Cloning And Functional Analysis Of M90 And MAPK10 Genes In Peronophythora Litchii

Litchi downy blight caused by Peronophythora litchii is the most serious disease during the litchee production in China.Currently available broad-spectrum fungicides have limited effect on disease control due to its far genetic relationship from fungi,as oomycete and diploid organism.Traditional gene knock-out technology is not working well in P.litchii either,hindering basic research on its pathogenicity.In this study,we utilized polyethylene glycol(PEG)-mediated protoplast transformation technique and double-stranded RNA(dsRNA)-mediated post-transcriptional gene silencing strategy,to study the roles and function of M90 and MAPK10 genes in the life cycle and pathogenicity of P.litchii,the main results are as follows:1.A homolog with 73% identity to Phytophthora infestans M90 was selected from the whole genome of P.litchii,namely P.litchii M90(PlM90).Alignment and phylogenetic analysis of the homologs of oomycetes Phytophthora sojae,Hyaloperonospora arabidopsidis,Plasmopara halstedii,Peronospora tabacina,and other model organisms including Saccharomyces cerevisiae and Arabidopsis thaliana,showed that M90 homologs were ubiquitous in eukaryotes and highly conserved in oomycetes,especially for their RNA-binding domain.2.Transcriptional levels of Pl M90 gene in the life cycle and infection stages were analyzed by quantitative real-time polymerase chain reaction(qRT-PCR).The result showed the relative expression in sporangium,zoospore and zoospore encyst were 2-3 fold to that in mycelia,and the relative expression in oospore raised to 24 fold comparing with mycelia stage,and significantly reduced in germination cyst stage and infection stage,which indicated that PlM90 gene may be involved in sexual reproduction and asexual reproduction of P.litchii,but dispensible for infection stages.3.Three PlM90-silenced transformants with silence efficiency as 98.3%,99.7% and 99.6% respectively,were obtained through PEG-mediated transformation strategy and confirmed by qRT-PCR.Phenotypic characterization was systematically performed,such as oospore production,mycelial growth rate,sporangia number,zoospore release rate and encyst rate,germination rate of encysts and pathogenicity,to analyze the function of PlM90 gene.Compared to the wild type,oospore number of three PlM90-silenced transformants were reduced upto 98.9%,99.7% and 99.6% respectively,and their mycelial growth rate was also significantly reduced although the colony became denser.Both progress of zoospore release and encyst were significantly delayed compared to the WT.On the other hand,there were no significant differences between the wild type and Pl M90-silenced transformants in sporangia number,germination rate of encysts or pathogenicity.4.Through a transcriptome analysis for PlM90-silenced transformants vs wild type strain,we identified 236 up-regulated genes and 101 down-regulated genes in two PlM90-silenced transformants.Gene Ontology(GO)analysis showed that about 40% up-regulated and 34% down-regulated genes could be classified into biological processes,about 28% up-regulated and 25% down-regulated genes into molecular function,and about 32% up-regulated and 41% down-regulated genes into cellular components.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed that tyrosine metabolism,cyanoamino acid metabolism,amino sugar and nucleotide sugar metabolism,arachidonic acid metabolism pathways were significantly affected in PlM90-silenced transformants.5.Based on the whole genome sequence analysis,a protein homologous with Phytophthora sojae MAPK10,in mitogen-activated protein kinase(MAPK)pathway,was identified and named as P.litchii MAPK10(PlMAPK10).PlMAPK10 was ubiquitously present in eukaryotes and highly conserved in the pathogenic oomycetes,especially for its STKc_MAPK domain.6.Transcriptional levels of PlMAPK10 gene in the life cycle and infection stages were analyzed by qRT-PCR and the result showed it was significantly induced in zoospore and zoospore encyst,but remained unchanged in the other life stages and infection stages,compared to the mycelia.7.Three PlMAPK10-silenced transformants with silence efficiency as 64.1%,69.7% and 80.8% respectively,were obtained though PEG-mediated transformation strategy,and verified by qRT-PCR.We characterized oospore production,mycelial growth rate,sporangia number,zoospore release rate and encyst rate,germination rate of encysts and pathogenicity in these PlMAPK10-silenced transformants to investigate the function of MAPK10 gene.The results showed that MAPK10-silenced transformants were significantly reduced in growth rate,sporangia number and pathogenicity,while with no obvious differences in zoospore activity,germination of encyst or oospore formation.8.To further study the reason of reduced pathogenicity resulted from MAPK10 gene silencing,we quantified the activity of two extracellar enzymes,extracellular oxidase and laccase.We found that the laccase activity is largely reduced,while the activity of extracellular oxidase is unaffected.qRT-PCR analysis of five putative laccase encoding genes showed that Pl137978 had significantly reduced expression compared to the wild type.