Functional investigations of the Lettuce infectious yellow virus (LIYV)-encoded proteins, P34, P5, P9 and the development of LIYV and Cucurbit yellow stunting disorder virus (CYSDV) immunity using RNA interference.

摘要

Lettuce infectious yellows virus is a crinivirus that belongs to the family Closteroviridae. Members in this family have the largest and most complex genomes among the positive sense, single-stranded RNA plant viruses. There are many criniviruses that current economic importance and therefore understanding the functions of their viral proteins and how they play a role in the virus life cycle is essential in order to develop new and more effective control strategies.;My objective was to gain a better understanding of the LIYV viral infection both at the level of protein function and the cell and molecular effects of LIYV infection. My research objectives were to establish the localization of the LIYV-encoded P34, to elucidate possible roles for LIYV-encoded P5, P9 in the virus life cycle and to develop control strategy using RNA interference against LIYV and against another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV), which currently causes great economic losses.;Since previous work showed that P34 binds non-specifically to single-stranded RNA molecules and its mutation dramatically decreased the accumulation of LIYV RNA 2, it was proposed that P34 is important in LIYV RNA 2 replication, hence raising the question where replication might occur in the plant cell. Chapter 2 describes my work in which I elucidated the localization of P34. Results of these localization experiments enabled me and previous lab members to demonstrate perinuclear/ER localization for P34 supporting previous work that it is required for efficient RNA 2 replication. Since there was very little known about the LIYV-encoded P5 and P9 proteins or the orthologs in other criniviruses, I attempted to gain an understanding of the possible roles of these proteins in the LIYV life cycle. Chapter 3 describes the generation of P5 and P9 mutants which were used for studies by agroinfiltration of whole plants (Figure 1.9.). To study both the replication as well as the systemic movement of wild type and P4 and P9 mutants these were inoculated to wild type and Hc-Pro (Hc-Pro is a strong silencing suppressor from Turnip mosaic virus) N. benthamiana plants and infections were characterized. Both mutants and wild type LIYV replicated in inoculated leaves, but the P9 mutant failed to establish systemic infections.;Lastly, transmission electron microscopy (TEM), immunosorbent electron microscopy and immunoblot experiments were performed in order to gain an understanding if P5, and/or P9 played a role in virion formation.;Although much has been studied and investigated for LIYV, so far no proteins that function as a silencing suppressor have yet been identified. I investigated and attempted to identify a silencing suppressor by testing each LIYV-encoded proteins, but have not yet found any silencing suppressor activity when agroinfiltrating these individually. One possibility could be that the silencing suppressor for individual LIYV proteins is not strong enough for the methods that I used here. However, small RNA Northern blots indicated that some LIYV proteins have potential silencing suppressor activity, and when co-agroinfiltrating these together I did observe silencing suppressor activity (Chapter 4).;Since members in the genus Crinivirus are emerging viruses and many of them cause major economic losses today, it is important to try to develop or attain resistance against these viruses. There have been many studies previously that have attempted to generate resistant lines utilizing RNA interference (RNAi) based approaches. RNAi is based on the introduction of double stranded, or hairpin RNAs that trigger the plant to induce RNAi by cleaving the dsRNA by Dicer and via the subsequent formation of RISC complexes that incorporate the small complementary RNAs. Subsequent infection that includes sequences that are the RNA sequences incorporated into the RISC complexes are recognized homologous to in planta and hence triggers immediate and robust RNA interference. Previous efforts both with LIYV as well as with another crinivirus, Sweet potato chlorotic stunt virus (SPCSV), have not succeeded to provide resistance. Chapter 5 describes my work on the generation of transgenic plant lines that express hairpin RNAs for the RNA 1-encoded replicase components of LIYV and for CYSDV, and the resulting immunity that was obtained when challenging these transgenic lines with LIYV and/or CYSDV.