Functional Analysis Of Receptor Like Protein Genes GbVDR5 And GbVDR6 In Gossypium Barbadense For Resistance To Verticillium Dahliae

Verticillium wilt,caused by Verticillium dahlia,is the major threat to the cotton production in China.Because there is no effective chemical means to combat the disease,the only effective way to control the outbreak of it is through the planting the resistant cultivars combined with comprehensive preventive measures.Major resistant resource of cotton exit in Gossypium barbadense L.and some wild species,but which can’t be utilized directly as the barriers of cross-selection and hybridization.Therefore,the identification of additional disease-resistance genes and elucidating their mechanisms will benefit a lot to the cotton breeding against verticillium wilt.Receptor like proteins(RLPs)have been proved played vital role in plant development and disease resistance,among which Vel of tomato was the first dahlia-resistant gene identified by map-based cloning.Based on screening of BAC library with partial Ve homologous fragment,40 positive clones were obtained.Then the Ve like proteins GbVDR5 and GbVDR6 were isolated from the Verticillium wilt-resistant cotton G barbadense cv.H7124 by genomewalking combined with RT-PCR.The ORF length of gene was 3234bp and encoding a protein with 1077aa.There is a cytosine deletion in the Gb VDR5 allelic genes at the nucleotide position 2766 downstream from the initiation codon in all susceptible upland cotton cultivars analyzed.This deletion causes the last 3 LRR domains and transmembrane domain at the C terminal missing in the Gb VDR5 allelic genes.GbVDR5 is localized on the plasma membrane.There were several systemic acquired resistance-related elements,light-sensitive elements and MYB binding sites located on the GbVDR5 promoter,which driving GUS expressed mostly in the root tip and stem growing point of transgenic Arabidopsis.The expression of GbVDR5 can be induced by the V.dahlia defoliating isolateV991 and non defoliating BP2 in H7124,but unchanged or repressed in the susceptible upland cotton cultivars.Silencing of Gb VDR5 in Verticillium wilt-resistant cotton H7124 compromised cotton resistance to V dahliae isolates V991 and BP2,and the overexpressed Arabidopsis and upland cotton plants possess the enhanced resistance to the 2 V dahliae isolates but has no function to tomato-hosting V dahliae JR2.More callose deposition and hydrogen peroxide were observed in the transgenic Arabidopsis than the wild type plants when inoculated by V dahliae.In addition,the overexpression of Gb VDR5 can also activate SA,JA and ET signal pathway.Another Ve like protein gene Gb VDR6 has an ORF of 3204bp and encoding a protein of 1067aa.The GbVDR6 was verified localized to the plasma membrane.There were several SAR-related elements and light-sensitive elements located on the promoter of Gb VDR6,which was observed driving the GUS expression in root,capsule and flower.The gene can be activated by V991 and BP2 in both H7124 and susceptible cultivars.The overexpression of GbVDR6 in Arabidopsis and cotton can only enhanced the resistance to non-defoliating V dahlia isolate BP2 but not to defoliating isolate V991.The SA,JA or ET signaling pathways were proved involved in the resistant pathway mediated by GbVDR6,among which the JA pathway was interfered the fiercest,and the transgenic Arabidopsis was less sensitive than the wild-type plants to MeJA.Furthermore,more reactive oxygen species and callose were accumulated in the transgenic than the wild type plants after V.dahliae infection.These results suggest that the antibacterial spectrum of Gb VDR6 was different with GbVDR5.The completion of cotton genome sequencing makes it possible to analyze RLP gene family at the whole genome level.There are 106 and 164 RLP genes were identified in the genome of Gossypium hirsutum L.and Gossypium barbadense L.respectively.These genes were assigned to the individual chromosome according to their location and the length of the chromosome.RLP gene clusters were found on A1 and D1 of both the Gossypium hirsutum L.and Gossypium barbadense L.The Gb VDR5 and Gb VDR6 were located on the A1 within the same gene cluster with Gbvel,which were 6Mb apart from the hotspot of verticillium wilt resistance QTL qVWA1-1.The results indicate GbVDR5 and GbVDR6 may be the key genes to Verticillium wilt resistance of cotton.