Function Dissection Of The ABC Transporter GCN4 In Resistance To Late Blight

Potato(Solanum tuberosum L.)is the world's most important food crop,and the introduction of potato staple food strategy in 2015 also shows that it occupies an increasingly important position in China.However,late blight caused by Phytophthora infestans is the most serious damage to potato production.Practice has proved that breeding potato varieties with durable resistance is the most economical and environmental solution to this problem.Because the pathogen associated molecular pattern(PAMP)is well conserved in different pathogens,while the effector evolves quickly,PAMP-triggered immunity(PTI)is considered to be more durable than effector-triggered immunity(ETI),but the mechanism of both immunity responses needs further study.Previous studies in our laboratory have shown that an ABC transporter in N.benthamiana,K4CBY0,is essential for the cell death response induced by a PAMP named INF1,secreted by P.infestans.The purpose of this study was to further study the function and mechanism of this protein in resistance to late blight,and to provide theoretical and technical support for the breeding of durable resistant potato to late blight.The main findings are as follows:1.The candidate ABC transporter K4CBY0 was confirmed to be GENERAL CONTROL NONREPRESSIBLE 4(NbGCN4)by sequence alignment.The over-expression vector of NbGCN4 was constructed.Results showed that the resistance to late blight was reduced by the over-expression of NbGCN4,while was strengthened by the silencing of NbGCN4,suggesting NbGCN4 may negatively regulate the resistance to late blight.2.After silencing of NbGCN4 by VIGS on N.benthamiana,the PTI-responsive genes Cf4/Avr4 and NIP,as well as the ETI-responsive genes R2/Avr2 and R3a/Avr3 a were transient overexpressed on newborn leaves.Results showed that the cell death responses induced by these genes were suppressed,indicating that NbGCN4 is involved in multiple PTI and ETI immune responses simultaneously,possibly as a hub gene.In addition,the RNAi vector of NbGCN4 was constructed,and the RNAi lines of N.benthamiana were obtained by genetic transformation.Three lines of them could not recognize INF1 and cause cell death,which could be used for further study on the resistance mechanism of late blight.3.The expression of StGCN4 in leaves of potato resistant accessions 09013,06HE13-1,08HE17 H and susceptible accessions 93045 and E-P-5 induced by P.infestans isolate 14-2 was analyzed by qRT-PCR.Results showed that StGCN4 could be up-regulated in all five accessions,and its expression was not significantly different in resistant and susceptible accessions,implying that StGCN4 may be involved in the basic immunity of plants.The expression of StGCN4 in potato E3 leaves induced by different PAMPs including CF,flg22,pep13 and chitin was also analyzed by qRT-PCR.Results showed that StGCN4 responded most rapidly to CF,significantly up-regulated at 0.5 h,and consistently up-regulated at the later stage.In addition,StGCN4 was also induced by Pep13,flg22 and chitin at about 1 h,which indicates that StGCN4 could respond to different PAMPs.The expression of StGCN4 was also analyzed by qRT-PCR in leaves of potato material AC142 subjected to abiotic stresses such as high salt(100 mM NaCl),high temperature(35 °C),low temperature(4°C),and drought.Results showed that StGCN4 was significantly up-regulated only at 24 h of drought stress,while did not change significantly under other induction conditions.The above results imply that StGCN4 may be mainly involved in the basal defense against biotic stress.4.The results of subcellular localization experiments showed that NbGCN4 was located on the cell membrane.However,the results of CO-IP experiments showed that NbGCN4 did not interact with Nb14-3-3,indicating that NbGCN4 may have different resistance mechanisms with Arabidopsis.Then,the interaction proteins of NbGCN4 were further screened by IP-MS.A total of 22 candidate proteins were identified,of which three proteins were predicted to be localized on the cell membrane.They were annotated as a class receptor with LRR domain(A0A1S4A328),RNA helicase(A0A0V0IKF4)and a DUF domain-containing protein(A0A2G2ZM45),which are most likely to interact directly with NbGCN4,but further validation is needed.