| Peronophythora litchii belongs to the Oomycetes.It is a homothallic diploid pathogen.The litchi downy blight caused by P.litchi is the most serious disease of litchi.P.litchii produces different types of spores through its life cycle for surviving in stress environments,source of initial infection or re-infection.This pathogen causes huge losses to litchi production by infecting the shoots,leaves,spikes and fruits of litchi.In order to provide a theoretical basis for developing the more effective control strategies and technologies for litchi downy blight,it is necessary to study the function of genes involved in their life cycle.In this study,CRISPR-Cas9 technology and polyethylene glycol(Polyethylene glycol,PEG)-mediated protoplast transformation technology was used to knock out PlRAB5 A,and then the mutants was analyzed by transcription analysis,fluorescence observation and phenotype analysis.A systematic study of the biological functions of PlRAB5 A was carried out.The main results are as follows:1.Two RAB5 homologous genes were selected in the P.litchii genome database through genome-wide bioinformatics analysis,and named P.litchii RAB5A(PlRAB5A)and P.litchii RAB5B(PlRAB5B).Through multiple sequence alignment analysis,it was found that the RAB5 A and RAB5 B protein are highly conserved among Phytophthora spp.Alignment and phylogenetic analysis RAB5 homologs of Eukaryotes Saccharomyces cerevisiae,Magnaporthe oryzae,Danio rerio,Homo sapiens,Oryza sativa and Arabidopsis thaliana,showed that the PlRAB5 A protein is closer to animals and fungi,and the PlRAB5 B protein is closer to plants..2.Relative quantification of P.litchii RAB5 A gene in 5 development stages(mycelia,sporangia,zoospores,cysts and germinating cysts)and 5 infection periods(infection of litchi young leaves for 1.5 hours,3 hours,6 hours,12 hours and 24 hours)by fluorescent quantitative PCR technology.Among them,the transcription level of PlRAB5 A in the sporangia stage was significantly higher than that in other stages,and the germinating cysts is the lowest.These results indicate that the PlRAB5 A might expressed in the life cycle of P.litchii,which is more important at the sporangia stage but dispensible at the germinating cysts stage.3.In order to observe the subcellular localization of the RAB5 A,we constructed the pTOR::GFP::PlRAB5 A gene fluorescent observation vector and introduced it into the protoplast of P.litchii by PEG-mediated transformation technology;GFP-PlRAB5 A nutants were verified by Sanger sequencing and purificated by single spore separation.Three green fluorescent stable expression transformants of P.litchii RAB5 A were obtained finally.Through FM4-64 dye staining and a fluorescent microscopy,it was found that green fluorescence was stably accumulated in the hyphae and sporangia stages,and the red light and the green light basically merge,indicating that the RAB5 A is located on vesicle membrane and early endosome on the mycelia and sporangia.4.In order to knockout the PlRAB5 A by CRISPR / Cas9,two sgRNA targets on the PlRAB5 A gene were designed by software,and 2 PlRAB5 A knockout mutants were obtained.Phenotype analysis revealed that the growth rate of the two PlRAB5 A knockout mutants was significantly reduced when compared to SHS3;Observation of mycelial morphology through Calcoflour white staining found that mutants showed swollen aerial hyphae;The number of sporangia did not change in the knockout mutants,but the rate of zoospores released was reduced.Further analysis revealed that the protoplasts in the sporangia of the knockout mutants could not be cleaved and formed zoospores;Compared with the wild-type SHS3,we found that the PlRAB5 A knockout mutants produced abnormal germ tube in germinating cysts period;while in sexual reproduction,it was mainly found that oospores could not be produced;the inoculation assays showed that the pathogenicity of PlRAB5 A knockout mutants was attenuated significantly. |
PDF Full Text Request