Figure 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+ concentrations. 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). Vertical bars
indicate standard deviations (n = 3).
Figure 4. CSL-gel triggers CML19 expression and silencingCML19 increases TMV infection in N. benthamiana .The leaves of N. benthamiana were inoculated with TMV‐GFP by
rubbing. The green GFP fluorescence signals were visualized at 6, 8, and
10 dpi under UV light. Representative pictures are presented. (a). qPCR
analysis showing the relative expression of CML19 in the silencedN. benthamiana. (b-d). qPCR analysis showed the expression level
of TMV‐CP in the inoculated leaves of CML19 silenced N.
benthamiana at 6, 8 and 10 dpi. (e). TMV-GFP was inoculated by rubbing
after CML19 silencing and representative pictures were taken at 2
and 7 dpi. The asterisk indicates a significant difference according to
Duncan’s multiple range test (*, p < 0.05; ***, p<0.001).
Vertical bars indicate standard deviations (n = 3).
Figure 5. Overexpression of CML19 inhibits TMV
infection. (a). Western blot analysis showed the accumulation of
GFP-CML19 protein in the overexpressed N. benthamiana and empty
vector GFP:00 control plants. (b). TMV-GFP infection was hindered
in CML19 -overexpressed plants compared to the control
plant.TMV-GFP was inoculated by rubbing after CML19overexpression and representative pictures were taken at 2 and 4 dpi.
(c). qPCR analysis showed the expression level of TMV‐CP in theCML19 overexpressed leaves of N. benthamiana at 2 and 4
dpi was significantly higher than that in the control plant (GFP:00).
The asterisk indicates a significant difference according to Duncan’s
multiple range test (*, p < 0.05; ***, p<0.001). Vertical
bars indicate standard deviations (n = 3).
Figure 6. 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 presented. (b). qPCR analysis showing the relative
expression of TMV-CP in the inoculated leaves of N.
benthamiana at 2 dpi (f). qPCR analysis showed the expression level ofTMV‐CP in the young leaves of N. benthamiana at 7 dpi.
Mean values displayed in each bar followed by different letters are
significantly different according to Duncan’s multiple range test (p
< 0.05). Vertical bars indicate standard deviations (n = 3).
Figure 7. 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 presented.
(b). The green GFP fluorescence signals were visualized at 12 and 16
days after TuMV inoculation under UV light. Representative pictures are
presented. (c). Disease symptoms caused by PVX were observed at 18 and
22 days after inoculation and representative pictures are presented.
(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. Mean
values displayed in each bar followed by different letters are
significantly different according to Duncan’s multiple range test (p
< 0.05). Vertical bars indicate standard deviations (n = 3).
All experiments were repeated three times and similar results were
obtained.
Figure 8. Schematic representation of a proposed action model
of CSL-gel on plants to improve broad-spectrum resistance to different
viruses.