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UBC Theses and Dissertations

Symptom recovery in Tomato ringspot virus infected Nicotiana benthamiana plants : investigation into the role of plant RNA silencing mechanisms Ghoshal, Basudev


Symptom recovery in virus-infected plants is characterized by the emergence of asymptomatic leaves after a systemic symptomatic phase of infection and has been linked with the clearance of the viral RNA due to the induction of RNA silencing. However, the recovery of Tomato ringspot virus (ToRSV)-infected Nicotiana benthamiana plants is not associated with viral RNA clearance in spite of active RNA silencing triggered against viral sequences. ToRSV isolate Rasp1-infected plants recover from infection at 27°C but not at 21°C, indicating a temperature-dependent recovery. In contrast, plants infected with ToRSV isolate GYV recover from infection at both temperatures. In this thesis, I studied the molecular mechanisms leading to symptom recovery in ToRSV-infected plants. I provide evidence that recovery of Rasp1-infected N. benthamiana plants at 27°C is associated with a reduction of the steady-state levels of RNA2-encoded coat protein (CP) but not of RNA2. In vivo labelling experiments revealed efficient synthesis of CP early in infection, but reduced RNA2 translation later in infection. Silencing of Argonaute1-like (NbAgo1) genes prevented both symptom recovery and RNA2 translation repression at 27°C. Also, translation repression was compromised in Rasp1-infected wild-type (WT) plants grown at 21°C. NbAgo1 and NbAgo2 mRNAs accumulated to similar levels at 21°C and 27°C in mock-inoculated WT plants. Both genes were induced during Rasp1 infection. Interestingly, the effect of silencing NbAgo2 on Rasp1 infection was only evident at low temperatures resulting in higher accumulation of CP. Taken together, our results suggest that although both NbAgo1 and NbAgo2 genes are induced, recovery of Rasp1-infected plants at 27°C is associated with an NbAgo1-dependent mechanism that represses the translation of viral RNA2. In contrast, recovery of GYV-infected plants is associated with a reduction of viral RNA and CP levels at both temperatures. Moreover, silencing of either NbAgo1 or NbAgo2 did not prevent recovery of GYV-infected plants at 21°C. However, both GYV-infected NbAgo1 and NbAgo2-silenced plants accumulated higher level of CP in recovered leaves compared to control plants. In conclusion, this study identifies translation repression as a novel regulatory mechanism in recovery and suggests that different mechanisms may operate during recovery in an isolate and/or temperature-dependent manner.

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