Characterization of SsHog1 and Shk1 Using Efficient Gene Knockout Systems through Repeated Protoplasting and CRISPR/Cas9 Ribonucleoprotein Approaches in Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is the causal agent of sclerotinia stem rot in over 400 plant species. In a previous study, the group III histidine kinase gene of S. sclerotiorum (Shk1) revealed its involvement in iprodione and fludioxonil sensitivity and osmotic stress. To further investigate the fungicide sensitivity associated with the high-osmolarity glycerol (HOG) pathway, we functionally characterized SsHog1, which is the downstream kinase of Shk1. To generate knockout mutants, split marker transformation combined with a newly developed repeated protoplasting method and CRISPR/Cas9 ribonucleoprotein (RNP) delivery approach were used. The pure SsHog1 and Shk1 knockout mutants showed reduced sensitivity to fungicides and increased sensitivity to osmotic stress. In addition, the SsHog1 knockout mutants demonstrated reduced virulence compared to Shk1 knockout mutants and wild-type. Our results indicate that the repeated protoplasting method and RNP approach can generate genetically pure homokaryotic mutants and SsHog1 is involved in osmotic adaptation, fungicide sensitivity, and virulence in S. sclerotiorum.

Plant and Fungal Genome Editing to Enhance Plant Disease Resistance Using the CRISPR/Cas9 System.

Crop production has been substantially reduced by devastating fungal and oomycete pathogens, and these pathogens continue to threaten global food security. Although chemical and cultural controls have been used for crop protection, these involve continuous costs and time and fungicide resistance among plant pathogens has been increasingly reported. The most efficient way to protect crops from plant pathogens is cultivation of disease-resistant cultivars. However, traditional breeding approaches are laborious and time intensive. Recently, the CRISPR/Cas9 system has been utilized to enhance disease resistance among different crops such as rice, cacao, wheat, tomato, and grape. This system allows for precise genome editing of various organisms via RNA-guided DNA endonuclease activity. Beyond genome editing in crops, editing the genomes of fungal and oomycete pathogens can also provide new strategies for plant disease management. This review focuses on the recent studies of plant disease resistance against fungal and oomycete pathogens using the CRISPR/Cas9 system. For long-term plant disease management, the targeting of multiple plant disease resistance mechanisms with CRISPR/Cas9 and insights gained by probing fungal and oomycete genomes with this system will be powerful approaches.