Tomato spotted wilt virus resistance in chrysanthemum expressing the viral nucleocapsid gene

Three tomato spotted wilt virus (TSWV) nucleocapsid (N) gene constructs were employed for Agrobacterium-mediated transformation of chrysanthemum (Dendranthema grandiflora) cv. Polaris. These constructs contained either a full-length N gene (pTSWVN+), a full-length N gene encoding a truncated N protein (pTSWVNt), or an antisense version of the full-length N gene (pTSWVN-), all derived from a dahlia isolate of TSWV (TSWV-D). Initial resistance screens were conducted on cuttings made from 152 pTSWVN+, 37 pTSWVNt, and 47 pTSWVN- transformed plants employing a highly virulent, heterologous strain of TSWV (TSWV-GB) isolated from chrysanthemum and vectored by thrips. This screening served to eliminate the majority of TSWV-susceptible transgenic lines. More rigorous resistance tests with three rounds of mechanical inoculation with TSWV-GB identified one pTSWVNt and two pTSWVN- transformed lines that exhibited a total lack of systemic symptoms and no virus accumulation. Six other lines, including some pTSWVN+, exhibited a lack of one or more of the destructive necrotic TSWV symptoms (stem canker and apical bud death) and a delay in symptom expression. Both sense and antisense constructs, therefore, were found to be effective at yielding TSWV resistance in chrysanthemum. Molecular analysis revealed that the highly TSWV-resistant pTSWVNt line had no detectable levels of N protein. All three resistant lines had low levels of N gene transcript and at least three transgene insertion sites within their genomes, although susceptible lines often had a similar number of insertion sites; The generation of Polaris lines resistant to TSWV transmitted either mechanically or by thrips represents the first time a major ornamental crop has been genetically engineered for disease resistance.