Resistance Mechanism Of Oxathiapiprolin And The Functional Characteristics Of ORPs In Phytophthora

Oxathiapiprolin is a novel fungicide with a new mode of action and chemical structure,and it was developed by Dupon in 2007.It has been shown to have substantial activity against Phytophthora and downy mildew pathogen with EC50 value of 10-4 ?g/ml.The results of binding assays and affinity chromatography have shown that the target of oxathiapiprolin is the oxysterol binding protein related protein(ORP).To date,data regarding the resistance risk,molecular mechanism,and functional characteristics of ORPs in Phytophthora are limited.In this study,the related studies were conducted and the results were as follow:First,the EC50 values of the 175 P.capsici isolates collected from 28 provinces,ranged from 3.19×10-4 ?g/ml to 9.86 ×10-4 ?g/ml and produced a unimodal distribution with a mean of 5.61×10-4 ?g/ml.These results provided strong evidence that no oxathiapiprolin-resistant subpopulations exist in wild populations of P.capsici.The results of the current study can be used to provide a baseline for monitoring changes in the sensitivity of P.capsici populations to oxathiapiprolin.Second,two kinds of oxathiapiprolin-resistant P.capsici mutants were obtained by fungicide adaption.The LP3-mutants exhibited strong fitness in a range of developmental stages,including mycelial growth,sporangium production,cystospore germination,and most importantly,pathogenicity.Combining the difficulty level of mutants' selection,the fitness of mutants and the soil-borne nature of P.capsici,we speculated that oxathiapiprolin possesses moderate resistant risk in P.capsici.Thirdly,point mutations,G839W,G770V and ?N837 in PcORP1 could cause high oxathiapiprolin resistant level in P.capsici were confirmed using traditional ectopic transformation and CRISPR/Cas9.Point deletion,?N837 in PsORP1 could also lead oxathiapiprolin resistance in P.sojae.Transformants with PcORP1?N837,PsORP1?N837,PcORP1G770V,obtained by CRISPR/Cas9,showed high fitness.Two molecular detection methods,AS-PCR and CAPS were established for the detection of PcORP1G839W in the field.Fourthly,in this study,we did not obtain transformants with high silence efficiency of PcORP1 or PsORP1.Homozygous PcORP1 knock-out transformants were also not be selected.Considering that oxathipiprolin exhibited super high activity by binding target protein ORP1,we speculated that PcORP1 and PsORP1 were essential protein for P.capsici and P.sojae,respectively.PcORP1 and PsORP1 exhibited typical plasma membrane and ER contact site localization in Nicotiana bentamihana.PH domain could bind with the ligand in PM,and the predicted transmembrane domain localized in ER.The same localization trait was observed in the mycelia cell of P.capsici or P.sojae.Therefore,we thought PcORP1 or PsORP1 play important role in the membrane contact sites.Last,deletion of PsORP2 using CRISPR/Cas9 had no significant effect on sporangia production,zoospore production,cyst germination,oospore production,virulence,or oxathiapiprolin sensitivity of P.sojae.PsORP1 also was not upregulated in ?PsORP2 transformants.Collectively our studies demonstrate that PsORP2 is not an essential protein for development or virulence in P.sojae under the conditions we tested.