Functional Analysis Of Ethylene Response Element GbERF1-like From Gossypium Barbadense In Response To Verticillium Dahliae

Verticillium wilt （VW） is an major disease for production in cotton. Ethylene signal pathway plays an important role for the interaction between cotton and V. dahliae. It is significant to demonstrate regulatory mechanism of ethylene on V. dahliae resistance in cotton, and it is valuable for improving cotton resistance and breeding by regulating ethylene signal pathway. We cloned an ethylene response factor GbERF1-like from the island cotton’7124’by SSH method in our laboratory, charactering its functional mechanism in cotton for VW resistance. The main results are as following:1. We obtained 880bp whole GbERF1-like sequence, ORF motif is 537bp, encoding 179 aa. It has a conserved AP2/ERF domain, consisting of 59 aa. Analyzing the phylogeny, GbERF1-like has the similar phylogeny with GhERF1 （AY181251.1）, GhERF8 （JN656957.1）, TbERF （XM₀07035031.1）, GbERF5 （JN003808.1） and AtERF1 （AB008103）. It expresses ubiquitously in all tissues in cotton, lower expression in vegetative organs, and higher in floral organs. GbERFl-like can be induced rapidly by ET、MeJA treatment, reaching the peak values after 0.5h and 1h treatment, respectively. It can also be up-regulated by V991 in resistant and susceptible cotton, with earlier and longer expression in resistant cotton.2. We got GbERF1-like over-expressing and RNAi lines in cotton（COV20, COV5, Ci8, Ci6）, over-expressing Arabidopsis lines（AOV12, AOV5） via agrobacterium-mediated transformation. The transgenic cotton and Arabidopsis were evaluated for VW resistance. Compared with WT, the over-expressing cotton and Arabidopsis lines had better resistance, meanwhile, more susceptible in RNAi cotton lines.GbERF1-like could positively regulate pathogenesis-related proteins PR3, PR4, negatively regulating PR1, PR5 in cotton. It could activate AtPR3, AtPR4 and AtPDF1.2 expression in Arabidopsis, but reduce the AtPR2. The lignin synthesis-related genes, GhPAL5, GhC4Hl, GhC3H1, GhHCT1, GhCCoAOMT1 and GhF5H2 had higher expression in OV lines, lower in RNAi lines. Similar expression also exist in Arabidopsis. These results confirmed that GbERFl-like may regulate the lignin synthesis pathway in cotton and Arabidopsis. Contrasting with Mock treatment, the lignin synthesis-related genes could be upregulated significantly in cotton and Arabidopsis lines treated with V991.Cotton stems were stained using Wiesner method, stems could be stained with magenta in the cotton lines treated with water and V991, however, deeper magenta in cotton lines treated with V991. In the Mock lines, the OV lines had the deepest magenta, and shallower in RNAi lines. After V991 treatment, the different lines had the similar magenta compared with Mock results. The measurements of total lignin content was consistant with stained results. After V991 treatment, all lines had more total lignin content compared to Mock lines. Within Mock and V991 treatments, the highest content existed in OV lines, increasing by 6.9%-44.3% versus WT, lowest in RNAi lines, reducing by 13.2%-23.4%. After Mock and V991 treatment, the lignin monomers were measured using cotton and Arabidopsis stems. Compared with Mock, G and S increased after V991 treatment. Within two treatments, cotton G monomer increased, but did not reach significance in OV lines versus WT, meanwhile G monomer in RNAi lines had significantly lower content compared with WT, reducing by 5.9%-12.4%. In Arabidopsis, G monomer in OV lines increased significantly versus WT, raising by 11.2%-31.6%. In WT and transgenic cotton lines, the Mock and V991 treatments could improve significantly S monomer content, the highest in OV lines, increased by 10.8%-33.6% versus WT, the lowest in RNAi lines, reducing by 7.4%-12.9%. In Arabidopsis, S monomer improved but not significantly.3. Cloning the promotors of GhHCTl and AtPAL3, their sequences were 1274bp and 1148bp, containing TAAT-box, CAAT-box, GCCGCC-box cis-elements. Yeast one hybrid verified the interaction between GhHCTl, AtPAL3 promotors and GbERF1-like. Dual luciferase report assays demonstrated that GbERFl-like protein could bind the two promotors and activate the transcriptional activities in cotton protoplasts, which provided the evidence for GbERF1-like regulating the lignin synthesis.4. To further confirm resistant enhancement was related to GbERFl-like mediated lignin synthesis in cotton. The GhHCT1 in cotton transgenic line COV20 was knocked down via VIGS method. Compared with the COV20-TRV:00 plants, the COV20-TRV: GhHCT1 plants had the following phenotypes as lower VW resistance, shallower red of stems, lower total lignin content of stems, shorter plant height. The expression of upstream lignin synthesis-related genes, like GhPAL1 （Gbscaffold40957.1）, GhPAL5 （Gbscaffold11559.9）, GhC4H（Gbscaffold12103.1）, reduced significantly. Meanwhile, its downstream genes expression, like GhCCR （Gbscaffold2152.13）, GhCAD （Gorai.009G1 92400.1）, GhF5H （Gbscaffold 12698.2） and GhCOMT （Gorai.004G125600.1）, increased significantly. These results demonstrated that lignin synthesis enhancement has an important role in GbERF1-like mediated VW resistance.5. The other resistant proteins which the GbERF1-like may interact with, were identified by the yeast two hybrid test. The MAPK, MLP, DIRIGENT have been reported in relation with disease resistance in cotton （Cai et al.,2014; Yang et al.,2015; Hosmani et al.,2013）. GbERF1-like protein might interact with such stress-related proteins as MAPK, MLP、DIRIGENT to regulate stress response in cotton. Such proteins as P450, HSP, ALLERGEN and GLYOXYLASE have not been studied in correlation with resistance, It need to be studied later whether the interactive proteins with GbERF1-like attend the lignin synthesis and resistance response.