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Gene‐disruption of the entomopathogenic fungus Beauveria bassiana incubated with dsRNA
Author(s) -
Shin Tae Young,
Lee Mi Rong,
Lee Se Jin,
Kim Jong Cheol,
Kim Sihyeon,
Park So Eun,
Kim Woo Jin,
Kim Jae Su
Publication year - 2021
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.202100049
Subject(s) - beauveria bassiana , biology , rna silencing , protoplast , entomopathogenic fungus , microbiology and biotechnology , green fluorescent protein , bassiana , gene , agar plate , gene expression , rna , rna interference , biological pest control , botany , genetics , bacteria
The species of Beauveria bassiana is widely used for the management of agricultural insect pests. In this study, we integrated egfp ‐double‐stranded RNA (dsRNA) to a previously generated egfp ‐expressing B. bassiana transformant (Bb‐egfp#3) using a protoplast integration method. The Bb‐egfp#3 protoplast was mixed with the dsRNA under PEG/CaCl 2 conditions and liquid‐cultured in Sabouraud dextrose broth for 5 days. A control culture followed the same procedure without dsRNA. Bb‐egfp#3/ egfp ‐dsRNA cultures showed very low fungal growth (OD 630 = 0.2) compared to the control culture, Bb‐egfp#3 only (OD 630 = 1.1). Screening of possible transformants on Sabouraud dextrose agar revealed a transformant T3, without egfp signal. T3 was confirmed as B. bassiana through sequencing of conserved genes and insect bioassays. Interestingly, the genomic egfp fragment of T3 was disrupted, and the egfp signal was not detected over four subcultures, which was also confirmed by RNA‐seq of Bb‐egfp#3 and T3. This study provides an interesting observation that protoplast integration with dsRNA could possibly generate significantly reduced gene expression in B. bassiana and it is stable across several generations.