The fungus Ustilaginoidea virens causes rice false smut, a major disease of
rice (Oryza sativa). However, there are only limited molecular studies on
this pathogen due to the lack of techniques for studying targeted gene
disruption mutants. The research team of Yafeng Liang from the Northwest A&F
University in China used the CRISPR-Cas9 system to generate mutants with
deleted USTA ustiloxin and UvSLT2 MAP kinase genes.



Three gRNAs targeting USTA, namely UA01, UA13, and UA21, were transformed
into U. virens. For all three, the gene replacement frequency was higher
when the Cas9 and gRNA constructs were transformed on the same vector. UA01
had the highest knockout frequency of 90% for generating ustA mutants. No
off-target mutations were detected in the ustA mutants generated via any of
the three gRNAs.



For UvSLT2, the gene replacement frequency was 50% with CRISPR-Cas9. While
ustA mutants had no detectable phenotypes, Uvslt2 mutants had slightly
reduced growth rate and had over 70% reduction in conidiation. Deletion of
UvSLT2 also increased sensitivity to cell wall stresses but tolerance to
hyperosmotic or oxidative stresses.



These results showed that the CRISPR-Cas9 system can be used as an efficient
gene editing approach in U. virens and could be used to develop varieties
resistant to false smut.



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