Cotton WRKY7 Cloning and Analysis of Plant Resistance to Verticillium wilt

doi:10.7668/hbnxb.20193069

Abstract

Abstract:

To eradicate Verticillium wilt of cotton,identification of resistant genes and breeding of resistant cotton varieties are the best methods at present.We used MEGA 5.2 and other related software to construct phylogenetic trees of the proteins encoded by WRKY7 genes in Gossypium hirsutum and Arabidopsis thaliana and Oryza sativa.The induction and response of GhWRKY7 gene to Verticillium dahliae and its mechanism were elucidate by subcellular localization,virus-induced gene silencing,qPCR and GUS reporting system analysis.The results showed that GhWRKY7 and AtWKRY7 were highly homologous and belonged to Group Ⅱ.GhWRKY7 was located in plant nucleus,and the relative expression of GhWRKY7 in cotton leaves and root organs was significantly higher than that in stems.GhWRKY7 gene expression was significantly upregulated after 24 h of inoculation.The GhWRKY7-silenced plants showed higher susceptibility to Verticillium dahliae infection compared to the control(Expression TRV empty vector plants),suggesting that GhWRKY7 gene positively regulated cotton disease resistance.Compared to the control,the expression levels of disease-resistance related genes,including GhPR1,GhPR3,GhPR4,GhPR5,GhPDF1.2,GhPAL1 and GhCYP71B36 in GhWRKY7-silenced plants significantly decreased after inoculation,indicating that GhWRKY7 improved the disease resistance of the plants due to increased the expression levels of disease-resistance related genes.Transient expression analysis of GhWRKY7 gene by constructing GUS reporter vector in tobacco cells revealed that GhCYP71B36 gene could specifically bound to cis-element of GhCYP71B36 promoter and transcriptionally activated downstream GhCYP71B36 expression,thus improved the disease resistance of cotton.In conclusion,GhWRKY7,as a transcription factor that positively regulates Verticillium wilt resistance in cotton,is involved in the expression of downstream disease-resistance related genes such as Camalexin synthesis,thereby improving plant disease resistance.Therefore,GhWRKY7 can be used as a candidate gene for cotton resistance breeding and provide security for cotton production.

Key words: Gossypium hirsutum, Verticillium dahliae, GhWRKY7, GhCYP71B36, Gene silencing

ZENG Yanming, HU Guang, JIA Pei, TANG Ye, WANG Bingting, WU Pan, LU Chengzhe, CHEN Aimin, PENG Qingzhong, WU Jiahe. Cotton WRKY7 Cloning and Analysis of Plant Resistance to Verticillium wilt[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 191-200. doi: 10.7668/hbnxb.20193069.

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Figures/Tables 8

Tab.1 Primer sequence

引物名称 Primer name	正向引物(5'—3') Forward primer sequence (5'—3')	反向引物(5'—3') Reverse primer sequence (5' —3')
GhWRKY7	ATGACCGTGGAATTGAAGAT	AGATGATTCCAGGATCAAAG
GhCYP71B36_pro	CTAGACTGTCAATCATAATA	CCATCATAACTGAGTTTTGCAAA
GhWRKY7	GCAAAGATTGGTGTTTTGGAA	AATGACCTTCTTCAATC
qGhWRKY7	GGTTCAGCAGAGTCAGCCAT	GCAACGGCGAGAGAATGAAC
qGhCYP71B36	ACATGGTCATGCTTGGCTCA	TGACGGTTCCCATGTACAGC
qGhPR1	GGGGCAGTGCTGACCTATCG	TTAGCACAACCAAGATGGACAGAGT
qGhPR3	GATGACTCCACAATCACCGAAGC	GCGGTCTTCTACCTGGGCATT
qGhPR4	GCCTATTGCTCAACTTGGGA	CAATCTGCTGGAACAAGGCT
qGhPR5	CGACAACTCTGGCTCAGGAA	CCATCATCTGTAACTCGGCG
qGhPDF1.2	CTGTGGTAGCGGATGGTGATAAG	GTGCAGACGCATTTGCGAAGGAA
qGhPAL1	CTGCTAAGGGTGGTGTCGTCG	CCAATGACCTCCTGTACTCGGC
qGhUBQ7	GAAGGCATTCCACCTGACCAAC	CTTGACCTTCTTCTTCTTGTGCTTG

Fig.1 Phylogenetic tree analysis of GhWRKY7 and WRKY proteins related to disease resistance in Arabidopsis thaliana and Oryza sativa Group Ⅰ consists of two WRKY domains and a C-X_4-5-C-X_22-23-H-X₁-H zinc finger structure;Group Ⅱ composed of a WRKY domain and a C-X_4-5-C-X_22-23-H-X₁-H zinc finger structure;Group Ⅲ consists of a WRKY domain and a C-X₇-C-X₂₃-H-X₁-C zinc finger structure.

Fig.2 Subcellular localization of GhWRKY7 A.Schematic diagram of 35S∶GFP expression frame;B.Schematic diagram of 35S∶GhWRKY7-GFP expression frame;C.The distribution of 35S∶GhWRKY7-GFP and control 35S∶GFP under laser confocal microscope.

Fig.3 Expression analysis of GhWRKY7 A.Tissue expression analysis of GhWRKY7;B.Induced expression analysis of GhWRKY7 by V.dahliae;C,D.Expression analysis of GhWRKY7 induced by SA and JA.The different alphabets stand for significant difference(P<0.05).The same as Fig.4—6.

Fig.4 Construction of GhWRKY7-silencing vector and development of silencing plants A.Construction schematic of pYL-156-GhWRKY7-silencing vector,which contains duplicated enhancers(35S_pro),NOS terminator(NOS_ter)and Multiple clone sites(MCS)for construction of various target gene fragments,the GhWRKY7 gene specific fragment size was 300 bp;B.Leaf albino phenotype of positive plants;C.Analysis of GhWRKY7 gene relative expression levels in negative control and silent plants.

Fig.5 The disease-resistant analysis of GhWRKY7-silenced plants A.Phenotype of GhWRKY7-silenced plants and control group;B.Rates of diseased plant;C.Disease index;D.Vascular bundle phenotypes on the stem section of inoculated plant and WT:WT is the normal plants without inoculation;TRV:00 and TRV:GhWRKY7 are the main stem section phenotypes of the infected plants after inoculation for 21 days;E.The recovery culture of V.dahliae from the stem segment of the inoculated plants.

Fig.6 Expression analysis of disease resistance marker genes in GhWRKY7-silenced plants and control plants A.Relative expression levels of three SA marker genes in GhWRKY7-silenced plants and control plants; B. Relative expression levels of three JA marker genes;C.Relative expression levels of GhCYP71B36. GhWRKY7-silenced plants and control group infection with V.dahliae at 21 days were sampled.

Fig.7 Schematic diagram of GhWRKY7 gene effector vector and GhCYP71B36_pro gene reporter vector construction and GUS staining results A.Schematic diagram of construction of pBI121-GhWRKY7 expression vector and pBI121-GhCYP71B36_pro-GUS reporting vector;B.GUS staining results of transient expression of tobacco on the left,on the right is the injection of each part of tobacco leaf.Positive control pBI121-GUS bacterial solution.

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