A Cytochrome B₅ Superfamily Protein,PlCytb₅-1,Regulates Mycelial Growth,Pathogenicity And Stress Tolerance In Peronophythora Litchii

Litchi downy blight caused by Peronophythora litchii is the most destructive disease in the litchi production in China.With the completion of genome sequencing and the transcriptome sequencing of P.litchii,understanding the pathogenesis of P.litchii at the molecular level could provide a theoretical basis for development of new control strategies.In this study,the cytochrome b₅ superfamily PlCytb₅-1 gene was successfully knocked out by polyethylene glycol（PEG）-mediated protoplast transformation technique and CRISPR/Cas9 gene editing system,to study the roles and function of PlCytb₅-1 gene in the life cycle and pathogenicity of P.litchii.The main results are as follows:1.Based on the analysis of the whole genome of P.litchii and the results of transcriptome sequencing,a highly expressed cytochrome b₅ superfamily gene was obtained,which was named PlCytb₅-1.Alignment and phylogenetic analysis of the homologs of oomycetes,such as Phytophthora infestans,Bremia lactucae,Albugo candida,and other organisms including Oryza sativa,Ustilago hordei,et al..The results showed that PlCytb₅-1 homologs were highly conserved in oomycetes,especially Cytb₅ superfamily domain.2.Transcriptional levels of PlCytb₅-1 gene in the life cycle and infection stages were analyzed by quantitative real-time polymerase chain reaction（q RT-PCR）.Comparing with that of mycelia stage,the PlCytb₅-1 had higher transcript levels（5-to 12-fold）in zoospores,cysts,cysts germination,and the initial stages of plant infection（1.5 hip,3 hip）.It suggested that PlCytb₅-1 gene may play an important role in colonizing host plants and in the early stage of infection of P.litchii.3.PlCytb₅-1 gene was successfully knocked out by CRISPR/Cas9 gene editing system.Phenotypic characterization was systematically performed,such as mycelial growth rate,sporangia number,zoospore release rate and germination rate of encysts,and oospore production to analyze the function of PlCytb₅-1 gene.Compared to the wild type,mycelial growth rate of three PlCytb₅-1 knockout mutants was significantly reduced.The growth promotion rate of wild type by exogenousβ-sitosterol was higher than that of PlCytb₅-1knockout mutant.On the other hand,there were no significant differences between the wild type and PlCytb₅-1 knockout mutants in sporangia number,zoospore release rate and the germination rate of encysts,and oospore production number.These showed that PlCytb₅-1affects the mycelial growth of P.litchii and the utilization of sterols.4.The tolerance of PlCytb₅-1 knockout mutants to cell wall stress,hyperosmotic stress and peroxide stress showed that PlCytb₅-1 was an important regulator for stress response.Reactive oxygen species（ROS）play a central role in plant defense response.The transcript level detection revealed that the PlCytb₅-1 was highly up-regulated under oxidative stress.5.To analyze the mechanism of PlCytb₅-1 regulating hydrogen oxide sensitivity of P.litchii,we quantified the activity of five peroxidase genes and six cytochrome P450 genes.We found that three peroxidase genes and four cytochrome P450 genes expression is largely reduced compared to the wild type by q RT-PCR analysis.PlCytb₅-1 gene is required for oxidative stress tolerance and virulence in P.litchii.6.The results of pathogenicity assay showed that PlCytb₅-1 knockout mutants were less virulent on litchi.However,no significant difference in pathogenicity between the PlCytb₅-1overexpressing mutants and the wild type.These indicated that PlCytb₅-1 is required for the pathogenicity of P.litchii and there may be a regulation mechanism of PlCytb₅-1 expression.