寄主诱导的大丽轮枝菌硫胺素转运蛋白基因（VdThit）沉默增强棉花对黄萎病的抗性

doi:10.1016/j.jia.2024.03.024

Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (10): 3358-3369.DOI: 10.1016/j.jia.2024.03.024

寄主诱导的大丽轮枝菌硫胺素转运蛋白基因（VdThit）沉默增强棉花对黄萎病的抗性

收稿日期:2023-10-16 接受日期:2024-01-17 出版日期:2024-10-20 发布日期:2024-09-11

Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene (VdThit) confers resistance to Verticillium wilt in cotton

Qi Wang^{1, 2*}, Guoqiang Pan^1*, Xingfen Wang³, Zhengwen Sun³, Huiming Guo¹, Xiaofeng Su^{1, 4#}, Hongmei Cheng^1#

¹National Key Laboratory of Agricultural Microbiology/Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
² Xinjiang Jinfengyuan Seeds Co., Ltd., Aksu 843100, China
³ College of Agronomy, Hebei Agricultural University, Baoding 071001, China
⁴Institute of Microbiology Applications, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China

Received:2023-10-16 Accepted:2024-01-17 Online:2024-10-20 Published:2024-09-11
About author:#Correspondence Hongmei Cheng, E-mail: chenghongmei@caas.cn; Xiaofeng Su, E-mail: suxiaofeng@caas.cn * These authors contributed equally to this study.
Supported by:
This research was supported by the National Key Research and Development Program of China (2022YFD1200300), the National Natural Science Foundation of China (32072376 and 32372515), Winall Hi-tech Seed Co., Ltd., China (GMLM2023), the Nanfan Special Project of Chinese Academy of Agricultural Sciences (CAAS) (ZDXM2303 and YBXM2415), the Natural Science Foundation of Hebei Province, China (C2022204205) and the Agricultural Science and Technology Innovation Program of CAAS.

摘要/Abstract

摘要：

黄萎病是由土传真菌大丽轮枝菌引起的，并且对多种作物都会构成极大威胁。利用分子育种技术创制高抗黄萎病的棉花品种是防治棉花黄萎病最有效的措施之一。在本研究中，我们利用寄主诱导的基因沉默技术，通过向棉花体内导入靶向大丽轮枝菌硫胺素转运蛋白基因（VdThit）的双链RNA，获得了两个稳定遗传的棉花转基因株系VdThit-RNAi-1和VdThit-RNAi-2。Southern印迹杂交的结果显示其均为单拷贝插入。对大丽轮枝菌Vd-mCherry侵染后的棉花根部进行显微观察，与野生型相比，转VdThit-RNAi基因植株根部病原菌的定殖显著减少，并且病情指数和真菌生物量也明显降低。实时定量PCR结果表明，在VdThit-RNAi棉花中，随着接种时间的延长，VdThit基因的表达受到显著抑制。小RNA测序结果进一步揭示了转VdThit-RNAi基因棉花中产生了大量靶向沉默VdThit基因的特异siRNA。此外，siVdThit靶向沉默VdThit基因会导致大丽轮枝菌中参与硫胺素生物合成途径的多个关键基因表达量显著升高。在田间条件下，转VdThit-RNAi基因棉花的抗病性和产量显著高于野生型植株。综上所述，我们的研究成果表明：利用寄主诱导的基因沉默技术靶向沉默VdThit基因，可抑制大丽轮枝菌在棉花中的致病力，为通过分子育种方法创制高抗黄萎病棉花新材料提供了新的策略和思路。

Abstract:

Verticillium wilt (VW), induced by the soil-borne fungus Verticillium dahliae (Vd), poses a substantial threat to a diverse array of plant species. Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures. In this study, we successfully generated two stable transgenic lines of cotton (Gossypium hirsutum L.), VdThit-RNAi-1 and VdThit-RNAi-2, using host-induced gene silencing (HIGS) technology to introduce double-stranded RNA (dsRNA) targeting the thiamine transporter protein gene (VdThit). Southern blot analysis confirmed the presence of a single-copy insertion in each line. Microscopic examination showed marked reductions in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type (WT). The corresponding disease index and fungal biomass of VdThit-RNAi-1/2 also exhibited significant reductions. Real-time quantitative PCR (qRT-PCR) analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton. Small RNA sequencing (sRNA-Seq) analysis revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines. Additionally, the silencing of VdThit by the siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd. Under field conditions, VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT. In summary, our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton, thereby potentially enabling the development of cotton breeding as a promising strategy for managing VW.

Key words: Verticillium dahliae , thiamine transporter , host-induced gene silencing (HIGS) , RNAi

Qi Wang, Guoqiang Pan, Xingfen Wang, Zhengwen Sun, Huiming Guo, Xiaofeng Su, Hongmei Cheng. 寄主诱导的大丽轮枝菌硫胺素转运蛋白基因（VdThit）沉默增强棉花对黄萎病的抗性[J]. Journal of Integrative Agriculture, 2024, 23(10): 3358-3369.

Qi Wang, Guoqiang Pan, Xingfen Wang, Zhengwen Sun, Huiming Guo, Xiaofeng Su, Hongmei Cheng. Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene (VdThit) confers resistance to Verticillium wilt in cotton[J]. Journal of Integrative Agriculture, 2024, 23(10): 3358-3369.

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寄主诱导的大丽轮枝菌硫胺素转运蛋白基因（VdThit）沉默增强棉花对黄萎病的抗性

Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene (VdThit) confers resistance to Verticillium wilt in cotton

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