Thermal-enhanced Antiviral RNA Silencing And Transgenic RNA Silencing

High temperature activates host's antiviral defense, resulting in developing mild symptoms or a quick recovery from viruses. This thermal activated antiviral defense has been described for many years, but its molecular mechanism has not been completely understood yet. In this study, we demonstrated the basic laws and molecular mechanisms of thermal activated RNA silencing triggered by virus (PVX or PVYN) or by co-infiltrating GFP in Nicotiana glutinosa and Nicotiana bethamiana respectively. The results are as follows.(1) 30℃continous heat therapy of PVX or PVYN infected N. glutinosa plants revealed, viral symptoms of the newly emerging leaves became more and more mild in a gradual manner which was hardly detectable at 12dpi, and viral titer tested by ELISA was approximate to that of mock-infected tissue. However, the symptoms of the lower leaves maintained with a subtle decline of viral titer. This observation indicates that high temperature can enhance the antiviral RNA silencing activity in N. glutinosa. Whether upper leaves develop viral symtoms or not depends on the balance between viral replication and viral RNA degradation. At high temperature, the plant-virus balance is disequilibrated due to the enhanced RNA silencing activity and viral RNA degradation, resulting in plants recovering from viral infection. When recovered plants were removed to 22℃, upper leaves developed viral symptoms again, indicating there is a dynamic balance between viral spread and antiviral RNA silencing. This results imply the possibility that host could overcome viral infecting as long as the RNA silencing is strong enough.(2) The heat therapy of N. glutinosa revealed that RDR1 and RDR6 involved in antiviral RNA silencing following different models. The SA-inducible and stress-correlating RDR1 respond to thermal regime in a gradual way with its mRNA accumulation reaching the highest level at 12dpi while upper leaves showed no symptoms. Besides, the accumulation of RDR1 mRNA of upper leaves was much higher than that of lower leaves. Thus, we postulate that RDR1 could participate in thermal-activated antiviral defense. In mock infected tissues, the accumulation of RDR6 mRNA was much lower than that of RDR1, And RDR6 seemingly did not respond to thermal regime but was correlated with PVX accumulation which was extremely high in PVX-infected lower leaves. When PVX titer went down to the level of mock infected tissues, RDR6 returned to the basic level. Thus, RDR6 possibly participates antiviral RNA silencing by responding to PVX accumulation in host cells. But the expession of RDR6 seemingly was not affected by virus in heat therapy against PVYN, indicating that RDR1 might work in heat therapy. This indicates that different RDRPs could participate in antiviral RNA silencing induced by different viruses.(3) The accumulation of viral siRNA was detected in the heat therapy experiments, and no obvious change was observed in viral infected lower leaves. Following the decreased viral accumulation the siRNA level reduced even to undetectable level (at 6dpi in PVX infected plants or at 9dpi in PVYN infected plants). This is inconsistent with the law of transgenic RNA silencing. This implies that siRNA accumulation might not a good indicator of the RNA silencing activity in antiviral RNA silencing.(4) The broad-spectrum pathogensis-related protein 1(PR1) aslo responds to thermal regime in heat therapy experiments. A 12-day duration heat therapy in N. glutinosa revealed high levels of PR1 accumulation in lower leaves but not in upper leaves. Besides, the accumulation of PR1 in PVX- or PVYN- infected tissues was visibly higher than in mock infected tissues, and PR1 expression reduced with the decrease of virus accumulation in upper or lower leaves. This suggests that PR1 respond to either thermal regime or virus infection. In the heat therapy experiment against PVYN, the increasing of PR1 in lower leaves of infected plants appeared earlier than that of mock infected plants. This might be due to the overlying effect of high temperature and PVYN. However, whether PR1 affect RNA silencing or plays a part directly by itself in antivirus defense is need to be further discussed.(5) After days of heat pretreatment at 30℃, N. glutinosa plants were inoculated with PVX and then kept at 22℃. The reults showed that plants pretreated at 30℃for 1-4 days still displayed obvious symptoms at 7dpi after inoculation, whereas plants pretreated at 30℃for 8-12 days displayed little or no viral symptoms whose PVX titer was close to that of mock infected tissues tested by ELISA. This indicated PVX resistance in N. glutinosa accumualted gradually under high temperature. That is high temperature enhanced the antiviral silencing activity even in the absense of virus. This suggests that thermal regime has an accumulative effect on antiviral RNA silencing in N. glutinosa, and a necessary duration of heat pretreatment will make plants get rid of viruses completely. Plants pretreated at 30℃for 12days were removed to 22℃for days of incubation and then inoclulated with PVX. The result revealed that 1-4 days incubation at 22℃could not bring much influence on viral resistance which was accumulated at high temperature, whereas the plants after 8-12 days incubation at 22℃developed visble symptoms in upper leaves, correlating well with the PVX titer tested by ELISA. This indicates that viral resistance enhanced by high temperature can last for a period of time with a gradual decline when ambient temperature declines.(6) The PVX resistance in N. benthamiana could also be enhanced at high temperature (30℃). However, after 12 days heat pretreatment at 30℃, PVX-inoculated plants were still developed severe symptoms. It is different from that tested in N. glutinosa. This indicates that the enhanced viral resistance cannot be maintained which will be dramatically compromised once ambient temperature decline. The simplex RNA silencing system in N. benthamiana might account for this results due to the lack of the inducible RDR1 compared with N. glutinosa. This indicates different pathways of RNA silencing might exist in these two plants.(7) As was revealed by the GFP transient infiltration in N. benthamiana or N. glutinosa, GFP expression in patches from plants kept at 22℃were slienced at 5dpi after infiltration, whereas those from plants kept at 30℃remained slienced at 3dpi after infiltration, indicating high temperature could enhance the transgene-induced RNA silencing evoked by GFP transient infiltration assay. The RNA silencing suppressor could delay the occurrence of GFP silencing but could not alter the trend of enhanced RNA silencing at high temperature. The occurrence of GFP silencing in patches from 30℃pretreated plants was no different from those of unpretreated plants. This was similar to the way of PVX resistance responding to high temperature in N. benthamiana, but different from that in N. glutinosa. Thus we may hypothesize that transgene-induced RNA silencing and virus-induced RNA silencing share the same pathway in N. benthamiana but different ones in N. glutinosa.