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\begin{document}

\title{A lentinan-loaded hydrogel with core-shell structure induces
broad-spectrum resistance against plant virus by activating the
expression of CML30}



\author[1]{Shunyu Xiang}%
\author[1]{Xue Chen}%
\author[1]{Jing Wang}%
\author[1]{Haoran Peng}%
\author[1]{Xing Lv}%
\author[1]{Long Jiang}%
\author[1]{Zhe Cao}%
\author[1]{Jin Huang}%
\author[2]{Daibin Wang}%
\author[3]{Lisong Ma}%
\author[1]{xianchao sun}%
\affil[1]{Southwest University}%
\affil[2]{Chongqing Tobacco Science Research Institute}%
\affil[3]{Agriculture and Agri-Food Canada}%


\vspace{-1em}



  
  \date{June 13, 2024}


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\selectlanguage{english}
\begin{abstract}
Control of plant virus disease largely depends on the induced plant
defense achieved by the external application of synthetic chemical
inducers with the ability to modify defense-signaling pathways. However,
most of the molecular mechanisms underlying these inducers remain
unknown. Here, we developed a lentinan-loaded hydrogel with the
core-shell structure and discovered how it protects plant from different
virus infections. The hydrogel was synthesized by adding a chitosan
shell on the surface of the sodium alginate-calcium ion-lentinan (LNT)
hydrogel (SL-gel) to form CSL-gel. CSL-gel exhibits the capacity of
prolonging the stable-release of lentinan and promoting calcium ions
release. Application of CSL-gel on the root of plants significantly
promotes plant growth and development and induces broad-spectrum
resistance against TMV, TuMV, PVX and TRV. Furthermore, we found that
the sustained release of calcium ions from the CSL-gel triggers the high
expression of cal-modulin-like protein 30 (CML30), and silencing of
CML30 enhances the susceptibility of tobacco to TMV. Our findings
provide evidence that the novel and synthetic CSL-gel with the
sustainable release of LNT and calcium ion strongly inhibits the plant
virus infection. This study uncovers a novel mode of action by which
CSL-gel with the stable release of calcium ion triggers CML30
expression.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Inducing and activating plant immune response is one of the most
effective ways to combat plant~virus diseases \textsuperscript{{[}1{]}}.
Plant virus that has a variety of infection modes, a wide range of host
and a long infection cycle causes a common systemic
infection\textsuperscript{{[}2{]}}. Control of plant virus disease
largely depends on the application of the virus passivation agents.
However, these agents are not able to provide complete control of the
plant viral diseases owing to their ability to partially reduce the
virus infection and inhibiting the replication and transfer of the virus
in the plant\textsuperscript{{[}3{]}}. Inducers with the ability to
activate plant resistance are becoming an increasingly important way to
control plant virus disease. Previous studies showed that they can
significantly~induce the expression of resistance-related genes and
enhance the immune response of plants to biotic
stresses\textsuperscript{{[}4{]}}. However, the application of the
existing inducers in the field is limited because their lasting time is
shorter than the existing period of virus infection. In addition, most
of the inducers consist of biological polysaccharides with a structure
that is easily degraded at the complicated environment in the field,
rustling in the reduced efficacy on the control of virus
disease\textsuperscript{{[}5-6{]}}. Therefore, there is an urgent need
to develop a novel inducer agent with the ability to last the release
time of loaded drugs for the field application.

Calcium ions (Ca\textsuperscript{2+}) as an essential nutrient element,
can promote plant growth, improve photosynthesis, and increase the
synthesis and accumulation of organic matter \textsuperscript{{[}7{]}}.
Ca\textsuperscript{2+} as a second messenger is also involved in the
signal transduction of plant to stress
responses\textsuperscript{{[}8-9{]}}. Previous studies showed that
Ca\textsuperscript{2+} signals are perceived by four types of calcium
signaling sensor proteins, including calmodulin (\emph{CaM} ),
calmodulin-like protein (\emph{CML} ), calcium-dependent protein kinase
(CDPK), and calcium-like protein Calcineurin B-like
(CBL)\textsuperscript{{[}10-11{]}}. However, \emph{CML} , CDPK, CBL are
found only in plants and some protists. \emph{CMLs} , as one of the
plant-specific Ca\textsuperscript{2+} receptors, participates in plant
biological stress, abiotic stress as well as many developmental
processes \textsuperscript{{[}12-13{]}}. It has been documented that
plant defense response, plant growth and development as well as cytokine
regulation, induce changes in intracellular calcium ion concentration,
which results in the induced expression of \emph{CMLs}
gene\textsuperscript{{[}14-15{]}}. Accumulated evidence showed that the
up-regulation of \emph{CML} gene expression is able to significantly
improve the plant resistance to biotic
stress\textsuperscript{{[}16-17{]}}. For example, \emph{CML41} was found
to reduce \emph{P. syringae} infection by regulating plasmodesmal
closure through mediating a Ca\textsuperscript{2+} signaling in response
to bacterial pathogens\textsuperscript{{[}17{]}}. Leba \emph{et al.}
showed that \emph{CML9} is involved in plant resistance to pathogenic
bacteria through a flagellin-dependent pathway in
plants\textsuperscript{{[}18{]}}. Tobacco calmodulin-like protein,
rgs-CaM, binds to the RNA silencing (RNAi) suppressor of the tobacco
etch virus to inhibit RNAi and increase tobacco resistance to the
virus\textsuperscript{{[}19{]}}. \emph{CML30} was found to be regulated
by abiotic stress and play diverse roles in plant hormone synthesis and
disease defense \textsuperscript{{[}20{]}}. Therefore, \emph{CML} can be
regarded as an important target site to stimulate plant defense
response.

Recent studies showed that sustained-release hydrogel as a drug carrier
has attracted more interest in delivering therapeutic agents to control
human diseases \textsuperscript{{[}21{]}}. Advantages of hydrogel
include a prolonged drug release and action time, increased stability of
loaded drugs and enhanced control efficiency of
disease\textsuperscript{{[}22-23{]}}. In addition, many studies showed
that sustained-release hydrogel exhibits the ability of carrying
multiple drugs and providing various control of multiple diseases. For
example, porphyrin photosensitizer sinoporphyrin sodium (DVDMS) and
Polylactic acid-copolymerized glycolic acid (PLGA) are loaded into the
sodium alginate-chitosan hydrogel that provides the dual action of
antibacterial and skin regeneration \textsuperscript{{[}24{]}}.
Therefore, the development of dual functions of hydrogel that could
deliver a range of drugs and provide the broad-spectrum resistance to
multiple diseases is required.

Our previous studies found that coating the surface of hydrogel with
amino oligosaccharides can prolong the release time of drugs loaded in
the hydrogel (ALA-gel)\textsuperscript{{[}25{]}}. However, amino
oligosaccharides are highly soluble in water, resulting in the slow
dissolve of the ALA-gel surface and the reduced drug release time from
the ALA-gel. To overcome the drawbacks of ALA-gel and improve the
sustained release of drugs loaded in the hydrogel, a multiple-functional
sodium alginate-lentinan hydrogel (SL-gel) with a dense chitosan shell
(CSL-gel) was developed aiming at improving the control efficacy against
plant virus diseases. As expected, CSL-gel exhibited stable and
sustained LNT release and broad-spectrum anti-virus activities. The
sustainable and controlled release of calcium ions activated the
expression of \emph{CML30} that enhances the resistance of tobacco
against virus with a lasting time up to 30 days after CSL-gel treatment.

\textbf{Materials and Methods}

\textbf{Chemical reagents}

The following chemical materials and reagents were used and purchased
from commercial suppliers without further purification: SA and chitosan
(viscosity of 200 \selectlanguage{ngerman}± 20 mPas, Shanghai Aladdin Biochemical Technology
Co., Ltd., Shanghai, China); CaCl\textsubscript{2} (Kaixiang fine
chemical co. LTD, Shanghai, China); LNT (purity\selectlanguage{english}:98\%, Watson
International Ltd).

\textbf{Synthesis of CSL-gel}

The SA, CaCl\textsubscript{2}, lentinan (LNT) and chitosan were used to
synthesis the CSL-ge following the method as described
previously\textsuperscript{{[}25-26{]}} : 1 g SA and 1 g LNT were
dissolved in 125 mL of distilled water and stirred until completely
dissolved. Subsequently, the SA-LNT mixed solution was slowly dropped
into 1000 mL of aqueous CaCl\textsubscript{2} (30 g/L) solution (stirred
at 400 revolutions/min for 30 min) to form a hydrogel precursor (SL-ge)
immediately (loading efficiency\textsuperscript{{[}27{]}} of 15.2\% and
encapsulation percentage\textsuperscript{{[}27{]}} of 89.4\%). Next, 10
g/L aqueous chitosan solution was added to the surface of the SL-gel and
stirred for 2 h (400 revolutions/min) to form the CSL-gel. The CSL-gel
was washed three times with deionized water.

\textbf{Characterization of the chitosan core-shell of CSL-gel}

Scanning Electron Microscopy (SEM), Zeta potential analysis, Fourier
Transform infrared spectrometry (FTIR) and elemental analysis were
performed by Beijing Zhongke Baitei Technical Service Co., LTD. Briefly,
SEM (SU-8020, Hitachi, Japan) was used for the observation of the
surface composition of the hydrogel according to manufactories
instruction. Zetasizer Nano ZS90 (Malvern Panalytical, Almelo,
Netherlands) was used to measure the surface charge of the hydrogel.
FTIR spectrometer Nicolet 6700 (Thermo Scientific, USA) was used to
collect infrared spectra in the range 4000--400\selectlanguage{english} cm\selectlanguage{english}-1 by 32 scans
following the manufacturer's instructions. Elemental analyzer (Vario EL
cube, Elementar Analysensysteme Madison, WI, U.S.A, Frankfurt, Germany)
were used to analyze the elemental composition on the formation of the
chitosan shell in the surface of the hydrogel.

\textbf{Measurement of LNT and calcium ion release from CSL-gel}

The standard curve of LNT was determined by UV-vis spectrophotometry
(UV-vis, PGEN-ERAL) according to the method as described
previously\textsuperscript{{[}25{]}}. The sustained-release ability and
rate of CSL-gel and SL-gel were tested according to the previous
methods\textsuperscript{{[}25{]}}: Briefly, 50 pieces of CSL-gel or
SL-gel were added into 10 mL of deionized water (pH 5) at 20 degC and an
UV\selectlanguage{english}-vis spectrophotometer was used to measure the absorbance of LNT in
solution at 24\selectlanguage{english}-336 h, respectively. The same method was used to measure
the cumulative release of calcium ions and investigate the effects of
different temperatures (Temperature: 10, 20, 30, 40, 50, and 60 degC,
pH=5), different pH values (pH: 3.0, 5.0, 7.0, and 9.0, adjusted by
Tris-HCl, at 20 degC), and different concentrations of cations (The
concentrations of NaCl solution were 0.1, 0.2, 0.4, and 0.8 mol/L,
respectively, pH=5, at 20 degC) on the sustained-release ability and rate
of CSL-gel as well. The cumulative release rate (CRR) of LNT was
calculated according to formula 3. The release kinetics of LNT from
CSL-hydrogel and SL-hydrogel were analyzed using Higuchi (Formula 4) and
Korsmeyer\selectlanguage{english}-Peppas models (Formula 5) \textsuperscript{{[}28{]}}.

CRR (\%) =\(\frac{\sum_{0}^{t}{C_{t}V_{\text{total}}}}{Q_{1}}\ \times 100\%\)(3)

C\textsubscript{t} represents the release LNT concentration at time t,
V\textsubscript{total} represents solution volume, and
Q\textsubscript{1} represents the total quality of loaded LNT.

Higuchi model: Q\textsubscript{t}=\(\text{Kt}^{1/2}\) (4)

Korsmeyer-Peppas model: Q\textsubscript{t}=\(\text{Kt}^{n}\) (5)

Q\textsubscript{t} represents the cumulative release rate of LNT until
time t, K represents the kinetic constant, and n is an index that
reflects the following release mechanisms: Fickian diffusion (n
\textless{} 0.43), non-Fickian or anomalous diffusion (0.43 \textless{}
n \textless{} 0.85), and case II transport (n \textgreater{} 0.85).

\textbf{Plant treatment with CSL-gel}

50 pieces of CSL-gels or SL-gels were evenly embedded near the root of
each plant at approximately four-leaf stage. Each treatment group has
twelve repeats. All plants were grown at the same condition with 12 h of
light and 12 h of darkness at 25 \selectlanguage{english} and were given 10 mL of water every
24 h.

\textbf{Virus inoculation and visualization of disease symptoms}

All viruses were inoculated 14 days after plants were treated with
different hydrogels. The rubbing method was used to inoculate TMV
labeled with a green fluorescent protein (GFP) in hydrogel treated
plants or \emph{CML30} silenced plants. The agroinfiltration was used to
inoculate TRV-GFP, TuMV-GFP and PVX in hydrogel treated plants. The
infection of virus-GFP in \emph{N. benthamiana} was observed under UV
lamp (Blak-Ray B-100AP, Upland, CA, U.S.A.) at the appropriate time
(TMV-GFP: 2 and 7 dpi, TRV-GFP: 5 and 9 dpi, TuMV-GFP: 12 and 16 dpi).
The infection of PVX in \emph{N. benthamiana} was observed under a
fluorescent lamp at 18 and 22 dpi. At the same time, the inoculated
leaves and young leaves of each group were collected in liquid nitrogen
and stored at -80 degC for a subsequent experiment.

\textbf{Quantitative real-time PCR (qPCR) and enzyme-linked
immunosorbent assay (ELISA)}

Total RNA was extracted from inoculated leaves and young leaves using
RNA extraction kit (Promega Biotechnology Co., Ltd., Shanghai, China).
Next, the reverse transcription kit (Shanghai Titan Technology Co.,
Ltd., Shanghai, China) was used to synthesize cDNA. Quantitative PCR
(qPCR) was performed according to the previous method using a CFX Touch
Real\selectlanguage{english}-time PCR machine (Bio\selectlanguage{english}-Rad, USA) and Quantinova\selectlanguage{english} SYBR Green PCR Kit
(Chongqing Shuguang Biotechnology, China)
\textsuperscript{{[}66{]}}.\emph{Actin} was used as an internal
reference. Quantification of the relative changes in gene transcript
levels was performed using the 2\selectlanguage{greek}\textsuperscript{-ΔΔCT}\selectlanguage{english} method. Each
sample has three biological replicates. All primers of qPCR were listed
in \textbf{Table 1 and S1} . The crude extracts prepared from leaf
samples at different inoculation times~were used to perform ELISA assay.
TMV-GFP antibodies and horseradish substrate coloration were used to
detect the protein accumulation of TMV-GFP
\textsuperscript{{[}29-30{]}}.

\textbf{Construction of \emph{CML30} silencing vector}

\emph{CML30} cDNA was amplified from leaf cDNA using primers\emph{CML}
-TRV-F and \emph{CML} -TRV-R (\textbf{Table 1} ). The fragment was then
inserted into VIGS vector Pash18 at the BamH I and Xhol I site. Finally,
the recombinant plasmids were introduced into\emph{Agrobacterium
tumefaciens} EHA105. The empty vector, TRV-GFP was used for control
treatment.

\textbf{Measurement of plant physiological index after CSL-gel
treatment}

A total of 50 pieces of CSL-gel or SL-gel were buried in the soil, and
each plant was given 10 mL of water every day for consecutive 14 days
and placed in a constant temperature growth room at 25 \selectlanguage{ngerman}°C (12 h of light
and 12 h of darkness). After 14 days, the plant height, plant width,
leaf width, fresh weight, and dry weight of \emph{N. benthamiana} were
measured. Each treatment group has twelve replicates.

\textbf{Biosafety evaluation of CSL-gel}

10 crucian carp with a length of 3-4 cm were placed into 1 L of pure
water (pH=5.5) with different amounts of CSL-gel (the number of CSL-gel
pieces per 1 L of water was 50, 100, 200, and 400). Pure tap water was
used as a blank control, and each group has three repeats. The life
activity and death of crucian carp were observed at 24 h intervals. Each
treatment group has four replicates.

\textbf{Statistical analysis}

All experimental data presented here involved at least three replicates.
The data are presented as means and standard deviations. The statistical
analysis was performed with SPSS software (version 17.0) and OriginPro
8.

\textbf{Results}

\textbf{Synthesis of CSL-gel and characterization of chitosan
core-shell}

To obtain the sustained release of lentinan and calcium ions, we
developed a novel and multiple-functional hydrogel by cross-linking the
mixed solution of sodium alginate and lentinan with calcium ions to form
a hydrogel precursor (SL-gel). A chitosan shell was built on the surface
of SL-gel to form a lentinan-loaded hydrogel with a core-shell structure
(CSL-gel) (\textbf{Figure 1a} ), which is expected to provide the stable
and sustained release of lentinan (LNT) and calcium ions (\textbf{Figure
1b} ). To assess the formation of chitosan shell on the surface of
CSL-gel, scanning electron microscopy (SEM) and Zeta potential were
employed. SEM images of SL-gel and CSL-gel were illustrated
in\textbf{Figure 1c} . SEMs showed that the surface of the SL-gel with
large pores was loose, but a dense chitosan shell covering the SL-gel
was observed on the surface of CSL-gel (\textbf{Figure 1c} ). Moreover,
the elemental analysis showed that elemental nitrogen was detected on
the surface of the CSL-hydrogel (\textbf{Table 2} ), indicating that
chitosan was added on the surface of CSL-gel. To avoid the influence of
LNT on the FTIR and zeta analysis, we performed the Fourier transform
infrared spectrometry (FTIR) and zeta analysis using the hydrogel
without LNT to characterize the chitosan shell. \textbf{Figure 1d}showed
a shared peak located at approximately 3340 cm\selectlanguage{english}\textsuperscript{-1} was
observed in the FTIR spectrum of the S-gel and CS-gel. However, the
CS-gel features a fully developed peak on 3340 cm\textsuperscript{-1},
which is attributed to the overlap associated with the peaks of the \selectlanguage{english}-OH
stretching vibration of SA and the \selectlanguage{english}-NH stretching vibration of the
chitosan in comparison to the S-hydrogel. Besides, the CS-gel exhibited
a peak at approximately 2927 cm\textsuperscript{-1}, which was formed by
the C\selectlanguage{english}-H stretching vibration of chitosan, suggesting that the reaction
of adding chitosan to the S-gel was successful. Sodium alginate is
composed of carbon, hydrogen, and oxygen, and the surface is negatively
charged. The zeta potential analysis showed that the surface of CS-gel
has a positive charge, but the S-gel with a negative charge
(\textbf{Figure 1e} ). Taken together, our findings strongly indicate
that a chitosan shell is added to the SL-gel, which has the potential to
stabilize the structure of CSL-gel and provide the sustained release of
LNT and calcium ions.

\textbf{Sustainable release of LNT and calcium ion of CSL-gel}

To assess the release property of loaded drugs in CSL-gel, we measured
the release rate of LNT at the corresponding time according to the
regression equation of LNT (y = 0.0028x + 0.0266, R\textsuperscript{2} =
0.9998, \textbf{Figure S1} ). As shown in \textbf{Figure 2a}
and\textbf{2b} , CSL-gel maintained a stable and cumulative release of
LNT from the first day to the 14 days after soaking. However, a
substantial amount of LNT was released from SL-gel on the first day and
the LNT release slightly increased with the extension of soaking time
and reached the maximum amount on the eighth day after soaking. At the
ninth day, a limited amount of LNT was released following the extension
of the soaking. Furthermore, we used Korsmeyer-Peppas and Higuchi models
to determine the release of LNT. \textbf{Figure 2c} showed that a clear
linear correlation (R\textsuperscript{2}=0.989) between the cumulative
release rate and the logarithm of time from CSL-gel in the
Korsmeyer-Peppas model fitting curve was observed. Similarly, a high
linear correlation between the cumulative release rate and the square
root of time during the release of LNT from the CSL-gel
(R\textsuperscript{2}=0.9823) was also observed in the Higuchi model
fitting curve (\textbf{Figure 2d} ). However, no linear correlation
between the cumulative release rate and the square root of time
(R\textsuperscript{2}=0.8683) and between the cumulative release rate
and the logarithm of time (R\textsuperscript{2}=0.933) of SL-gel was
observed. The corresponding dispersion coefficient (n) of SL-hydrogel
was 0.3089 (n \textless{} 0.43), indicating that the release of LNT from
SL-gel under these conditions followed Fickian diffusion (\textbf{Table
3} ). In contrast, the dispersion coefficient (n) of CSL-gel was 0.6305
(0.43 \textless{} n \textless{} 0.85), which indicates the CSL-gel
followed non-Fick diffusion mechanism (\textbf{Table 3} ). These
findings demonstrated that the chitosan shell controls the release of
LNT from the CSL-gel, resulting in the cumulative and sustainable
release of LNT.

Soil is a complex system in which the properties, such as pH,
temperature, and ion concentration, are dynamic due to the impact of
external factors such as fertilization, irrigation, and
rainfall\textsuperscript{{[}31{]}}. The drug release from hydrogel will
be influenced by different soil environments. To assess the release rate
of LNT at different soil properties, we measured the release of LNT from
hydrogel at different pH, temperatures, and ion concentrations. As shown
in \textbf{Figure 2e} , the cumulative LNT release rate of CSL-gel
highly increased following the increased temperature, which is caused by
the high swelling of the hydrogel at a high temperature. Similarly, the
corresponding release rate of LNT from CSL-gel largely increased from
7.54\% to 41.35\% when the pH increased from 3 to 9 (\textbf{Figure 2f}
). This observation follows the fact that at low pH most of the carboxyl
groups on alginate are deionized and form strong hydrogen bonds with
hydroxyl groups, resulting in a tight shrinkage of the calcium alginate
pellet that prevents the LNT release from the CSL-gel. Interestingly, we
found that ion concentration present positively regulated the release
LNT from CSL-gel. As shown in \textbf{Figure 2e} , the cumulative
release rate of LNT highly increased from 33.7\% to 99.5\% following the
increased Na\textsuperscript{+} concentration from 0 to 0.8 M. The ion
exchange between Na\textsuperscript{+} and Ca\textsuperscript{2+}
strengthens the electrostatic repulsion between -COO\textsuperscript{-},
which ultimately increases the swelling of the hydrogel to stimulate the
release of LNT \textsuperscript{{[}32{]}}.

\textbf{CSL-gel promotes plant growth and enhances the resistance
against plant virus}

The chitosan present on the surface of CSL-gel can form a robust
electrostatic force with sodium alginate, resulting in the accelerated
release of calcium ions from the gel. To test our hypothesis, we soaked
the SL-gel or CSL-gel in water and measured the concentration of
Ca\textsuperscript{2+} in the water solution at 2-day intervals. As
shown in \textbf{Figure 3a} , the amount of
Ca\textsuperscript{2+}released from the CSL-gel was significantly higher
than that of SL-gel. To assess whether the released
Ca\textsuperscript{2+} can promote plant growth and development, we
measured the plant height, leaf width, dry weight, and fresh weight of
\emph{N. benthamiana} at 14 days after SL-gel or CSL-gel treatment.
\textbf{Figure 3b-g} showed CSL-gel-treated plants displayed a
significant increase in plant growth, including height, plant width,
leaf width, dry weight compared to that of the water-treated plant. In
addition, the growth indexes of plants treated with CSL-gel were higher
than those of the S-gel-treated and control plants (\textbf{Figure 3b-g}
). Moreover, After 30 days of treatment, the growth trend of
CSL-gel-treated plants was significantly higher than that of the
water-treated and SL-gel-treated group (\textbf{Figure S2} ). These
results demonstrated that CSL-gel promotes plant development.

To examine the anti-virus activity of the CSL-gel with the sustained
release of LNT and calcium ion, we applied the CSL-gel into the soil
surrounding the plant root. SL-gel serves as a control. We inoculated
the TMV-GFP by rubbing on the leaves of the plant at 7 day after CSL-gel
application. As shown in \textbf{Figure 3h} , at 2 dpi the number of
green fluorescent spots in CSL-gel-treated \emph{N. benthamiana} was the
same as that on the SL-gel-treated plants. At 7 dpi, the green
fluorescent signals present in the young leaves of the CSL-gel-treated
plant were significantly weaker than that of the water and SL-gel
treatment groups (\textbf{Figure 3h} ). qPCR analysis showed that the
number of TMV-\emph{CP} transcripts between the two treatment groups was
no significant differences, but at 7 dpi the expression level of
TMV-\emph{CP} in the CSL-gel-treated plant was significantly lower than
that in the SL-gel-treated and control plants (\textbf{Figure 3i-j} ).
Taken together, we can conclude that the CSL-gel with the sustainable
release of LNT and calcium ion significantly enhances the tobacco
resistance against TMV as well as promoting the plant development.

\textbf{CSL-gel induces \emph{CML30} expression and silencing
of\emph{CML30} enhances the susceptibility to TMV}

The increased Ca\textsuperscript{2+} concentration in plant cells
promotes the expression of \emph{CML} that plays a role in plant
development and resistance in response to biotic
stresses\textsuperscript{{[}33{]}}. To examine the impact of CSL-gel on
the expression of \emph{CML} genes, the relative expression of
\emph{CML30}in plants treated with CSL-gel was quantified by qPCR.
SL-gel treated plant severs as a positive control. \textbf{Figure}
\textbf{4a and 4b}showed that the \emph{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 \emph{CML30} expression by
CSL-gel contributes to the TMV resistance, we generated the
\emph{CML30}RNAi silencing construct to transiently knockdown the
expression of\emph{CML30} in tobacco by agroinfiltration. As shown in
\textbf{Figure 4c} , PCR analysis showed that the expected silencing
fragment was present in the tested 5 Agrobacterium strains. qPCR
analysis showed that\emph{CML30} expression level in the silenced plant
was 80\% reduced compared with the wild-type plant at 8 days after
agroinfiltration (\textbf{Figure 4e} ), indicating that the silencing
construct is effective in reducing \emph{CML30} expression. Eight days
after infiltration, all infiltrated leaves were inoculated with
TMV-GFP.\textbf{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 \textbf{(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 (\textbf{Figure 4d} ). In
addition, the number of TMV-\emph{CP} transcripts was quantified by
qPCR. \textbf{Figure 4f}showed that the expression of TMV-\emph{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 (\textbf{Figure 4g} ). Based on our findings, we can
conclude that silencing \emph{CML30} promotes the TMV infection on
\emph{N. Benthamian} , indicating that CSL-gel induces \emph{CML30}
expression that contributes to the TMV resistance.

\textbf{Anti-TMV activity of CSL-gel mainly depends on the chitosan
shell and calcium ion}

CSL-gel promotes the expression of \emph{CML30} and the silencing
of\emph{CML30} enhances TMV infection. To further determine
that\emph{CML30} induced by calcium ion released from the CSL-gel
contributes to the resistance against TMV infection, we generated the
CS-gel and S-gel without LNT to eliminate the role of LNT on the
anti-TMV activity. As shown in \textbf{Figure 5a} , at 2 dpi, the number
of green fluorescent spots in the inoculated leaves of \emph{N.
benthamiana} treated with CS-gel was lower than that of the S-gel and
water-treated plants. qPCR analysis showed that the expression levels of
TMV-\emph{CP} were significantly lower in the inoculated leaves
of\emph{N. benthamiana} treated with CS-gel than that in the water and
S-gel-treated plants (\textbf{Figure 5b and 5c} ). At 7 dpi, the green
fluorescence signals and the number of TMV-\emph{CP} transcripts of the
water control group and S-gel treatment group were significantly higher
than that of CS-gel control. However, there was no significant
difference between the water-treated and the S-gel-treated plans on the
TMV infection at 2 and 7 dpi. These findings indicated that
\emph{CML30}induced by CSL-gel also plays an important role in anti-TMV.

\textbf{CSL-gel induces broad-spectrum resistance against different
plant virus}

To assess whether CSL-gel induces broad-spectrum resistance against
different virus, \emph{N. benthamiana} plants treated with CSL-gel were
separately inoculated with the tobacco rattle virus (TRV), potato virus
X~(PVX) and turnip mosaic virus (TuMV) by rubbing. SL-gel and water
treated plants were included as controls. At the early stage of
infection (TRV: 5 dpi, TuMV: 12dpi), the number of green fluorescent
spots in CSL-gel-treated\emph{N. benthamiana} was similar to
SL-gel-treated plants. However, in the late stage of infection (TRV: 9
dpi, TuMV: 16 dpi), the green fluorescent signals present in the young
leaves of the CSL-gel-treated plant were significantly lower than that
of the water and SL-gel treatment groups (\textbf{Figure 6a-b} ).
\textbf{Figure 6c} showed that 18 days after inoculation, symptoms
caused by PVX were relatively mild in the young leaves of and
CSL-gel-treated plants, while PVX caused severe symptoms in the young
leaves of water-treated plants. At 22 dpi, PVX infection developed
severely curly young leaves in the water-treated plants, and plants
treated with SL-gel showed mild symptoms. However, no symptoms caused by
PVX infection were observed in the CSL-gel-treated plants
(\textbf{Figure 6c} ). Furthermore, qPCR analysis showed that the number
of virus-CP transcripts in the CSL-gel-treated plant was significantly
lower than that in the SL-gel and water-treated plants (\textbf{Figure
6d-i} ). Therefore, we can conclude that CSL-gel inhibits the infection
caused by the majority of viruses in \emph{N. benthamiana}plants.

\textbf{Safety test of CSL-gel}

The toxicity test of pesticides on aquatic organisms is an important
measure strategy for the safety evaluation of
pesticides\textsuperscript{{[}34{]}}. Our previous studies showed that
the LC\textsubscript{50} of LNT to crucian carp at 24, 48, 72, and 96 h
was about 1.6, 0.8, 0.4, and 0.4 mg/mL,
respectively\textsuperscript{37}. To evaluate the CSL-gel safety, the
number of deaths of \emph{crucian carp} was examined in the water
immersed with series of numbers of CSL-gel at 24 h, 48 h, 72 h and 96 h.
The pure tap water was used as a negative control. As shown
in\textbf{Table 4} , no dead \emph{crucian carp} was observed in the
CSL-treated water with LNT concentration lower than 336 mg as well as in
the water control. However, dead \emph{crucian carp} was observed at 48
h in the water treated with 400 pieces of CSL-gel with an LNT
concentration at 336 mg, indicating that CSL-gel maintained a relatively
high safety to environmental organisms.

\textbf{Discussion}

Plant viruses cause a common systemic disease named plant cancers that
differ from other plant diseases \textsuperscript{{[}1-2{]}}. They can
entirely rely on the host plant to acquire nutrients for replication,
movement, and other life activities, resulting in damage to plant
chloroplasts and photosynthesis, reduced accumulation of plant
carbohydrates, and inhibiting plant growth \textsuperscript{{[}1-3{]}}.
Traditional methods depend on virus deactivators and plant immunity
inducers to minimize the infection \textsuperscript{{[}35{]}}. However,
most virus deactivators and plant immunity inducers are not able to
completely inhibit the proliferation and movement of the virus in the
plant after the virus infection \textsuperscript{{[}3{]}}. In this
study, a lentinan-loaded hydrogel with the core-shell structure was
developed, which features the stable and sustainable release of lentinan
and calcium ions. The prolonged-release of LNT and calcium ions
significantly promotes plant growth and development and provides
broad-spectrum resistance against TMV, TuMV, PVX and TRV. In addition,
we found that the sustained release of calcium ions from the CSL-gel
activates the expression of calmodulin-like protein 30 (\emph{CML30} ),
and the silencing of\emph{CML30} enhances the susceptibility of tobacco
to TMV. We summarize the action of CSL-gel in \textbf{Figure 7} .
Therefore, the low-cost and easily synthesized CSL-gel with a novel mode
of action triggering\emph{CML30} expression has the potential to be
utilized in the field against severe virus disease and increase the
yield of crop plants.

Our results showed that the chitosan shell prevents the rapid bursting
of the hydrogel, resulting in the controlled and stable release of LNT
and calcium ions and the extended-release time (\textbf{Figures 1 and 2}
). Polysaccharide immunity inducer, such as amino oligosaccharides,
chitosan, chitin, and lentinan, has been widely used in anti-plant virus
diseases \textsuperscript{{[}36{]}}. Lentinan, as a biological
polysaccharide, has multiple disease-resistant functions, such as
closing plant stomata, inducing the expression of plant resistance genes
and improving plant disease-related enzyme
activity\textsuperscript{{[}25{]}}. However, previously generated
hydrogel loaded with LNT is confined in field application due to the
limited induction time of lentinan and its instability in the complex
field environment \textsuperscript{{[}25{]}}. The newly synthesized
hydrogel with a core-shell structure that functions as a slow-release
carrier prolongs the action time of lentinan on plants, promotes the
plant growth and induces the resistance of \emph{N. benthamiana} to TMV
(\textbf{Figure 3} ). At present, plant immunity inducers as an
alternative agent are more effective in controlling plant virus
disease\textsuperscript{{[}35{]}}. Many studies reported that plant
immunity inducers can induce the expression of plant disease-related
genes, and promote the activities of stress-related enzymes to
comprehensively improve the resistance of plants to
pathogens\textsuperscript{{[}35{]}}. For example, Lentinan has been used
as a common inducer in the prevention and treatment of plant virus
diseases in the field. It can significantly promote the activity of SOD,
POD and CAT and stimulate the expression of \emph{PR1} and \emph{PR3} to
improve plant resistance\textsuperscript{{[}36-37{]}}. Previous studies
showed that chitosan was able to increase the activity of plant
defense-related enzymes (PAL, PPO, POD, CAT, SOD), and induce the
production of secondary metabolites associated with disease resistance
and induce phenolic metabolic pathways to improve the antagonism of
plants to fungi, bacteria and viruses \textsuperscript{{[}38-39{]}}.

Calcium ion (Ca\textsuperscript{2+}), as one of the important nutrient
elements, can increase the germination rate of plant seeds, promote the
development of plant roots and leaves, and increase the absorption of
nutrients to enhance plant growth \textsuperscript{{[}7, 40{]}}.
Previous studies showed that the application of calcium ion at a
concentration in the range of 0-14mmol/L increases plant growth, root
growth, and dry matter accumulation \textsuperscript{{[}40{]}}.
Ca\textsuperscript{2+,} as a universal second messenger, can regulate
the plant response to biotic and abiotic stresses. For example, it can
regulate plant cell membrane protective enzyme systems to alleviate the
effects of drought, salt stress, and water stress on
growth\textsuperscript{{[}41-42{]}}. The activities of antioxidant
enzymes, such as SOD, POD, and CAT, in soybeans and apples treated with
calcium ions were significantly improved \textsuperscript{{[}43{]}}.
Furthermore, the application of calcium fertilizer effectively prevents
the occurrence of brown spot disease \textsuperscript{{[}44{]}}. Our
results showed that the chitosan shell promotes the CSL-gel to stably
and sustainably release Ca\textsuperscript{2+} owing to the strong
electrostatic interaction between SA and chitosan (\textbf{Figure 2} ).
The plants treated with CS-gel exhibit significantly enhanced growth
than those treated with the SL-gel (\textbf{Figures 3 and 5} ). This
evidence is consistent with previous reports, indicating that CSL-gel
with the stable and sustainable release of calcium ions promotes plant
growth and improves plant resistance against viruses.

Previous studies showed that Ca\textsuperscript{2+} is perceived by
calmodulin (CaM) and calmodulin-like (\emph{CML} ) proteins to
participate in physiological and biochemical reactions in
plants\textsuperscript{{[}8-9{]}}. As a type of plant-specific
Ca\textsuperscript{2+} receptor, \emph{CML} involves various
physiological activities in the process of plant growth and development,
such as regulating plant defense responses, enhancing plant anti-stress
responses, and controlling plant hormone levels \textsuperscript{{[}22,
45-46{]}}. For example, overexpression of \emph{Arabidopsis~CML8}confers
enhanced resistance to~\emph{Pseudomonas syringae} in an SA-dependent
process\textsuperscript{{[}47{]}}. \emph{ArabidopsisCML9} is rapidly and
strongly induced by \emph{Pseudomonas syringae} and abiotic stress and
abscisic acid (ABA), which acts as a positive regulator in response to
\emph{Pseudomonas syringae} and a negative regulator to salt
stress\textsuperscript{{[}48-49{]}}. Tomato plants overexpressing
\emph{CML44} exhibit higher antioxidant enzyme activity and greater
tolerance to abiotic stresses\textsuperscript{{[}42{]}}. Our results
showed that CSL-gel strongly induces the expression of \emph{CML30} and
the silencing of\emph{CML30} enhances the infection of TMV
(\textbf{Figure 4} ), suggesting that \emph{CML30} plays a positive role
in the resistance to TMV. The CSL-gel maintains a stable and cumulative
release of calcium ions into the soil, which activates plant
\emph{CML30} to adapt to the increased calcium ions in the environment.
In turn, the accelerated expression of \emph{CML30} enhances the
resistance against TMV. However, the mechanisms underlying this
observation remain unknown. Because\emph{CML} as a sensor of
Ca\textsuperscript{2+} regulates diverse plant processes, the
identification of \emph{CML30} interactors will aid us in understanding
the anti-virus activity of \emph{CML30} in the subsequent studies.

\textbf{Conclusions}

A hydrogel with a core-shell structure and sustainable release of
lentinan and calcium ions based on the polycationic properties of
chitosan was developed in this study. This hydrogel has multiple
functions including the promotion of plant growth, continuous induction
of plant resistance against the different virus, and enhancement
of\emph{CML30} expression to improve TMV resistance. These findings
presented here form a solid basis for us to understand the resistance
against TMV, highlight the importance of \emph{CML} in plants against
TMV infection and expand our tools to control plant virus diseases as
well in the future.

\textbf{Author contributions}

S. X., X. C., J .W., L. M. and X. S. conceived and designed the
experiments. S.X., X. C., J. W., H. P., X. L., L. J., Z. C., J. H.
performed the experiments, S. X., X.C., J. W., D. W., L.M., and X.S.
analyzed the data. S.X., L.M. and X.S.drafted the manuscript.

\textbf{Funding}

This study was partly supported by the National Natural Science
Foundation of China (31670148 and 31870147), the Fundamental Research
Funds for the Central Universities (XDJK2020B064 and XDJK2017C015), the
Science and Technology Projects of Chongqing Company of China Tobacco
Corporation (NY20180401070001, A20201NY02-1306 and NY20180401070010).

\textbf{Conflict of interest statement}

The authors declare no competing financial interest.

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\textbf{Legends:}

\textbf{Figure 1.} \textbf{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.

\textbf{Figure 2.} \textbf{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\textsuperscript{+} concentration. Double asterisks indicate
separation among CSL-gel and SL-gel at the same amount by Duncan's
multiple comparisons (\selectlanguage{english}*\selectlanguage{english}*, p \textless{} 0.01). Vertical bars indicate
standard deviations (n = 3).

\textbf{Figure 3.} \textbf{CSL-gel with the sustainable release of
calcium ions promotes plant growth and significantly enhances the
resistance of \emph{N. benthamiana}} \textbf{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 \emph{N. benthamiana} plants display
enhanced resistance against TMV. \emph{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 \selectlanguage{english}\emph{TMV-CP} in the inoculated leaves of \emph{N.
benthamiana} at 2 dpi. (j). qPCR analysis showing the expression level
of TMV\selectlanguage{english}-CP in the inoculated leaves of \emph{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 \textless{} 0.05).

\textbf{Figure 4. CSL-gel triggers \emph{CML30} expression and
silencing\emph{CML30} increases TMV infection in \emph{N. benthamiana}}
\emph{.}(a-b). CSL-gel treatment triggers the expression of \emph{CML30}
at 2 days and 7 days. (c). The PCR results showing the presence of
recombinant plasmids in Agrobacterium. (d). Silencing
\emph{CML30}increases TMV infection. The leaves of~\emph{N. benthamiana}
~were inoculated with TMV\selectlanguage{english}-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 \emph{CML30} in the silenced \emph{N.
benthamiana.} (f). qPCR analysis showed the expression level of TMV\selectlanguage{english}-CP
in the inoculated leaves of~\emph{CML30} silenced \emph{N. benthamiana}
~at 3, 4 and 5 dpi.~(g). The accumulation of TMV-CP protein was detected
by ELISA.

\textbf{Figure 5. Comparison of the anti-TMV activity of S-gel and
CS-gel.} (a). \emph{N. benthamiana} plants treated with CS-gel display
increased resistance against TMV. The leaves of \emph{N.
benthamiana}were inoculated with TMV\selectlanguage{english}-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 \emph{TMV-CP} in the inoculated leaves of
\emph{N. benthamiana} at 2 dpi. (c). qPCR analysis showed the expression
level of \selectlanguage{english}\emph{TMV-CP} in the young leaves of~ \emph{N. benthamiana} ~at
7 dpi.

\textbf{Figure 6.} \textbf{CSL-gel treated \emph{N. benthamiana} plants
exhibit significantly increased resistance against TRV, TuMV and PVX} .
The leaves of \emph{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 \emph{TRV-CP} in the
inoculated leaves of \emph{N. benthamiana} at 5 dpi and in the young
leaves at 9 dpi. (f-g). qPCR analysis showing the relative expression of
T\emph{uMVV-CP} in the inoculated leaves of \emph{N. benthamiana} at 12
dpi and in the young leaves at 16 dpi. (h-i). qPCR analysis showing the
relative expression of \emph{PVX-CP} in the inoculated leaves of\emph{N.
benthamiana} at 18 dpi and in the young leaves at 22 dpi.

\textbf{Figure 7.} Schematic representation of a proposed action model
of CSL-gel on plants to improve broad-spectrum resistance to different
viruses.

\textbf{Table 1.}Primers used in this study.

\textbf{Table 2.} Elemental analysis of the SL-gel and CSL-gel.

\textbf{Table 3.} Kinetic parameters associated with LNT released from
the SL-gel and CSL-gel.

\textbf{Table 4.} Toxicity of CSL-gel to Crucian Carp.

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