Screening Of Peronophythora Litchii Effectors And The Functional Analysis Of PlPAE5

Litchi downy blight is the most destructive disease on litchi,causing by Peronophythora litchii,which greatly affected the growth and development of litchi.Effectors always play a decisive role in plant-pathogen interaction.However,the research on effectors in P.litchii was only bioinformatics analysis but no functional investigation.In this study,Agrobacterium-mediated transient expression technology was used to screen the effectors,it was found that Pl111352 and Pl108620 can cause cell death on tobacco;PlPAE5 can promote P.capsici infection to tobacco.A further study was carried out on the PlPAE5 of pectin acetylesterase activity,it was elucidated that PlPAE5 was a pathogenic factor which up-regulated at the early infection stage.The results are as follows:1.Screening of the effectors of P.litchii:71 effectors were constructed on plant expression vectors PVX::HA and pBIN::RFP,which was selected from the previous bioinformatics analysis.then the proteins were transiently expressed in tobacco cells.Then 10 proteins were randomly selected to verify the stability in tobacco by Western-blot technique.Three effectors with obvious phenotypes were obtained,two proteins Pl111352and Pl108620 can cause HR reactions in tobacco,one protein can significantly promote the infection of P.capsici to tobacco--pectin acetylesterase PlPAE5.2.Transcriptional pattern analysis of PlPAEs:by bioinformatics,five homologous genes?Pectin acetylesterase,PAEs?of PlPAE5 were found in P.litchii genome,and those was conserved in Phytophthora sp.and Pythium sp.but functional studies had not been reported.Through transcriptional pattern analysis of PAEs,PlPAE4 and PlPAE5 with signal peptide increased significantly at the early infection stage than that of in hyphal stage.3.PlPAE5 plays a role in pathogenicity:knockout PlPAE4 and PlPAE5 by CRISPR/Cas 9 technology,it was found that mutants were not found only in the presence of sgRNA and Cas9 vectors but found when adding the replacement vector pBSSK::PlPAE5.The phenotype obtained of PlPAE4 and PlPAE5 mutants,it was found that PlPAE4 mutants had not affected P.litchii's growth rate and pathogenicity;PlPAE5also had no effect on growth rate and cell wall stress,but pathogenicity was significantly reduced on litchi leaves.However,it was found that the overexpression of PlPAE5resulted in a significant decrease of pathogenicity,the inference was probably that overexpression triggers the expression of plant defense genes and induces defensive responses.4.The toxicity of PlPAE5 can be dependent on the PAE domain:The conserved domain of PlPAE5 was analyzed by bioinformatics,and the deletion mutants PVX::PlPAE5^1-80aa?PVX::PlPAE5¹-165aa-165aa were designed to express in tobacco.The results showed that the deletion mutants PVX::PlPAE5¹-80aa-80aa could not promote the infection of tobacco with P.capsici,indicating that the PAE domain of PlPAE5 was the key area of PlPAE5 for P.capsici infection.