Study On The Biocontrol Mechanisms Of Pythium Oligandrum And Pythium Periplocum Based On Multi-omics Technologies

Oomycetes,fungi-like eukaryotic organisms,share similar nutritional strategies and morphological structures with fungi,but fall within the kingdom Stramenopiles according to evolutionary analysis.Until now,most of oomycete species were found that belong to Phytophthora spp and Pythium spp,which infect many plants,animals and insects.Many Pythium species were verified infect several plant pathogens since 1940s.Thus,these Pythium species are important material for controlling plant disease.However,little is known about mycoparasite Pythium,such as the genomic characteristics,mechanisms of interacting with plant pathogens and the development of oospores.This limits in-depth researches about the biocontrol mechism of these mycoparasitic Pythium species.Based on the high-throughput sequencing technologies,two kinds of mycoparasite Pythium species,Pythium oligandrum and Pythium periplocum were selected to analyze questions mentioned above.The main result we found list as follows:Genomic feature analysis of P.oligandrum and P.periplocum.In this study,we performed de novo genome sequencing and analysis of P.oligandrum Drechsler ATCC 38472 and P.periplocum CBS 532.74.According to phylogenic analysis based on housekeeping genes,P.oligandrum and P.periplocumare are under close relationship with P.aphanidermatum,P.arrhenomanes and P.insidiosum.The genome size of P.oligandrum is 39 Mb,which encodes 14488 genes.The genome size of P.periplocumis 35.89 Mb which encodes 13987 genes.We found some genes,such as oxidoreductases and polyketide synthetase,have high collinearity between P.oligandrum and P.periplocum,which suggests link to adaption of mycoparasite Pythium.Compare to plant pathogenic Pythium,specific expansion occurred in some gene families of mycoparasite Pythium,which include ABC transporter,kinase and protease,especially subtilisin proteases and transmembrane kinases.Interestingly,The NLP（Nep1-like protein）proteins of mycoparasite Pythium were assigned to Type-2,which is close to NLP proteins of fungi but not NLP proteins of other oomycetes.Thus,we speculate that NLP protein of mycoparasite Pythium were obtained via horizontal gene transferring from fungi.Unlike plant pathogenic oomycetes,the enzymes with biosynthesis activity of NH3 are absent in mycoparasite Pythium but Enzymes with biosynthesis activity of aryl polyene are present in mycoparasite Pythium specifically.mycoparasite Pythium can also encode CRN effectors（Crinkling and necrosis induced protein）,which display little diversity with plant pathogenic CRN effectors.The CAZymes（Carbohydrate-active enzymes,CAZymes）expanded in the genome of P.oligandrum and P.periplocum.CAZymes is polysaccharide degrading enzyme including massive families and with function of cell wall degrading,which play an important role in plant pathogenicity.Compare to plant pathogenic oomycetes,the expansion of CAZymes occurred in the genomes of two mycoparasite Pythium.Three expanded CAZy families expanded which include cellulase（GH5₁4 and AA9）and chitinase（GH19）.Take AA9 as example,there are 16 and 17 members in two mycoparasite Pythium separately,which were under tandem duplication in gene sparse region.Five CAZy families are unique,which include glucanase（GH55 and GH71）,chitinase（GH46）and hemicellulose（GH76 and GH43₂6）.We speculate that they were obtained via horizontal gene transferring from fungi and bacteria.Using transcriptome of next chapter,some genes from GH5 14 and AA9 were induced during interaction with Ph.infestans,which uncover these genes play an important role in mycoparasitism.Taken together,we infer that tandem duplication and horizontal transferring gene events are crucial factor that shape CAZymes of mycoparasite Pythium.The transcriptome data of mycoparasite Pythium interacting with Ph.infestans preliminarily reveal the biological mechanism of mycoparasite Pythium.Mycoparasite Pythium can inhibit many plant pathogens through mycelium coiling.However,little is known about the mechanism.In this study,we collected three samples for transcriptome sequencing:1)As a control case,samples of growth of P.oligandrum and P.periplocum for 3 days;2)samples of interaction of Ph.infestans for 12 hours（hours post infection,hpi）;3)samples of interaction of Ph.infestans for 24 hours（hours post infection,hpi）.Here we found:1)expression level of mycoparasite Pythium genes were under dramatical changes during interaction procession.2)Compare to samples of control case versus 12 hpi and 12 hpi versus 24 hpi,we made annotation of differential expressed genes（DEGs）,which uncover highly expressed genes were enriched in pathway of antibiotics biosynthesis and second metabolites during interaction procession.Take alcohol dehydrogenase as example,ten genes were signiticantly induced among 128 genes,which suggest second metabolites involved in interaction with Ph.infestans.Some transmembrane,such as sugar transporter and ABC transporter,were also significantly induced,which infer a complex procession of material exchange during interaction.Some secreted proteins,such as proteases,cell wall degrading enzymes,glucose-methanol-choline oxidoreductase,and Thaumatin were also induced during interaction,which implicated these secreted proteins play a certain role in antibacterial procession.Transcrip tome data analysis of mycoparasite Pythium oospore growth preliminarily reveal physiological metabolism during oospore development.mycoparasite Pythium can produced oospore easily,which is the main reproduction mode of mycoparasite Pythium.During biological control trials,the oospore of mycoparasite Pythium is an ideal material.Thus,researches about mechanism of oospore development are important for biocontrol.In this study,we collect two samples for transcriptome sequencing:1)Culturing P.oligandrum and P.periplocum for 3 days,in which oospores were not formed.2)Culturing P.oligandrum and P.periplocum for 12 days,in which oospores were observed.We investigate annotation of differential expressed genes during oospore development.We found some ABC transporters and ATPases were significantly induced,which indicate that osmotic pressure is important during oospore development.Some highly expressed intracellular proteins include kinase,Myb transcription factor and proteins with Ankyrin repeats domain.Thus,we think that complex signal transmission and gene regulation may involve in oospore development.Some secreted proteins such as cell wall degrading enzymes were significantly induced and enriched in pathway of glycoprotein and nucleotide sugar metabolism,which suggest potential cell wall modification of oospore cell wall.