Multiple point mutations in PsORP1 gene conferring different resistance levels to oxathiapiprolin confirmed using CRISPR–Cas9 in Phytophthora sojae

Abstract

BACKGROUND: Oxathiapiprolin is among the first commercial oxysterol-binding protein inhibitors (OSBPIs) developed by DuPont Corporation and shows excellent activity against plant-pathogenic oomycetes. Although more than 21 target site mutations have been identified in insensitive oomycetes, only G770V, G839W, and ΔN837 have been verified to confer oxathiapiprolin resistance in Phytophthora capsici or P. sojae. The effect of other mutations on OSBPIs sensitivity requires urgent investigation. RESULTS: P. sojae transformants containing 16 mutations of PsORP1 were recovered using the CRISPR-Cas9 system. Transformants containing L733W, S768F, S768Y, N837Y, N837F, P861H, L863W, or I877Y showed high oxathiapiprolin resistance, with resistant factors (RFs) > 3000. Point mutations S768K, S768I, G770L, G770P, G770A, ΔG818/F819, N837I, and I877F exhibited low resistance, with RFs < 80. Phenotype assays revealed that the most highly resistant transformants showed enhanced or similar pathogenicity, oospore production, and cyst gemination. However, most transformants displayed decreased sporangia and zoospore production compared with parental wild-type P6497.CONCLUSION: This study demonstrated that L733W, S768F, S768Y, N837Y, N837F, P861H, L863W, and I877Y in PsORP1 confer high oxathiapiprolin resistance in P. sojae.

Transcriptional Variability Associated With CRISPR-Mediated Gene Replacements at the Phytophthora sojae Avr1b-1 Locus

“…However, our results are unusual in that only a single copy of the transgene is inserted into the genome, at the Avr1b-1 locus. Our results are also unusual because other studies have reported normal expression of incoming transgenes after gene replacements at other Phytophthora loci (Ma et al, 2017;Miao et al, 2020;Ochola et al, 2020).…”

Genetic mechanism, baseline sensitivity and risk of resistance to oxathiapiprolin in oomycetes

“…10 Phytophthora sojae transformants containing 16 mutations in the OSBP gene were recovered using the CRISPR-Cas9 system half of which associated with high resistance factors. 22 A deletion at position N837 was also reported to confer resistance to oxathiapiprolin in Phytophthora capsici and Phytophthora sojae. More recently, seven heterozygous point mutations in the target protein were found in Phytophthora capsici mutants resistant to fluoxapiprolin, another OSBP inhibitor.…”

Activity and Resistance Assessment of a New OSBP Inhibitor, R034-1, in Phytophthora capsici and the Detection of Point Mutations in PcORP1 that Confer Resistance

“…Furthermore, P. sojae transformants containing amino acid changes of L733W, S768F/Y/K/I, G770V/L/P/A, N837Y/F/I, I877Y/F, G839W, P861H, L863W, and ΔG818/F819/837 in the target protein also showed oxathiapiprolin resistance to various extents. , A similarity of structure and host range, as well as strong cross-resistance, have been found between R034-1 and oxathiapiprolin. In addition, oxathiapiprolin-resistant mutants HNJZ10-E and LP3-M were also less sensitive to the compound R034-1, and R034-1-resistant mutants also had stably inherited resistance to oxathiapiprolin.…”

“…We speculate that different amino acid substitutions or a deletion at the same position could cause different changes in the protein structure, thereby affecting the binding of fungicide to the target protein. Miao et al also found that P. sojae transformants containing S768F/Y, G770V, N837Y/F, and I877Y in PsORP1 conferred high oxathiapiprolin resistance, but transformants containing S768I, G770L/P/A, N837I, and I877F exhibited low oxathiapiprolin resistance.…”

“…We speculate that different amino acid substitutions or a deletion at the same position could cause different changes in the protein structure, thereby affecting the binding of fungicide to the target protein. Miao et al 41 also found that P. sojae transformants containing S768F/Y, G770V, N837Y/F, and I877Y in PsORP1 conferred high oxathiapiprolin resistance, but transformants containing S768I, G770L/P/A, N837I, and I877F exhibited low oxathiapiprolin resistance. The same phenomenon was also observed in other fungicides; for example, an H272L mutation in succinate dehydrogenase B (SdhB) in Botrytis cinerea led to higher resistance to boscalid than the H272R/Y mutation, 43 and A577T in myosin5 caused lower resistance to phenamacril in Fusarium asiaticum than A577G.…”