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Transgenic Wheat Harboring an RNAi Element Confers Dual Resistance against Synergistically Interacting Wheat Streak Mosaic Virus and Triticum Mosaic Virus.

Wheat streak mosaic virus (WSMV) and triticum mosaic virus (TriMV) are economically important viruses of wheat, causing significant yield losses in the Great Plains region of the USA. These two viruses are transmitted by wheat curl mites, which often leads to mixed infections with synergistic interaction in growers' fields that exacerbates yield losses. In this study, a genetic resistance strategy, employing an RNA interference (RNAi) approach, was implemented by assembling a hairpin element composed of a 202-bp stem sequence of the NIb (replicase) gene, from each of WSMV and TriMV in tandem coupled with an intron sequence in the loop. The resultant plasmid was used to transform spring wheat genotype CB037. Phenotyping of T1 lineages across eight independent transgenic events for resistance revealed that two of the transgenic events provided resistance to WSMV and TriMV. T2 populations derived from the two events, classified as dual resistant, were subsequently monitored for stability of the resistance phenotype through the T4 generation. The resistance phenotype in these events was temperature dependent, with a complete dual resistance at temperatures ≥25°C and increasingly susceptible response at temperatures below 25°C. Northern blot hybridization of total RNA from transgenic wheat revealed that virus-specific small RNAs (vsRNAs) accumulated progressively with increase in temperature with no detectable levels of vsRNAs accumulation at 20°C. Thus, the resistance phenotype of wheat harboring RNAi element was correlated with accumulation of vsRNAs, and generation of vsRNAs provides molecular markers for the prediction of resistant phenotype of transgenic plants at a specific temperature.