Resistance to Grapevine leafroll associated virus-2 is conferred by post-transcriptional gene silencing in transgenic Nicotiana benthamiana
Ling, Kai-Shu ; Zhu, Hai-Ying ; Gonsalves, Dennis
In: Transgenic Research, 2008, vol. 17, no. 4, p. 733-740
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- Grapevine leafroll-associated virus-2 (GLRaV-2) is an important component of the leafroll disease complex in grapevine. We have previously sequenced the GLRaV-2 genome and identified the coat protein (CP) gene. The objective of this study is to test the concept of pathogen-derived resistance against a closterovirus associated with grapevine leafroll disease. Because GLRaV-2 is capable of infecting Nicotiana benthamiana, we decided to test the concept on this herbaceous host. Thirty-seven T0 transgenic N.benthamiana plants expressing the GLRaV-2 CP gene were regenerated following Agrobacterium-mediated transformation. Disease resistance was evaluated in greenhouse-grown T1 and T2 plants by mechanical inoculation with GLRaV-2. Although all the inoculated non-transgenic plants showed symptoms 2-4weeks post inoculation, various numbers of transgenic plants (16-100%) in 14 of 20 T1 lines tested were not infected. In these resistant plants, GLRaV-2 was not detectable by enzyme linked immunosorbent assay. Although virus resistance was confirmed in T2 progenies, the percentage of resistant plants was generally lower (0-63%) than that of the corresponding T1 lines (0-100%). Northern blot and nuclear run-off results showed that virus resistance in the transgenic plants was consistently associated with the low level of transgene RNA transcript suggesting a post-transcriptional gene silencing. The success of pathogen-derived resistance to GLRaV-2 in transgenic N.benthamiana plants represents the first step towards eventual control of the leafroll disease in grapevines using this strategy