Proteomic Analysis Of Potato Defense Response To Herbivore Stress And Functional Study Of Key Regulatory Genes

Potato(Solanum tuberosum)is the fourth largest crop in the world.Potato tuber moth(Phthorimaea operculella)belongs to the Lepidoptera: Gelechiidae,is a main pest in potato industry worldwide.The larvae can feed on leaves and drill into tubers,which is destructive to potato industry.Due to these characteristics,the efficiency of traditional chemical control to this pest is limited,and it is easy to result in pesticide resistance and environmental pollution.Researches on resistance mechanisms and digging out the inherent herbivore resistance characteristics of potato can provide a new approach for the integrative management of this potato pest.This study used mechanical damage and oral secretion of tuber moth to simulate herbivore damage,and then analyzed the changes of protein contents,membrane protein contents and protein phosphorylation statuses of potato leaves which were treated with simulated herbivore damage,found out the proteins that may regulate the defense against this herbivore,and explored the function of key regulatory proteins.The main findings are as follows:1.Comparative proteomic study was carried out for potato leaves after 5 h simulated herbivore damage,by using the quantitative proteomic analysis technique.The contents of 60 proteins were significantly changed in potato leaves at 5 h after potato tuber moth damage,among which 53 were upregulated.Functional enrichment analysis showed that the changed proteins mainly participate in the functional groups of secondary metabolism and REDOX reaction.In the early defense signal and hormone pathway,26 proteins involved in the calcium signal pathway showed changed levels,of which 4 proteins were up-regulated.9 proteins in SA pathway showed changed levels,of which 2 were up-regulated.8 proteins in JA pathway showed changed levels,of which 2 were up-regulated.2.Comparative membrane proteomic study was carried out for potato leaves after 5 h simulated herbivore damage,by using the quantitative proteomic analysis technique.Membrane proteins were extracted from potato leaves after 5 h simulation of herbivore damage,and the proteins were quantified.Meanwhile,based on results of subcellular localization prediction,a total of 72 differentially expressed membrane proteins were screened out,52 of which were significantly down-regulated.GO enrichment analysis showed that 31 membrane proteins were enriched in the functional groups of protein phosphorylation and protein kinase activity regulation,and 45 membrane proteins were riched in the functional group of ATP binding.KEGG enrichment analysis showed that each of the 3 pathways,oxidative phosphorylation,plant-pathogen interaction and plant hormone signal transduction,has 3 significantly down-regulated membrane proteins.Among these differentially expressed proteins,putative wall-associated receptor kinase-like 16,which is enriched in calcium ions signal pathway,has been reported to participate in plant and pathogen interaction;and serine/threonine-protein kinase,which could regulate plant hormone signal transduction,has been reported to participate in sucralose metabolism.3.Comparative phosphorylation proteomic study was carried out for potato leaves after 5 h simulated herbivore damage,by using the quantitative proteomic analysis technique.After 5h simulation of herbivore damage,56 proteins were significantly changed in protein phosphorylation level,of which 52 were down-regulated.The changed proteins were enriched in biological processes such as exonuclease activity,phosphatidylinositol metabolism,arginine and proline metabolism.Further analysis revealed the significant down-regulation of calcium-dependent protein kinase 2(CDPK2)and mitogen‐activated protein kinase phosphatase 1(MKP1).Previous studies have revealed that these two proteins participate in plant response to pathogen,but their roles in plant-insect interaction remain to be explored.4.The function of CDPK2 and MKP1 genes were characterized by using the VIGS technique in tobacco system.Phylogenetic analysis showed that St CDPK2 and St MKP1 were conserved to homologous genes in tobacco and tomato species.Based on previous work,we studied the functions of CDPK2 and MKP1 genes in Nicotiana benthamiana,a model species.These two genes were silenced by VIGS,bioassay study showed that the body weight and feeding leaf area of larvae fed on CDPK2 silenced tobacco plants were significantly higher than those on control group,while the body weight and feeding leaf area of larvae fed on MKP1 silenced tobacco plants were similar to those on control group.In addition,after simulated herbivore treatment,the induction of JA pathway related genes in CDPK2 silenced plants were suppressed,while the SA pathway related genes were significantly induced.These results suggested that CDPK2 may regulate plant defense against potato tuber moth through regulating JA and SA pathways,and the role of MKP1 in potato induced defense needs to be further studied.In summary,quantitative proteomics analysis was conducted on the changes of total protein content,membrane protein content and protein phosphorylation modification of potato exposed to simulated herbivore damage.Proteins that may be involved in potato response to tuber moth pests were identified,and the functions of CDPK2 and MKP1 in pest defense were explored.This study is helpful to understand the mechanism of plant-insect interaction,and provides a basis for further research on plant pest resistance,development of potato tuber moth resistant varieties and integrative management of potato tuber moth.