Legends:
Figure 1. Synthesis of CSL-gel and characterization of chitosan shell . (a). schematic representation of the synthesis of CSL-gel. (b). Diagram of CSL-gel and SL-gel drug release simulation. (c). SEM images of surface composition of SL-gel and CSL-gel. (d). Zeta potential of the S-gel without LNT and chitosan shell and CS-gel without LNT. (e). FTIR spectra of the S-gel and CS-gel.
Figure 2. CSL-gel exhibits sustainable and cumulative release of LNT . (a). Comparison of the cumulative release of LNT from the SL-gel and CSL-gel. (b). The cumulative release rate of LNT from CSL-gel and SL-gel. (c). The fitting curves for the Korsmeyer-Peppas model. (d). The fitting curves for the Higuchi model. (e). The cumulative release rate of LNT from CSL-gel at different temperatures. (f), The cumulative release rate of LNT from CSL-gel at different pH. (g). The cumulative release rate of LNT from CSL-gel at different Na+ concentration. Double asterisks indicate separation among CSL-gel and SL-gel at the same amount by Duncan’s multiple comparisons (∗∗, p < 0.01). Vertical bars indicate standard deviations (n = 3).
Figure 3. CSL-gel with the sustainable release of calcium ions promotes plant growth and significantly enhances the resistance of N. benthamiana against TMV . (a). Comparison of calcium ions cumulative release between SL-gel and CSL-gel. (b-f).Comparison of plant height, plant width, leaf width, dry weight, and fresh weight between CSL-gel and SL-treated plants. (g). Representative pictures showing the plant growth between different treatments. (h). CSL-gel treated N. benthamiana plants display enhanced resistance against TMV. N. benthamiana plants were inoculated with TMV-GFP constructs by rubbing and representative pictures were photographed at 2 and 7 dpi. (i) qPCR analysis showing the expression level of TMV‐CP in the inoculated leaves of N. benthamiana at 2 dpi. (j). qPCR analysis showing the expression level of TMV‐CP in the inoculated leaves of N. benthamiana at 7 dpi. The expression level was normalized to Actin. Mean values displayed in each bar followed by different letters, are significantly different according to Duncan’s multiple range test (p < 0.05).
Figure 4. CSL-gel triggers CML30 expression and silencingCML30 increases TMV infection in N. benthamiana .(a-b). CSL-gel treatment triggers the expression of CML30 at 2 days and 7 days. (c). The PCR results showing the presence of recombinant plasmids in Agrobacterium. (d). Silencing CML30increases TMV infection. The leaves of N. benthamiana  were inoculated with TMV‐GFP by rubbing.The green GFP fluorescence signals were visualized at 2, 3, 4 and 5 dpi under UV light. Representative pictures are showed. (e). qPCR analysis showing the relative expression of CML30 in the silenced N. benthamiana. (f). qPCR analysis showed the expression level of TMV‐CP in the inoculated leaves of CML30 silenced N. benthamiana  at 3, 4 and 5 dpi. (g). The accumulation of TMV-CP protein was detected by ELISA.
Figure 5. Comparison of the anti-TMV activity of S-gel and CS-gel. (a). N. benthamiana plants treated with CS-gel display increased resistance against TMV. The leaves of N. benthamianawere inoculated with TMV‐GFP by rubbing. The green GFP fluorescence signals were visualized at 2 and 7 dpi under UV light. Representative pictures are showed. (b). qPCR analysis showing the relative expression of TMV-CP in the inoculated leaves of N. benthamiana at 2 dpi. (c). qPCR analysis showed the expression level of TMV‐CP in the young leaves of  N. benthamiana  at 7 dpi.
Figure 6. CSL-gel treated N. benthamiana plants exhibit significantly increased resistance against TRV, TuMV and PVX . The leaves of N. benthamiana were inoculated with TRV, TuMV and PVX by rubbing or agroinfiltration 14 days after CSL-gel treatment. (a). The green GFP fluorescence signals were visualized at 5 and 9 days after TRV inoculation under UV light. Representative pictures are showed. (b). The green GFP fluorescence signals were visualized at 12 and 16 days after TuMV inoculation under UV light. Representative pictures are showed. (c). Disease symptoms caused by PVX were observed at 18 and 22 days after inoculation and representative pictures are showed. (d-e). qPCR analysis showing the relative expression of TRV-CP in the inoculated leaves of N. benthamiana at 5 dpi and in the young leaves at 9 dpi. (f-g). qPCR analysis showing the relative expression of TuMVV-CP in the inoculated leaves of N. benthamiana at 12 dpi and in the young leaves at 16 dpi. (h-i). qPCR analysis showing the relative expression of PVX-CP in the inoculated leaves ofN. benthamiana at 18 dpi and in the young leaves at 22 dpi.
Figure 7. Schematic representation of a proposed action model of CSL-gel on plants to improve broad-spectrum resistance to different viruses.
Table 1.Primers used in this study.
Table 2. Elemental analysis of the SL-gel and CSL-gel.
Table 3. Kinetic parameters associated with LNT released from the SL-gel and CSL-gel.
Table 4. Toxicity of CSL-gel to Crucian Carp.