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.

BACKGROUND: Oxathiapiprolin, developed by DuPont, is the only commercial oxysterol-binding protein inhibitor (OSBPI) of oomycete pathogens. Although the activity of oxathiapiprolin on some Pythium spp. and Phytopythium spp. has been reported, it has not been tested on many other species, and little is known about the mechanisms of Pythium spp. that are tolerant to it. RESULTS: Oxathiapiprolin exhibited a strong inhibitory effect on mycelial growth of Phy. litorale, Phy. helicoides and Phy. chamaehyphon, with EC 50 values ranging from 0.002 to 0.013 ∼g mL −1. It also showed good effectiveness against Py. splendens and two Py. ultimum isolates, with EC 50 values ranging from 0.167 to 0.706 ∼g mL −1 , but showed no activity against 14 other Pythium spp. Oxathiapiprolin provoked a slight upregulation of PuORP1 in Py. ultimum, but it did not lead to PaORP1-1 or PaORP1-2 overexpression in Py. aphanidermatum. Transformation and expression of PuORP1, PaORP1-1 or PaORP1-2 in the sensitive wild-type Phytophthora sojae isolate P6497 confirmed that either the PuORP1, PaORP1-1 or PaORP1-2 was responsible for the observed oxathiapiprolin tolerance. CONCLUSION: This study showed that oxathiapiprolin had excellent activity against Phytopythium spp. but displayed a differentiated activity against different Pythium spp. ORP1s in Pythium spp. are positively related to the tolerance of Pythium species to oxathiapiprolin.

Proteins that contain the FYVE zinc-finger domain are recruited to PtdIns3P-containing membranes, participating in numerous biological processes such as membrane trafficking, cytoskeletal regulation, and receptor signaling. However, the genome-wide distribution, evolution, and biological functions of FYVE-containing proteins are rarely reported for oomycetes. By genome mining of Phytophthora sojae, two proteins (PsFP1 and PsFP2) with a combination of the FYVE domain and the PX domain (a major phosphoinositide binding module) were found. To clarify the functions of PsFP1 and PsFP2, the CRISPR/Cas9-mediated gene replacement system was used to knock out the two genes respectively. Only heterozygous deletion mutants of PsFP1 were recovered, and the expression level of PsFP1 in the heterozygous knockout transformants was significantly down-regulated. These PsFP1 mutants showed a decrease in mycelial growth and pathogenicity and were more sensitive to hydrogen peroxide. These phenotypes were recovered to the level of wild-type by overexpression PsFP1 gene in the PsFP1 heterozygous knockout transformant. In contrast, deletion of PsFP2 had no significant effect on vegetative growth, asexual and sexual reproduction, pathogenicity, or oxidative stress sensitivity. PsFP1 was primarily localized in vesicle-like structures and both the FYVE and PX domains are important for its localization. Overall, our results indicate that PsFP1 plays an important role in the vegetative growth and virulence of P. sojae.