CSL-gel induces CML19 expression and silencing ofCML19 enhances the susceptibility to TMV
The increased Ca2+ concentration in plant cells
promotes the expression of CML that plays diverse roles in plant
development and resistance in response to biotic stresses[40]. To examine the impact of CSL-gel on the
genome-wide expression of plant genes, we performed RNA-seq on the
CSL-treated, SL-treated and water-treated plants at 10 days after
treatment, respectively. Transcriptomic analysis showed that a total of
514 differentially expressed genes (DEGs) were upregulated in N.
benthamiana treated with CSL-gel compared to SL-treated and
water-treated plants (Figure S3). CML genes in the DEGs list were
checked and we found that CML19 was specifically upregulated
after CSL-gel treatment. To confirm the RNA-Seq results, the relative
expression of CML19 in plants treated with CSL-gel was quantified
by qPCR. SL-gel treated plant severs as a positive control.Figure S4 showed that the CML19 expression in the
CSL-gel treated plants was significantly higher than that in the SL-gel
treated plants at 7 days after treatment.
To further determine whether upregulated CML19 expression by
CSL-gel contributes to TMV resistance, we generated the CML19RNAi silencing construct to transiently knockdown the expression ofCML19 in tobacco by agroinfiltration. qPCR analysis showed that
the CML19 expression level in the silenced plant was 40% reduced
compared with the wild-type plant at 8 days after agroinfiltration
(Figure 4a ), indicating that the silencing construct is
effective in reducing CML19 expression. Eight days after
infiltration, all infiltrated leaves were inoculated with TMV-GFP.Figure 4e showed that at 6 dpi, the level of GFP signals
present in silenced plants and WT plants were similar. At 8 dpi, strong
GFP signals were observed in the inoculated leaves of silenced plants
compared with that in WT plants. Similarly, in the young leaves of the
silenced plant, increased GFP signals were visualized, while in the WT
plant limited GFP signals were observed in the young leaves(Figure 4e ). At 10 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 the WT plant
(Figure 4e ). In addition, the number of TMV-CPtranscripts was quantified by qPCR. Figure 4b-c showed that the
expression of TMV-CP in the silenced plant was significantly
higher than that in the WT plant at 6, 8 and 10 dpi. Based on our
findings, we can conclude that silencing CML19 promotes TMV
infection in N. Benthamian , indicating that CSL-gel inducesCML19 expression that contributes to TMV resistance.