| [Objective] Phelipanche aegyptica Per.has seriously hindered the development of the melon industry in some regions of China,and cultivating resistant melon cultivars is the most economical and effective method to control the harm of P.aegyptica.The study focuses on the interaction parasitism between melon cultivars that are resistant or susceptible to P.aegyptica parasitism,analyzes the parasitism of P.aegyptica and resistance mechanisms of melon,explores key genes related to P.aegyptica parasitism and melon resistance,and provides reference for finding control targets for P.aegyptica parasitism and providing a basis for resistant melon breeding.[Methods] The pot experiment and rhizotron experiment were employed to evaluated the parasitic resistance of two selected melon cultivars,’KR1326’(resistant)and ’K1237’(susceptible),the resistance phenotype and the period of the parasitism relationship between P.aegyptiaca and melon were determine.The mechanism of P.aegyptiaca parasitism was analyzed based on transcriptome sequencing analysis results.Differential expression genes in melon were analyzed by comparative transcriptome,and the key candidate genes of melon resistance were screened and verified.Based on the transcriptome data of P.aegyptiaca,the secreted proteins were screened according to specific screening channels.Candidate secretory effector proteins were screened from the P.aegyptiaca secretory group and their functions were further analyzed by transient expression in N.benthamiana and HIGs assay.[Results](1)Evaluation of ‘KR1326’ and ‘K1237’ melon cultivars P.aegyptiaca resistance: compared with ’K1237’,’KR1326’ showed obvious higer resistance to P.aegyptiaca,which could be used for subsequent tests.At 14 dpi,the roots of ’KR1326’ showed slight browning at the attachment site.At 20 dpi,the tubercles of P.aegyptiaca around the ’KR1326’ roots showed browning and necrosis.However,the tubercles of P.aegyptiaca was successfully connected with the vascular system of ’K1237’ root systemthe.The parasitic relationship between P.aegyptiaca and melon occurred during the attachment stage.(2)Mechanism of parasitism of Melon by P.aegyptiaca: Through the comparative transcriptome analysis of early and late attachment after inoculation with ’KR1326’ and ’K1237’,it was preliminarily confirmed that peptidase was involved in the formation of its invasion cells.A series of cell wall degrading enzymes were produced to participate in the modification and degradation of melon cell wall to promote invasion.At the same time,transferase,stimulin,toxin,effector etc were involved in the immune regulation of the host melon to ensure the successful establishment of parasitic relationship.Combined phenotype and transcriptome analysis,the key period of parasitism of P.aegyptiaca on melon was in the early stage of attachment.(3)Analysis of differentially expressed genes in Melon and functional verification of anti-P.aegyptiaca candidate genes: Transcriptome analysis of melon showed the activation of ’KR1326’ defense response,and enrichment analysis showed richer biochemical metabolic activities and immune responses in resistant melon.The up-regulated expression genes include many genes involved in anti-P.aegyptiaca-related,including genes encoding calcium channel proteins,transcription factors,receptor kinases,TIR-NBS-LRR resistance proteins,etc.The continuous high expression of CmNLR gene in the resistant melon variety’KR1326’ indicated that TIR type NLR protein was an important molecule involved in melon immunity,which was basically consistent with the mechanism of plant resistance to pathogen microorganism invasion.Overexpression of intact CmNLR and CmNLRh proteins in ’K1237’ can prevent the connection between P.aegyptiaca and the vascular system of melon root,and the transformed root system showed stronger ROS outbreak and clearance ability.Thus,TIR type NLR protein can confer melon tolerance to guarodan.(4)Prediction of P.aegyptiaca secreted proteins: A total of 2740 secreted proteins were obtained from P.aegyptiaca,which contained signal peptides and lacked transmembrane domains and ER localization domains.Secretion group contained a large number of cell wall degrading enzymes(CWDEs),including cellulases,hemicellulases,pectinases,keratases,esters,etc,and other secreted proteins,such as proteases,and protease inhibitors,etc.Various categories of secreted proteins showed specific expression patterns in the parasitism relationship between P.aegyptiaca and melon.Different secreted proteins played different roles in the invasion and proliferation of host melon,the regulation of host cell proliferation and metabolism,and the regulation of host cell immune response.We hypothesize that secreted proteins are key to the successful development and host affinity of guarodan.(5)Prediction and functional verification of candidate secretory effector proteins(CSEPs): A total of 209 CSEPs were predicted from the P.aegyptiaca secretion set,including different classes of secretory proteins.Twenty PVX recombinant vectors were successfully constructed and verified by the transient expression system of tobacco.The results showed that the transient expression of gene Cluster-15140.0 inhibited the programmed cell death(PCD)induced by BAX,and Cluster-15140.0 encoded the elicitin protein,which involved in pathogenesis and defense response biological processes.In addition,25 TRV recombinant vectors were constructed,and their function was verified by host-induced gene silencing.Among them,silencing of genes Cluster-107894.0,Cluster-11592.0 and Cluster-12482.0 significantly reduced the parasitization rate of P.aegyptiaca on the host N.benthamiana,tobacco plants expressing TRV:Cluster-107894.0 had the least amount of parasitises.Cellulase encoded by Cluster-107849.0 has hydrolase activity,cyclin dependent kinase inhibitor encoded by Cluster-11592.0 has phosphoprotein phosphatase activity,and glucan 1,3-β-glucosidase encoded by Cluster-12482.0 has hydrolase activity.[Conclusion] Melon cultivar ’KR1326’ showed obvious resistance to P.aegyptiaca,and P.aegyptiaca could establish compatible interaction with ’K1237’,and the parasitism relationship occurred in the attachment stage.The key period of parasitism was in the early stage of attachment,peptidase may be involved in the formation of its invading cells,CWDEs may be involved in the modification and degradation of melon cell wall,and transferases,stimulins,toxins,effectons,etc may be involved in the melon immune regulation.’KR1326’ showed richer biochemical metabolic activity and immune response to P.aegyptiaca parasitism,and TIR type NLR protein could confer melon tolerance to P.aegyptiaca.2740 secreted proteins make up the P.aegyptiaca secretory set,which contains a large number of CWDEs,such as cellulase,hemicellulase,pectinase,keratase,ester,and a variety of proteases and protease inhibitors,etc.The elicitin protein could inhibited Bax-induced PCD,and the silencing of cellulase gene,kinase inhibitor and glucosidase gene could significantly reduced the parasitization rate of P.aegyptiaca on N.benthamiana. |
PDF Full Text Request