Characterisation of tospovirus resistance in transgenic plants.

Over the past two decades tomato spotted wilt virus (TSWV) has become increasingly important as a pathogen in many crops. This can be attributed to intensified world trade and concomitant spread of one of the most important vectors of the virus, the thrips Frankliniella occidentalis. Moreover, this vector species has become resistant to most insecticides. Efforts using conventional breeding to include resistance into major crops generally are laborious and time-consuming, and moreover, suitable sources of natural resistance against TSWV are very limited. Alternative strategies for conferring virus resistance to plants are therefore urgently needed.The main topic of the research described in this thesis concerns the development and improvement of transgenic resistance in crop plants against tomato spotted wilt virus (TSWV) and related tospoviruses. From previous investigations it was known that resistance against TSWV could be obtained by expressing the nucleoprotein (N) gene of the virus in transgenic tobacco plants. In the initial hypothesis, the observed resistance was attributed to the expressed viral protein. In contrast to this general theory, however, plants expressing the highest amounts of protein were not most resistant. In Chapter 3 evidence is presented that the expression of the viral N protein in transgenic plants is not essential for resistance, since expression of a translationally defective N gene RNA results in plants with identical resistance phenotypes, indicating a novel, RNAmediated, form of resistance.The specificity of this RNA-mediated resistance appeared to be high and was only functional against the homologous virus (TSWV), not against the related tospoviruses TCSV and GRSV. By simultaneous expression of the three nucleoprotein genes from these viruses, it was demonstrated that it is possible to introduce a broad resistance against tospoviruses by expressing multiple sequences from a single insertion in the genome (Chapter 4).To answer the question whether any part of the TSWV genome is capable of inducing RNA-mediated resistance in transgenic plants and thereby further expanding the possible use of tospoviral sequences for transgenic resistance, a large array of viral genome parts was expressed in transgenic plants. This proved to be successful only when sequences derived from the previously mentioned N gene and the viral movement protein gene, NS _m , were used as transgenes (Chapter 5). In contrast, all other parts of the TSWV genome, when expressed in transgenic plants, did not induce resistance, suggesting gene-specific resistance induction (Chapter 6). More detailed studies revealed that nuclear transcription rates of transgenes in resistant and susceptible plants differed considerably, while their steady state cytoplasmic RNA levels were the same. This suggested that the expressed sequences were actively broken down in resistant plants by a mechanism that could also degrade incoming viral RNAs (with sequences identical to the transgene). This mechanism is similar to the "co- suppression" phenomenon observed in other transgenic plants where endogenous genes could be silenced by transgenes. By studying the effect of virus inoculation on protoplasts it appeared that virus replication could be blocked in N gene transgenic protoplasts, whereas this was not observed in protoplasts isolated from NSm transgenic plants. Considering these plants are resistant at the tissue level, this implies inhibition of virus transport. Differences in resistance mechanisms at the cellular level support the explanation that the resistance specifically operates on the (N or NS,) mRNA level in the respective transgenic plants (Chapter 7).In Chapter 8 it is shown that transgenic expression of viral proteins can have unwanted side-effects, that are nonetheless informative for the biochemical activity and function of the expressed protein. Plants expressing the NS _m protein to detectable levels showed aberrations in growth, probably as a result from specific accumulation of this transgenically expressed protein in plasmodesmata, cytoplasmic channels connecting neighbouring cells. Specific association of this protein with plasmodesmata gave further evidence that this protein is involved in cell-to-cell transport of the virus, and moreover that part of the typical TSWV symptoms may be attributed to this protein.Some of our preliminary data have shown that RNA-mediated resistance can compete with - and even beat - the scarcely available sources of natural resistance. Transgenic tomato plants expressing N gene sequences were challenged with a TSWV isolate capable of overcoming a natural source of resistance (Sw-5) in tomato (kindly provided by Dr. G. Thompson, Pretoria, South Africa). It was shown that despite the capacity to break natural resistance genes in tomato plants, this TSWV isolate was unable to infect transgenic plants. Anticipating the breaking of transgenic resistance by mutant TSWV isolates, transgenic resistance provides a more flexible approach when compared to natural resistance, since genes derived from future resistance-breaking isolates can be swiftly and efficiently used to breed a new generation of resistant plants.Exploiting transgenic resistance based on co-suppression-like RNA-mediated resistance as described in this thesis, minimizes chances of unwanted genetic exchange between transgenes and incoming viruses. First of all, no transgenic protein is produced, and second, the -produced transgenic RNA is rapidly broken down already in the transgenic plant cell, reducing any possible recombination to the utmost minimum.In conclusion, forms of transgenic resistance as described in this thesis, provide a useful tool to combat tospovirus diseases in crop plants in the future