Sharma Veerandra Kumar, Basu Saumik, Chakraborty Supriya
Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India.
Plant Cell Rep. 2015 Aug;34(8):1389-99. doi: 10.1007/s00299-015-1795-8. Epub 2015 Apr 28.
Two RNAi constructs were designed targeting chilli-infecting begomoviruses and associated betasatellites. Broad-spectrum resistance was achieved against multiple begomoviruses associated with leaf curl disease of chillies in India. Chilli leaf curl disease (ChiLCD) caused by begomoviruses (family: Geminiviridae) has emerged as one of the most devastating viral diseases of chilli, especially in the Indian sub-continent. The severity of disease incidence is expanding at an alarming rate due to the emergence of new begomoviruses with greater ability to infect this crop in almost all the major chilli producing regions of India. In this study, we applied the RNA interference (RNAi) based strategies to control infection of chilli-infecting begomoviruses (CIBs). For this, we have generated transgenic Nicotiana benthamiana plants harboring two different intron hairpin RNAi constructs [designated as TR1 (AC1/AC2) and TR2 (AC1/AC2/βC1)] using conserved regions of viral genome and associated betasatellite. During our study, we observed that, two lines harboring TR1 construct (13-1 and 2-4) and one line harboring TR2 construct (5-1) have shown resistance to the most predominant Indian CIBs like Chilli leaf curl virus-Pakistan isolate Varanasi, Tomato leaf curl New Delhi virus-isolate chilli, and a newly identified begomovirus species, Chilli leaf curl Vellanad virus. Resistant lines accumulated transgene-specific siRNAs, confirming RNAi-mediated resistance against these viruses. Furthermore, these resistant lines also displayed delayed symptom appearance and milder symptoms, as compared to virus-inoculated non-transgenic plants. Average viral DNA accumulation in the resistant lines was reduced up to 90% as compared to non-transgenic plants. Thus, our study demonstrated the application of RNAi-mediated approach in providing resistance against diverse monopartite and bipartite begomoviruses associated with ChiLCD.
设计了两种RNA干扰构建体，靶向感染辣椒的双生病毒及相关的卫星β病毒。实现了对印度多种与辣椒卷叶病相关的双生病毒的广谱抗性。由双生病毒（双生病毒科）引起的辣椒卷叶病已成为辣椒最具毁灭性的病毒病之一，尤其是在印度次大陆。由于新的双生病毒出现，其在印度几乎所有主要辣椒产区感染这种作物的能力更强，病害发生率的严重程度正以惊人的速度扩大。在本研究中，我们应用基于RNA干扰（RNAi）的策略来控制感染辣椒的双生病毒（CIBs）。为此，我们利用病毒基因组的保守区域和相关卫星β病毒，培育出了携带两种不同内含子发夹RNAi构建体[命名为TR1（AC1/AC2）和TR2（AC1/AC2/βC1）]的转基因本氏烟草植株。在我们的研究过程中，我们观察到，携带TR1构建体的两个株系（13 - 1和2 - 4）以及携带TR2构建体的一个株系（5 - 1）对印度最主要的CIBs具有抗性，如辣椒卷叶病毒 - 巴基斯坦分离株瓦拉纳西、番茄卷叶新德里病毒 - 辣椒分离株，以及一种新鉴定的双生病毒种，辣椒卷叶韦拉纳德病毒。抗性株系积累了转基因特异性小干扰RNA（siRNAs），证实了RNAi介导的对这些病毒的抗性。此外，与接种病毒的非转基因植株相比，这些抗性株系还表现出症状出现延迟且症状较轻。与非转基因植株相比，抗性株系中病毒DNA的平均积累量减少了90%。因此，我们的研究证明了RNAi介导的方法在提供对与辣椒卷叶病相关的多种单分体和双分体双生病毒的抗性方面的应用。
