Functional Analysis Of Phytophthora Sojae Apoplastic Effector PsLac1

Soybean root rot disease caused by Phytophthora sojae(P.sojae)is the destructive diseases to soybean production,which causes annual losses in soybean production worldwide.During infection,P.sojae secretes a large number of virulence factors to promote infection in soybean.These virulence factors are in general called effectors.According to their own characteristics,these effectors can be classified as enzymatic proteins,non-enzymatic proteins,secondary metabolites and small molecule RNAs.Depending on the subcellular localizations,the P.sojae effectors are divided into intracellular effectors and apoplastic effectors.Studies on the function of effectors are instrumental for understanding the pathogenesis of P.sojae,which will provide new research ideas for designing control strategies on the soybean root rot.Previously work in our lab identified multiple apoplastic effectors secreted by P.sojae.In this study,we focus on an effector named PsLac1 which encodes a lactonase.We combined bioinformatics analysis,genetic and biochemical experiments to analyze the biological functions of PsLac1.The contents are as follows:PsLac1 is a virulent effector of Phytophthora sojae: q RT-PCR showed that PsLac1 was greatly up-regulated at the early stage,suggesting that PsLac1 may play an important role during P.sojae infection.In order to further study whether PsLac1 has the virulent function of promoting P.sojae infection in soybean,we used CRISPR/Cas9 gene editing technology to knock-out PsLac1.Two PsLac1 knock-out mutants were obtained,which showed no significant differences in hyphae growth compared to the wild type.Infection assays showed that the PsLac1 knock-out mutants are less virulent on the susceptible soybean hypocotyl compared to control,suggesting that PsLac1 is involved in the pathogenicity of P.sojae.PsLac1 has 6-phosphogluconolactonase activity: The SMART website predicts that PsLac1 contains a lactonase domain.In order to confirm the lactonase activity of PsLac1,the protein was expressed using Pichia pastoris heterologous expression system.In vitro enzyme activity experiment proved that PsLac1 was a 6-phosphogluconolactonase.Sequence alignment between PsLac1 and 6-phosphogluconolactonase in other species have been reported,we predicted six potential active sites required for enzyme activity.Pure proteins of the seven mutants were obtained using Pichia pastoris heterologous expression system.But mutations at these sites did not affect PsLac1 lactonase activity.These results indicated that PsLac1 is a secreted 6-phosphogluconolactonase from P.sojae,and mutations of D202,D287,H289,G227,L235 and P237 do not affect PsLac1 lactonase activity.Due to 6-phosphogluconolactonase protein sequence is conserved in the oomycetes,this study is important for further research on the function of secreted 6-phosphogluconolactonase.PsLac1 antagonists the inhibition of gluconic acid-δ-lactone on PsXEG1: In this study,we found that gluconic acid-δ-lactone could inhibit the xylanase activity of PsXEG1,an important virulence factor of P.sojae,by in vitro enzyme activity assay,and this inhibition occurs in a dose-dependent manner.Meanwhile,PsLac1,an apoplastic effector secreted by P.sojae,could remove the inhibitory effect of gluconic acid-δ-lactone on PsXEG1 enzyme activity.These results indicate that P.sojae secretes PsLac1 could protect PsXEG1 to promote the virulent function of PsXEG1.Collectively,this result provides a basis for the study of the synergistic effect of apoplastic lactonase and glycosyl hydrolases in Phytophthora and is instrumental for our understanding of P.sojae pathogenesis.