Screening And Verification Of Genes Related To PAMP Triggered Immune Response Against Potato Late Blight

Potato is the fourth largest food crop in the world,while late blight is the most serious disease affecting potato production.In the double-layer immune system of plants,pathogen-associated molecular pattern(PAMP)-triggered immunity is considered to be more broad-spectrum and durable.Elicitins are the most well-studied oomycete PAMP.The culture filtrate(CF)of Phytophthora infestans mainly contains elicitins including INF1,which can induce cell death immune response in Nicotiana benthamiana leaves.But the immune signal components are mostly unknown.Our lab previously used CF to treat Nicotiana benthamiana leaves and took samples at five time points for analysis.By SWATH proteomics technology,a total of 1771 differentially expressed proteins(DEPs)were identified.In this study,bioinformatic analysis was carried out to select some candidate genes for functional verification.The aim was to identify the key genes involved in the INF1 triggered PTI response and late blight resistance,which will lay foundation for further research on the mechanism of late blight resistance.The main results are as follows:1.By GO annotation,KEGG pathway annotation and STRING interaction analysis,a total of 110 DEPs were selected from 1771 as candidate genes and constructed to a VIGS vector TRV2.Among them,105 have completed preliminary functional verification in Nicotiana benthamiana,and for most of them,the gene silencing efficiency after VIGS is above 70%.The effect of silencing 105 candidate genes individually on the cell death induced by CF and INF1 was verified,and it was found that two silences completely inhibited the cell death induced by INF1,namely ATP synthase δ subunit(No.75)and HSP90(No.86),and the silence of 75 simultaneously down-regulated late blight resistance.In addition,two silences partially inhibited the cell death induced by INF1,namely RNAbinding protein(No.7)and methyltransferase(No.39).All gene silencing did not affect CF induced cell necrosis.2.The effect of 105 candidate genes on the reactive oxygen species(ROS)burst response induced by flg22 was further verified.And it was found that a total of 47 candidate genes significantly affected the ROS response after being silenced.After 24 candidate genes silenced,ROS response was up-regulated by 25%-50%,after 12 candidate genes silenced,ROS response was up-regulated by more than 50% and the largest increase was cytb561(No.17).In contrary,after 9 candidate genes silenced,ROS response was down regulated by 25%-50%,after 2 candidate genes silenced(No.39 and No.75),ROS response was down regulated by more than 50%.3.According to the above results,three genes namely cytb561(No.17),methyltransferase(No.14)and ATP synthase δ subunit(No.75)were constructed to an overexpression vector.Then they were transiently overexpressed in Nicotiana benthamiana leaves to test if they affect INF1-induced cell death and late blight resistance.The results showed that overexpression of ATP synthase δ subunit and methyltransferase gene had no effect,while overexpression of cytb561 gene could inhibit the cell death induced by INF1,and negatively regulates resistance to late blight.In conclusion,two new genes ATP synthase δ subunit(No.75)and cytb561(No.17)were identified to play a key role in the INF1 induced PTI pathway and late blight resistance in this study.Other results also provide important basis for further analysis of resistance mechanism of late blight.