Hygromycin B resistance (UF1-oah1-3-6), tolerance (UF1-oah1-3-6➀, ➁), and sensitivity (UF1, UF1-oah13-6-➀,-➁) were assayed at 3 and 8 days after inoculation (DAI) of PDA-HygB medium.
Oxalate production (acidification) is indicated by a change in PDA-BPB medium from blue to yellow. (A) Single-ascospore progeny (➀, ➁,-➀, and-➁) of primary transformant UF1-oah1-3-6 (wild-type "UF1" background, Ssoah1 target site 3, strain number 6) were assayed for OA production and hygromycin B (HygB) sensitivity on potato dextrose agar (PDA) medium supplemented with bromophenol blue (BPB) and HygB, respectively. Applying this technology to create mutations in a second previously uncharacterized gene allowed us to determine the requirement for melanin accumulation in infection structure development and function.Ĭharacterization of CRISPR-Cas9-inserted sequences following meiosis. Targeting the previously characterized Ssoah1 gene allowed us to confirm the loss-of-function nature of the CRISPR-Cas9-mediated mutants and explore new aspects of the mutant phenotype. sclerotiorum and in a second independent gene. This result was confirmed in multiple target sites within the same gene in three independent wild-type isolates of S. We observed that 100% of the mutants integrated large rearranged segments of the transforming plasmid at the target site facilitated by the nonhomologous end joining (NHEJ) repair pathway. A key finding of this research is that transformation with a circular plasmid encoding Cas9, target single guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted, insertional gene mutation. Here, we report on the development of a clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (CRISPR-Cas9)-mediated strategy for creating gene disruption mutants and the application of this technique for exploring roles of known and hypothesized virulence factors. To improve this understanding, methods for efficient functional gene characterization that build upon the existing complete S. Understanding the basis of this broad-host-range and aggressive pathogenicity is hampered by the quantitative nature of both host resistance and pathogen virulence. The necrotrophic fungal plant pathogen Sclerotinia sclerotiorum is responsible for substantial global crop losses annually resulting in localized food insecurity and loss of livelihood.
0 kommentar(er)
