Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (11): 1858-1869.doi: 10.3864/j.issn.0578-1752.2019.11.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Functional Characterization of Gene CRVW Involved in Cotton Resistance to Verticillium Wilt

WANG QiuYing,WANG WeiQiao,ZHANG Yan,WANG GuoNing,WU LiQiang,ZHANG GuiYin,MA ZhiYing,YANG Jun(),WANG XingFen()   

  1. College of Agronomy, Hebei Agricultural University/North China Key Laboratory for Crop Germplasm Resources, Ministry of Education, Baoding 071001, Hebei
  • Received:2019-01-08 Accepted:2019-03-22 Online:2019-06-01 Published:2019-06-11
  • Contact: Jun YANG,XingFen WANG E-mail:yang22181@163.com;cotton@mail.hebau.edu.cn

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Abstract:

【Objective】 Verticillium wilt is an important disease in cotton production, and it seriously affects the yield and quality of cotton. Genome sequences of Gossypium hirsutum provide valuable information resources for searching for resistance genes. In this study, an uncharacterized gene, designed as CRVW (cotton resistance to Verticillium wilt), was cloned and identified for disease resistance. The results will lay a foundation for upgrading cotton genomic information, further studying the resistance mechanism and molecular breeding. 【Method】 The open reading frame (ORF) of CRVW was cloned from upland cotton cultivar ND601 using the primers, which were designed according to the reference genome sequence. The online software ProtParam was used to predict protein properties, including amino acid composition, molecular weight, the theoretic isoelectric point, instability index and grand average of hydropathicity. PSIPRED v3.3 was used to predict the protein secondary structure. The prediction of protein subcellular localization and cis-acting elements in the promoter was performed using ProtComp v. 9.0 and PlantCARE, respectively. To elucidate the subcellular localization of the CRVW protein, the CRVW-GFP fusion construct was transformed into onion epidermal cells by particle bombardment. qRT-PCR was performed using normal cotton tissues and tissues that were treated with exogenous application of salicylic acid (SA) and Verticillium dahliae stress. The function of CRVW involving in cotton resistance to V. dahliae was further verified by the technology of virus-induced gene silencing (VIGS). To preliminarily analyze the disease resistance pathway mediated by CRVW, the expression of some marker genes related to plant disease resistance was assayed in CRVW-silenced plants.【Result】 A 780 bp ORF of CRVW was successfully cloned from G. hirsutum ND601. CRVW encodes a putative protein of 259 amino acids with a molecular mass of 30.2 kD and an isoelectric point of 9.59. The protein secondary structure of CRVW contains 69.50% random coil, 17.76% α-helical, 11.20% extension and 1.54% β-sheet. By bioinformatics prediction and fluorescence observation, we found that CRVW was mainly located in the cell membrane and cytoplasm. CRVW was expressed in the roots, stems and leaves of cotton, but the highest expression occurred in the roots. The upstream sequence of CRVW ORF (CRVW-P) contains cis-acting elements in response to four kinds of hormones, including ethylene, SA, auxin and abscisic acid. Additionally, CRVW-P includes a few other elements relating to injury, defense, stress, disease, drought and low temperature. The expression of CRVW was significantly upregulated in the leaves sprayed with SA. After inoculated with V. dahliae, CRVW was dramatically upregulated both in resistant cultivar ND601 and susceptible cultivar CCRI8, but the upregulated expression in susceptible cultivar lagged behind in the resistant cultivar. After 20 days inoculated with V. dahliae, CRVW-silenced cotton seedlings showed more clearly chlorosis, wilting and defoliating comparing to CK. Further statistical analysis showed that CRVW-silenced cotton seedlings had higher disease index than the CK, suggesting that the silence of CRVW significantly reduced the resistance of cotton seedling to V. dahliae. Endogenous SA content in CRVW-silenced cotton seedlings was significantly lower than in CK. The expression of marker genes related to SA accumulation and signal regulation, including ICS1 (isochorismate synthase 1), EDS1 (enhanced disease susceptibility 1), PAD4 (phytoalexin deficient 4), NPR1 (nonexpresser of PR gene 1) and PR1 (pathogenesis- related protein 1), were significantly down-regulated after silencing CRVW.【Conclusion】 CRVW is located in the cytoplasm and the cell membrane, mainly expressed in cotton roots, and involved in the process of cotton resistance to Verticillium wilt, perhaps through SA-mediated defense pathway.

Key words: cotton, Verticillium wilt, CRVW, clone, virus-induced gene silencing, resistance

Fig. 1

RNA extraction from cotton seedlings and PCR amplication of CRVW"

Fig. 2

Subcellular localization of CRVW in onion epidermal cells"

Fig. 3

Expression analysis of CRVW in different organs of cotton plants"

Table 1

The prediction of cis-acting elements in CRVW-P"

名称 Name 序列 Sequence 位置 Position 功能 Function
ERE ATTTTAAA +175,+725,+1470,+1530 乙烯响应Ethylene responsiveness
TCA TCATCTTCAT +1935 水杨酸响应Salicylic acid responsiveness
AuxRR-core GGTCCAT +441 生长素响应Auxin responsiveness
ABRE ACGTG +67,-566 脱落酸响应Abscisic acid responsiveness
WUN-motif AAATTTCTT -305 伤害响应Wound responsiveness
WRE3 CCACCT +1761,-1987 伤害响应Wound responsiveness
STRE AGGGG -911 胁迫响应Stress responsiveness
TC-rich repeats ATTCTCTAAC +550 防御和胁迫响应Defense and stress responsiveness
box S AGCCACC +1759 伤害和病菌响应Wound and pathogen responsiveness
LTR CCGAAA -347 低温响应Low-temperature responsiveness
MBS CAACTG +1494 干旱响应Drought responsiveness

Fig. 4

Analysis of salicylic acid-induced expression of CRVW in cotton CK: Control group; SA: Salicylic acid induced group; *** indicate that the differences are significant at 0.001 level. The same as below"

Fig. 5

Expression analysis of CRVW in ND601 roots under Verticillium dahliae stress VD: Verticillium dahliae treatment group; CK: Control group; * indicates that the difference is significant at 0.05 level; ** indicate that the differences are significant at 0.01 level. The same as below"

Fig. 6

Expression analysis of CRVW in CCRI8 roots under Verticillium wilt stress VD: Verticillium dahliae treatment group; CK: Control group"

Fig. 7

Silencing of CRVW reduced cotton seedlings resistance to Verticillium dahlia A: Albino phenotype of CLA1-silenced cotton plants; B: Wilt symptom of CRVW-silenced cotton plants inoculated with V. dahliae; C: The disease indices of CRVW-silenced cotton plants with infection by V. dahliae. WT: Wild type group; CK: Control group; CRVW: CRVW-silenced group"

Fig. 8

Accumulation of SA in V. dahliae-infected cotton seedlings"

Fig. 9

Expression analysis of SA biosynthesis and signaling pathway-related genes in the CK and CRVW-silenced cotton seedlings after inoculation with the V. dahliae"

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