Coat protein-mediated resistance to cucumber mosaic virus subgroup IB in black pepper (Piper nigrum L.)

Abstract

Cucumber mosaic virus (CMV) infects a large number of plant species including Piper nigrum L. and related species. As natural resistance to CMV is absent in Piper spp., the study was undertaken to produce transgenic P. nigrum plants harboring the complete coat protein (CP) gene of CMV via Agrobacterium-mediated transformation and their evaluation for resistance against the virus. Among one hundred and nine hardened transformed plantlets, eight revealed the presence of the transgene in PCR. The production of transcript in these plants was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and buildup of CMV CP by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). Screening of all eight transgenic lines against CMV through cleft grafting revealed that all lines except one were symptomless or showed mild or moderate symptoms. The transgenic line with the highest resistance was vegetatively propagated and integration of transgene in these clones was validated by Southern hybridization. The presence of transcript in clones was affirmed by Northern blotting– and Western blotting–ratified translation of transgene. Furthermore, relative expression studies proved manifold expression of transgene compared to actin gene as analyzed by RT-qPCR. These studies validate the stable integration and expression of transgene which might be inhibiting the movement of virus to the scions in graft inoculated plants. This is the first report on CP-mediated resistance in P. nigrum and paves the way to the production of transgenic CMV–resistant P. nigrum using CP and other desirable genes, the only effective method to combat CMV attack in the crop.