistance cassette derived from the vector pBHt2 [30] and an expression cassette, in which the MCS for insertion of targeted genes was driven by the Aspergillus nidulans oliC promoter and terminated by the Aspergillus nidulans trpC terminator
The T-DNA region of pCBg1 contains two cassettes: an expression cassette that comprises the same modules as pCBh1 and a glufosinate-resistance cassette, which consists of the Aspergillus nidulans trpC promoter derived from the vector pBHt2, the basta-resistance gene derived from the vector pLOB7 [37] and the CaMV35S polyA terminator derived from the vector pCAMBA-1300-221
Summary
Botrytis cinerea is a notorious phytopathogenic ascomycete that causes gray mold disease in more than 200 host plant species and significantly damages large amounts of pre- and postharvest crops around the world every year [1,2]. The B. cinerea T4 and B05.10 genome sequences predicted 43 genes probably encoding key enzymes for secondary metabolite (SM) biosynthesis [5]. This fungus has been found to synthesize several phytohormones: IAA [6], ethylene [7], cytokinins [8], and ABA [9]. These secondary metabolites and phytohormones may have potential economical value, and together with B. cinerea have been used for the fermentative production of ABA at the industrial scale [10,11,12]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
