Geminivirus-resistant Tomato Plants: Combining Transgenic and Conventional Strategies for Multi-viral Resistance

Abstract

Begomoviruses, which constitute one genus of the Geminiviridae family, are single-stranded DNA viruses that infect many dicotyledonous crops important to large agricultural industries as well as to subsistence growers. Although all begomoviruses are transmitted by whiteflies (Bemisia tabaci), they have proven difficult to manage even with heavy insecticide applications. The begomovirus, Tomato yellow leaf curl virus (TYLCV), has been a problem in tomato production in Israel since the 1950s and in the United States since 1997. Approximately 89 begomoviruses have now been reported to infect tomato. Crop losses due to begomoviruses such as TYLCV and Tomato mottle virus (ToMoV), are limiting factors in tomato cultivation in Israel, the U.S., and many tomato-growing regions throughout the world. To overcome these limitations, we proposed a two-step strategy that combines transgenic and conventional resistance in order to develop tomato plants that are resistant to multiple begomoviruses. In the first step, we have developed transgenic tomato plants expressing trans-dominant interfering mutants Rep and C3 from TYLCV and ToMoV, and tested whether these plants are resistant to infection by these two viruses. In the second step we have tested whether pyramiding transgenic and conventional resistance is superior to either strategy alone. The specific objectives of the proposal were: 1. Design and test trans-dominant interfering constructs for TYLCV and ToMoV Rep and C3 in transient replication interference assays. 2. Generate and test transgenic tomato plants expressing mutant Rep and C3 in resistance assays. 3. Generate and test conventional resistant lines that also express mutant Rep and C3. Two viral replication interfering constructs, expressing the trans-dominant interfering mutants Rep and C3, were designed and constructed during this project. One construct, pNSB1630 was based on TYLCV sequences and the other, pNSB1682, based on ToMoV sequences. The TYLCV transformation construct was tested in a protoplasts replication assay, and was found to inhibit TYLCV replication. The ToMoV transformation construct is yet to be tested in a protoplast assay. Both transformation vectors, pNSB1630 and pNSB1682, were used to transform four different tomato lines, and generate transgenic plants. The tomato lines used for transformation were: FL7613, MM, TY172, TY199. FL7613 and MM are susceptible to both TYLCV and ToMoV. TY172 and TY199 are breeding lines developed at Volcani Center. TY172 is resistant to TYLCV but susceptible to ToMoV, while TY199 is resistant to both TYLCV and ToMoV. When transgenic T1 plants expressing the pNSB1630 constructed were screened for TYLCV resistance, it was found that these plants showed very low level of TYLCV resistance, if any. However, some of these lines showed high level of resistance to ToMoV. Only five transgenic T1 lines expressing the pNSB1682 construct were tested (so far) for resistance to ToMoV. It was found that all five lines express very high level of resistance to ToMoV. Although we haven’t finished (yet) the screen of all the transgenic lines, it is already clear that we were able to successfully combine genetic resistance for TYLCV with transgenic resistance to ToMoV.