Title : Expression of hairpin RNA of root knot nematode pola1 gene in solanaceae hosts reduced nematode multiplication and improved agronomic characters
Abstract:
Engineering nematode resistance via in planta expression of double stranded RNA (dsRNA) having sequence homology with essential root knot nematode genes promises a comparatively more beneficial and lower risk management strategy than integrated use of nematicides, resistant crop varieties and good cultural practices currently employed. Expression of hairpin RNA of a targeted essential nematode gene in suitable host plants is known to trigger gene-specific RNAi pathway in the plants and/or infecting nematode parasites which can ultimately lower transcript levels of such targeted gene in the nematodes. Silencing the expression of essential nematode genes may produce lethal effects in the parasites and confer protection on the expressing host plant. Thus, the selection of such appropriate target gene is a key consideration for success of this strategy. Plant parasitic nematodes such as Meloidogyne spp. are among the most significant constraints to sustainable agriculture and responsible for huge yield losses in various crop species running into several million dollars worldwide but particularly in developing countries. Our study, therefore, evaluated the potential effectiveness of root knot nematode PolA1, an essential single copy nuclear gene encoding the largest subunit of RNA polymerase I enzyme as a target in conferring nematode resistance on Agrobacterium tumefaciens-transformed tobacco plants. We cloned a 472 bp sequence of M. incognita PolA1 via an entry vector into pANDA35HK RNAi binary vector which had the target gene in sense and anti-sense orientations. We transformed tobacco and tomato explants with Agrobacteria EHA 105 cells carrying this construct and characterized resulting putative transgenics. Selected T0 plants and their T1 progeny lines expressing double stranded RNA of the PolA1 target gene were then subjected to M. incognita infection at the end of which nematode parasitic success and key agronomic traits were evaluated. Nematode challenge bioassay showed reduction in nematode galling, female nematode fecundity and amount of nematode juveniles in soil among the transgenic plants. Transgenic plants also showed reduced progression of overall disease condition as well as improved agronomic characters compared to wild type plants. Transcript level of PolA1 was reduced in adult female nematodes feeding on transgenic roots suggesting that RNAi machinery may have been activated in the nematodes following ingestion of either PolA1 dsRNA or resulting siRNAs. Put together, our results indicate that M. incognita PolA1 gene is a potentially effective target for host induced silencing-mediated reduction of root knot nematode damage on the Solanaceae host plants although further molecular evaluation and resistance assessment are necessary.
