| Pythium porphyrae is an important waterborne oomycete pathogen that can colonize Neopyropia yezoensis thalli and cause serious red rot disease in Southeast Asia.Little known about pathogen virulence factors and pathogenic mechanisms hinder the development of prevention and control methods for red rot disease.Therefore,in this study,candidate gene related to environmental adaptability and pathogenicity of Pyt.porphyrae were screened by comparative genomic analysis.Moreover,the expression stability of Pyt.porphyrae reference genes in different environmental stress,developmental stages,and infection stages were also evaluated.On the basis of above analysis,the transcription level of glycoside hydrolase family 5(GH5)genes,the potential pathogenic genes,were investigated during different developmental stages and infection stages in Pyt.porphyrae.Of these,pp07886,the highest transcription gene during the early infection stage,was chosen for further functional and pathogenicity analysis.In addition,we tried to establish a genetic transformation system of Pyt.porphyrae based on the mature genetic transformation system of Phytophthora sojae,but it was failed.The results of this paper could provide basic data and new ideas for the pathogenic mechanism research of Pyt.porphyrae.Following is the specific contents and results:1.Comparative genomic analysis of Pyt.porphyrae.In this study,on the basis of genome sequencing,assembly,and annotation of Pyt.porphyrae and Pyt.chondricola in our laboratory,the average nucleotide identity(ANI),core-specific genes,gene family,phylogenetic tree,and single nucleotide polymorphism(SNP)were analyzed in oomycetes to evaluate the taxonomic status of Pyt.porphyrae.In order to obtain the genes related to environmental adaptation and pathogenicity in Pyt.porphyrae,evolutionary selection pressure and the expansion and contraction of gene family were investigated between terrestrial oomycetes and Pyt.porphyrae as well as between Pyt.porphyrae and nonpathogenic oomycete Pyt.oligandrum.The results indicated that ANI values between Pyt.porphyrae and Pyt.chondricola was 98.19%-98.65%,which was much higher than its in different Pythium strains(64.68%-71.76%),suggesting that the relationship between Pyt.porphyrae and Pyt.chondricola should act as different subspecies rather than as species.GO enrichment,KEGG enrichment and Pfam annotation were performed to reveal the predicted function on the potentially environmental adaptation and pathogenicity genes of Pyt.porphyrae.The genes related to adaptation to the marine environment were mainly enriched in the pathway of antibiotic synthesis,mitoghagy and autophagy,ABC transporters,MAPK signaling,inositol phosphate metabolism,spliceosome,nucleocytoplasmic transport,and protein processing in the endoplasmic reticulum during cell progression and metabolism.The predicted gene function included calcium-activated chloride channel,transient receptor potential(TRP)ion channel,ion channel,polycystin cation channel,Ectoine synthase,helicase,endonuclease/exonuclease,ATPase,transporters,transposase,etc..The potential pathogenic genes of Pyt.porphyrae were mainly enriched in the pathway of amino acid metabolism,antibiotic synthesis,spliceosome,endocytosis,ubiquitin mediated proteolysis,phosphatidylinositol signaling system,and sphingolipid metabolism during cell progression and metabolism.The predicted gene function included glycosyl hydrolases family 5,7,and 11,cutinase,lipase,elicitin,necrosis inducing protein(NPP1),NRDE-2,Jacalin-like lectin,Concanavalin A-like lectin,etc..These results provided directions for further studies on the mechanisms of environmental adaptation and pathogenicity in Pyt.porphyrae.2.Exploration of genetic transformation system in Pyt.porphyrae.In this study,we tried to establish the genetic transformation of Pyt.porphyrae based on the transformation approach of protoplasts using polyethylene glycol/calcium chloride in Phy.sojae.Firstly,mycelial growth rate of Pyt.porphyrae in different medium(NPB,PM,V8,NPBS,PBS,PMS,V8S,and CMYSWA)was compared,and the mycelial were used to prepare protoplasm.The result showed that mycelial grown in different media could prepare protoplasts with quantity of 10~6CFU/m L without cell wall residue.Secondly,the lowest concentration of antibiotic that could inhibit the growth of different Pyt.porphyrae(NBRC 30800(00),NBRC 33126(26),NBRC 100633(33),NBRC 33253(53),JS151205(BR),HT201801(HT),RZ201902(RZ),and LS201903(LS))were screened.The result revealed that different Pyt.porphyrae strains showed various susceptibility to G418.00 and 26 showed a high tolerance to G418 with 50μg/m L completely inhibiting its growth,following by HT with 20μg/m L and the rest of Pyt.porphyrae strains with 10μg/m L.Finally,the effects of different Pyt.porphyrae strains,medium,protoplast concentrations,plasmid concentrations,PEG incubation times,and germination times during the p TOR plasmid transformed into Pyt.porphyrae protoplasts were explored,but none positive transformants were screened.This study conducted a beneficial exploration on the genetic transformation system of Pyt.porphyrae,and the results provided the basic data for further establishment of the genetic transformation system of Pyt.porphyrae.3.Evaluation of reference genes for quantitative real-time PCR normalization in the Neopyropia oomycete pathogen Pyt.porphyrae.Little is known about the pathogenetic mechanisms involved because of a lack of a genetic transformation system for this pathogen.Understanding the gene expression patterns of pathogens in response to different conditions is vital;however,this requires suitable reference genes for quantitative real-time polymerase chain reaction data normalization.In this study,11reference genes of Pyt.porphyrae were evaluated at different developmental stages(vegetative growth,sporangia formation,zoospore formation,and zoospore release),temperatures(4,15,25,and 37℃),salinities(0,20,and 35),p Hs(4,7.5,and 10)and infection stages[5,7,9 days post infection(dpi)].Various mathematical algorithms(delta-C_Tcomparison method,Best Keeper,Norm Finder,and ge Norm)were used to evaluate the stability of the reference gene expression.Of the 11 genes,those encodingγ-tubulin andα-tubulin2 showed the best stability across the different developmental stages,actin2 and 18s under different temperature conditions,gapdh and ef1αunder different salinity conditions,gapdh and eif2αunder different p H conditions,and gapdh andα-tubulin2 across different infection stages.Under all the tested conditions,gapdh showed the best stability,indicating a reliable candidate as a universal reference gene for q RT-PCR analysis.Limited to our knowledge,this is the first study to evaluate reference genes in Pyt.porphyrae,with the aim of finding suitable candidate genes for the standardization of gene expression studies in this algal oomycete pathogen,and potentially other related pathogens.4.Glycoside hydrolase family 5 gene pp07886 in Pyt.porphyrae:Identification,characterization,expression pattern,and activation of host immunity.Glycoside hydrolases are involved in penetration of the host cell wall to allow oomycete invasion,as well as in nutrition and in counteracting the innate immune response of the host.Comparative genomic analysis revealed that some glycoside hydrolase genes were expanded during the evolution,which may be associated with pathogenic pathogenicity.In this study,we performed a preliminary investigation of a GH gene from Pyt.porphyrae.pp07886,encoding a GH5 family member,was cloned from the Pyt.porphyrae genome and phylogenetically identified as GH5＿23 subfamily.The recombinant Pp07886(r Pp07886)was found to be a polyspecific enzyme with xylanase and hesperidinase activity;thus,it might be involved in breaking down the algal cell wall and hydrolyzing hesperidin to counteract host immune defenses.The q RT-PCR and western blot results showed that Pp07886 expression was strongly induced(187 to 1496fold compared to vegetable mycelia)during the early infection stage(30 min to 1 day post inoculation).Furthermore,transcripts of pp07886 were strongly induced(4 and 9fold compared to vegetable mycelia grown in SGG medium)by both xylan and hesperidin.Finally,r Pp07886 was able to activate expression of host innate immune genes(C-lectin,Hsp20,peroxidase,flavonol 3-O-glucosyltransferase,flavonoid3’-hydroxylase,ubiquitin-conjugating enzyme E2,and ubiquitin ligase E3).These results suggested that Pp07886 was involved in early infection and induction of innate immunity in N.yezoensis.Thus,this research provided important data for further functional study of GH5 proteins in Pyt.porphyrae,and increased our understanding of the molecular mechanism of action of GH5 proteins in oomycetes.5.Pathogenicity analysis of the glycoside hydrolase family 5 gene pp07886 in Pyt.porphyrae.Targeted attenuation,enhancement,and termination of individual gene expression using mature genetic transformation system is the most effective method to explore the pathogenicity of pathogenic genes.Although the genetic transformation of Pyt.porphyrae was fail established,evaluating gene function using the transformation systems of model organisms was the viable alternative approach.Therefore,In this study,The genetic transformation system of Phy.sojae was used to generate pp07886overexpression transformant Pp11,and to obtain Ps4041(a homolog of pp07886 in Phy.sojae)mutant transformants Ps-pp07886(Ps4041 replaced by pp07886)and Ps-RFP(Ps4041 replaced by red fluorescent protein gene(rfp))via CRISPR/Cas9 system,to evaluate its biological function.Compared to wild-type strain P6497,overexpression of pp07886 in Phy.sojae significantly increased virulence.Although mutant transformants showed reduced mycelium growth and virulence,Ps-pp07886 displayed higher mycelium growth and virulence than Ps-RFP indicating pp07886 contributed to pathogen growth and virulence.The q RT-PCR and western blot results showed that Pp07886 expression in Pp11 and Ps-pp07886 were strongly induced within 3 h of infection.Furthermore,the recombinant Pp07886(r Pp07886)was able to elicit soybean defense mechanism.These results suggested that Pp07886 contributed to pathogen virulence,growth,and induction of host immunity.Thus,pp07886 may be a crucial virulence factor for Pyt.porphyrae to invade N.yezoensis.This research provided new ideas for gene function study in Pyt.porphyrae,and increased our understanding of the pathogenicity of GH5 protein in oomycete. |
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