Investigating The Function Of Chitin Synthase Genes In Phytophthora Capsici And Phytophthora Sojae

Oomycetes exhibit a filamentous growth morphology that is very similar to that of many fungi,but they are not fungi or even not related to fungi.A low content or lack of chitin in oomycetes cell walls has been taken as a criterion for that differentiates them from true fungi.Phytophthora capsici and P.sojae are both representative oomycetes species and have a worldwide distribution.The cell wall composition and the function of putative chitin synthases in these two Phytophthora species were investigated in this study.The main results are as follows:We identified one CHS gene named PcCHS from P.capsici,and two CHS genes,PsCHS1 and PsCHS2,from P.sojae.Sequence analysis showed that the three CHS proteins all contained the yeast and fungal conserved chitin synth motifs,and they also have their unique domains.The transcript patterns of PcCHS and PsCHS1 were similar in different stages;both of them had higher expression level in zoospores,cysts and infection stages.However,the expression level of PsCHS1 remained stable in the detected developmental and infection stages.No chitin was detected in Phytophthora cell walls by GC-MS,but the chitin synthase competitive inhibitor nikkomycin Z showed sensitivity differentiation in P.capsici and P.sojae.It had no effect on the mycelial growth in P.capsici at the concentration 10 ?M whereas the inhibition ratio reached 90%in P.sojae.Moreover,the expression level of PsCHS2 was down-regulated with the treatment of nikkomycin Z.Therefore,we supposed that PsCHS2 was involved in the metabolism of N-acetylglucosamine(GlcNAc)rather than the biosynthesis of chitin.With PEG/CaCl2 mediated protoplast transformation,the CHS knockout mutants were obtained by using CRISPR/Cas9,and the silenced and overexpression transformants were created by RNAi.The CHSs could be divided into two types by systematic characterization of the impact of individual gene knockout,silencing,and overexpression on asexual and sexual growth,cell wall structure and chitin composition,chemical sensitivity,virulence on plants as well as subcellular localization in Phytophthora.Type ? CHS,including PcCHS and PsCHS1,was mainly involved in sporangial development,zoospore release and pathogenicity on plants;the genus-specific Type ? CHS,mainly referred to PsCHS2,played an important role in the oospore production.To further verify the functions of Phytophthora CHS genes,PcCHS,PsCHS1 and PsCHS2 were individually expressed in Saccharomyces cerevisiae mutants,in which ScCHSl,ScCHS2,and ScCHS3 genes were single,double,or all knocked out.The chitin defects were restored by genetic complementation of yeast CHS mutant with PcCHS or PsCHS1 but not PsCHS2 from Phytophthora.However,the chitin content was not recovered when PcCHS or PsCHS1 expressed in the ScCHS23 or?ScCHS123 yeast mutants.The experiments conducted in vitro indicated that PcCHS or PsCHS1 could complement the chitin biosynthesis in yeast,which remained the catalytic activity of chitin synthase,but its functional expression needed the participation of ScCHS2 or ScCHS3.RNA-Seq was used to further explore the regulation pathway of PsCHS2,and the disruption of PsCHS2 mainly affected transport,aromatic amino acid family metabolic,and pyruvate metabolism.The content of aromatic amino acids and GlcNAc was detected,and the results showed that they all reduced in PsCHS2 silenced and knockout mutants compared to the wild-type parental isolate.This result,combined with the results from transcriptomics,further revealed the evolution of a novel phosphoenolpyruvate-dependent phosphotransferase system in oomycetes.We supposed that the system was used for the transport of GlcNAc,and we also proposed a model for the regulation of PsCHS2 in Phytophthora.