Molecular mechanism of resistance to CAA and OSBPI fungicides in Phytophthora capsici and P. sojae
Xili Liu: China Agricultural University
<div>Oomycetes are among the most problematic group of disease-causing organisms in agriculture. Flumorph is a CAA fungicide that has high activity against oomycete pathogens. Sequencing analysis indicated that CesA3, which is one of the four cellulose synthase enzymes, contained three substitutions in the amino acid sequence in the resistant <em>P. capsici</em> isolates. Back-transformation confirmed that the F1073L, G1105A, V1109L substitutions were linked to CAA resistance, and each mutation could make different contributions to the resistance phenotype. Meanwhile, five stable mutant types were obtained on flumorph-amended media in <em>P. sojae</em>. Further research found that the stepwise evolution of<em> CesA3</em> resulted in increasing resistance to CAA fungicides. The Type I mutants were found to have a single point mutation (I1027V) in CesA3 protein which is the target protein of CAA fungicides, while the other mutant types not only carried the I1027V mutation but also possessed a second mutation V1025L, G1020A, G1020S or Q992H, respectively. Site-direct mutagenesis using CRISPR/Cas9 technology completely validated the role of each mutation, both individually and in combination with I1027V, with the transformants exhibiting similar resistance patterns to the original mutants. Oxathiapiprolin is the first of OSBPI fungicides. In our previous study, point mutations of G770V and G839W in oxysterol binding protein-related protein-1 (ORP1) were detected in oxathiapiprolin-resistant <em>P</em>.<em> capsici</em> mutants. In the current study, the CRISPR/Cas9 system was used to verify the effect of these two point mutations on characteristics of <em>P</em>. <em>capsici</em>. Transformants containing heterozygous G770V and G839W mutations in PcORP1 showed high levels of oxathiapiprolin resistance. These results will be useful for the development of the anti-resistance novel OSBPI fungicides.</div>
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