Introduction of Large Sequence Inserts by CRISPR-Cas9 To Create Pathogenicity Mutants in the Multinucleate Filamentous Pathogen Sclerotinia sclerotiorum

• DOI: 10.1128/mbio.00567-18
• Publication Date: 2018-06-25T13:36:13Z
• Authors: Jingtao Li; Yanhua Zhang; Yucheng Zhang; Pei-Ling Yu; Hongyu Pan; Jeffrey A. Rollins
• Keywords: Sclerotinia Sclerotiorum; Functional Genomics

The necrotrophic fungal plant pathogen Sclerotinia sclerotiorum is responsible for substantial global crop losses annually resulting in localized food insecurity and loss of livelihood. Understanding the basis this broad-host-range aggressive pathogenicity hampered by quantitative nature both host resistance virulence. To improve understanding, methods efficient functional gene characterization that build upon existing complete S. genome sequence are needed. Here, we report on development a clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (CRISPR-Cas9)-mediated strategy creating disruption mutants application technique exploring roles known hypothesized virulence factors. A key finding research transformation with circular plasmid encoding Cas9, target single guide RNA (sgRNA), selectable marker resulted high frequency targeted, insertional mutation. We observed 100% integrated large rearranged segments transforming at site facilitated nonhomologous end joining (NHEJ) repair pathway. This result was confirmed multiple sites within same three independent wild-type isolates second gene. Targeting previously characterized Ssoah1 allowed us to confirm loss-of-function CRISPR-Cas9-mediated explore new aspects mutant phenotype. Applying technology create mutations uncharacterized determine requirement melanin accumulation infection structure function.IMPORTANCE Fungi cause diseases rotting or blighting tissue limited specificity remain among most difficult control. largely due understanding pathogenicity. mechanistic requires ability manipulate candidate genes test hypotheses regarding their disease development. notorious these so-called pathogens. work described here provides method rapidly constructing vectors efficiency compared methods. characterize functions sclerotiorum, oxalic acid production as factor fungus demonstrate not required infection. Using approach, pace related will increase.