Downloaded from www.microbiologyresearch.org by IP: 54.237.14.251 On: Wed, 26 Oct 2016 15:56:35 Journal of General Virology (2000), 81, 587–595. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Transgene translatability increases effectiveness of replicase-mediated resistance to Cucumber mosaic virus William M. Wintermantel and Milton Zaitlin Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA Transgenic tobacco plants expressing an altered form of the 2a replicase gene from the Fny strain of Cucumber mosaic virus (CMV) exhibit suppressed virus replication and restricted virus movement when inoculated mechanically or by aphid vectors. Additional transformants have been generated which contain replicase gene constructs designed to determine the role(s) of transgene mRNA and/or protein in resistance. Resistance to systemic disease caused by CMV, as well as delayed infection, was observed in several lines of transgenic plants which were capable of expressing either full-length or truncated replicase proteins. In contrast, among plants which contained nontranslatable transgene constructs, only one of 61 lines examined exhibited delays or resistance. Once infected, plants never recovered, regardless of transgene translatability. Transgenic plants exhibiting a range of resistance levels were examined for transgene copy number, mRNA and protein levels. Although ribonuclease protection assays demonstrated that transgene mRNA levels were very low, resistant lines had consistently more steady-state transgene mRNA than susceptible lines. Furthermore, chlorotic or necrotic local lesions developed on the inoculated leaves of transgenic lines containing translatable transgenes, but not on inoculated leaves of lines containing nontranslatable transgenes. These results demonstrate that translatability of the transgene and possibly expression of the transgene protein itself facilitates replicase-mediated resistance to CMV in tobacco. Introduction The mechanisms responsible for replicase-mediated re- sistance to various plant viruses have been studied extensively for a number of years (reviewed by Palukaitis & Zaitlin, 1997). There is evidence that different mechanisms may be responsible for engendering replicase-mediated resistance to different virus species. Studies on replicase-mediated resistance to Tobacco mosaic virus (TMV) and Pea early browning virus concluded that translatability of the transgene, encoding the 54 kDa ORF of each virus, was necessary for resistance (Carr et al., 1992 ; MacFarlane & Davies, 1992). Similarly, translation of the 2a replicase protein of Alfalfa mosaic virus was required for replicase-mediated resistance to this virus (Brederode et al., 1995). In contrast, transgenic tobacco plants containing potyvirus coat protein sequences (Lindbo et al., 1993 ; Smith et Author for correspondence : William Wintermantel. Present address : USDA-ARS, 1636 E. Alisal Street, Salinas, CA 93905, USA. Fax 1 831 755 2814. e-mail bwinterpwa.ars.usda.gov al., 1994), and replicase sequences of Potato virus X (Mueller et al., 1995) engender resistance through silencing of transgene mRNA and homologous viral RNA. In some cases, transgenic plants exhibit more than one resistance phenotype, which can complicate analysis of mechanisms. For example, Pang et al. (1994) found that coat protein-mediated resistance against tospoviruses was RNA- mediated when directed against closely related isolates, but protein-mediated when directed against more distantly related tospoviruses. Tenllado et al. (1995) found that transgenic plants containing the 54 kDa gene of Pepper mild mottle virus exhibited two different resistance responses, one complete and the other delayed. Further studies have suggested protein involvement in this system as well (Tenllado et al., 1996). In addition, although TMV replicase-mediated resistance was shown to be protein-mediated in protoplasts (Carr et al., 1992), research by Marano & Baulcombe (1998) found that transgenic tobacco plants containing a gene encoding the TMV 54 kDa protein exhibited transgene silencing. The silencing was shown to contribute to virus resistance. 0001-6718  2000 SGM FIH