Kumar Sanjeev, Tanti Bhaben, Patil Basavaprabhu L, Mukherjee Sunil Kumar, Sahoo Lingaraj
Department of Bioscience and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, India.
Department of Botany, Gauhati University, Guwahati, Assam, India.
PLoS One. 2017 Oct 27;12(10):e0186786. doi: 10.1371/journal.pone.0186786. eCollection 2017.
Cowpea is an important grain legume crop of Africa, Latin America, and Southeast Asia. Leaf curl and golden mosaic diseases caused by Mungbean yellow mosaic India virus (MYMIV) have emerged as most devastating viral diseases of cowpea in Southeast Asia. In this study, we employed RNA interference (RNAi) strategy to control cowpea-infecting MYMIV. For this, we generated transgenic cowpea plants harbouring three different intron hairpin RNAi constructs, containing the AC2, AC4 and fusion of AC2 and AC4 (AC2+AC4) of seven cowpea-infecting begomoviruses. The T0 and T1 transgenic cowpea lines of all the three constructs accumulated transgene-specific siRNAs. Transgenic plants were further assayed up to T1 generations, for resistance to MYMIV using agro-infectious clones. Nearly 100% resistance against MYMIV infection was observed in transgenic lines, expressing AC2-hp and AC2+AC4-hp RNA, when compared with untransformed controls and plants transformed with empty vectors, which developed severe viral disease symptoms within 3 weeks. The AC4-hp RNA expressing lines displayed appearance of milder symptoms after 5 weeks of MYMIV-inoculation. Northern blots revealed a positive correlation between the level of transgene-specific siRNAs accumulation and virus resistance. The MYMIV-resistant transgenic lines accumulated nearly zero or very low titres of viral DNA. The transgenic cowpea plants had normal phenotype with no yield penalty in greenhouse conditions. This is the first demonstration of RNAi-derived resistance to MYMIV in cowpea.
豇豆是非洲、拉丁美洲和东南亚一种重要的食用豆类作物。由印度绿豆黄花叶病毒（MYMIV）引起的卷叶病和金色花叶病已成为东南亚豇豆最具毁灭性的病毒病。在本研究中，我们采用RNA干扰（RNAi）策略来防治感染豇豆的MYMIV。为此，我们培育了携带三种不同内含子发夹RNAi构建体的转基因豇豆植株，这些构建体包含七种感染豇豆的双生病毒的AC2、AC4以及AC2与AC4的融合体（AC2+AC4）。所有这三种构建体的T0和T1代转基因豇豆品系都积累了转基因特异性小干扰RNA（siRNA）。利用农杆菌感染性克隆对转基因植株进一步检测至T1代，以检测其对MYMIV的抗性。与未转化对照和用空载体转化的植株相比，表达AC2-hp和AC2+AC4-hp RNA的转基因品系对MYMIV感染表现出近100%的抗性，未转化对照和空载体转化植株在3周内出现严重的病毒病症状。接种MYMIV 5周后，表达AC4-hp RNA的品系出现较轻症状。Northern杂交显示转基因特异性siRNA积累水平与病毒抗性之间存在正相关。抗MYMIV的转基因品系积累的病毒DNA滴度几乎为零或非常低。转基因豇豆植株在温室条件下具有正常表型，产量无损失。这是首次证明RNAi介导的豇豆对MYMIV的抗性。
