Abstract
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.
Highlights
Twenty-seven putative transgenic T0 lines derived from RNA interference (RNAi)-AC2 construct, 34 lines from RNAi-AC4 construct, and 36 lines from RNAi-AC2+AC4 stacked construct (Figs 1 and 2; Tables 1 and 2) were confirmed for presence of transgenes by polymerase chain reaction (PCR) (Fig 3A–3C)
Chosen five PCR-positive transgenic plant lines generated from RNAi-AC2, RNAi-AC4 and RNAi-AC2+AC4 stacked constructs were analyzed for T-DNA copy number by Southern hybridization using nptII probe
The results revealed that three PCR-positive transgenic lines
Summary
Walp.) is one of the most important warm-season food and forage legumes cultivated across sub-Saharan Africa, Central and South America, Europe, Southeast Asia, and the United States [1,2]. Cowpea grains and green peas provide a valuable revenue-source for resource poor farmers of the developing world [3,4]. It is an important source of nutrition due to high protein content, palatability, and relative freedom from anti-.
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