CSL-gel induces CML30 expression and silencing ofCML30 enhances the susceptibility to TMV
The increased Ca2+ concentration in plant cells promotes the expression of CML that plays a role in plant development and resistance in response to biotic stresses[33]. To examine the impact of CSL-gel on the expression of CML genes, the relative expression of CML30in plants treated with CSL-gel was quantified by qPCR. SL-gel treated plant severs as a positive control. Figure 4a and 4bshowed that the CML30 expression in the CSL-gel treated plants was significantly higher than that in the SL-gel treated plants at 2 and 7 days after treatment.
To further determine whether upregulated CML30 expression by CSL-gel contributes to the TMV resistance, we generated the CML30RNAi silencing construct to transiently knockdown the expression ofCML30 in tobacco by agroinfiltration. As shown in Figure 4c , PCR analysis showed that the expected silencing fragment was present in the tested 5 Agrobacterium strains. qPCR analysis showed thatCML30 expression level in the silenced plant was 80% reduced compared with the wild-type plant at 8 days after agroinfiltration (Figure 4e ), indicating that the silencing construct is effective in reducing CML30 expression. Eight days after infiltration, all infiltrated leaves were inoculated with TMV-GFP.Figure 4d showed that at 3 dpi, the GFP signals increased compared to that at 2 dpi, but the level of signals present in silenced plants and WT plants were similar. At 4 dpi, strong GFP signals were observed in the inoculated leaves of silenced plants compared with that in the WT plants and in the young leaves of the silenced plant increased GFP signals were visualized, while in WT plant limited GFP signals were observed in the young leaves (Figure 4d ). At 5 dpi, pronounced green fluorescence signals were visualized in the young leaves of the silenced plant, but slightly expanded GFP signals were observed in the young leave of WT plant (Figure 4d ). In addition, the number of TMV-CP transcripts was quantified by qPCR. Figure 4fshowed that the expression of TMV-CP in the silenced plant was significantly higher than that in the WT plant at 3, 4 and 5 dpi. Similarly, ELISA analysis showed that a significant amount of TMV was accumulated in the silenced plant compared to that in the WT plant (Figure 4g ). Based on our findings, we can conclude that silencing CML30 promotes the TMV infection on N. Benthamian , indicating that CSL-gel induces CML30 expression that contributes to the TMV resistance.