Discussion
Plant
viruses cause a common systemic disease named plant cancers that differ
from other plant diseases [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 [1-3]. Traditional methods
depend on virus deactivators and plant immunity inducers to minimize the
infection [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 [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 (CML30 ), and the silencing ofCML30 enhances the susceptibility of tobacco to TMV. We summarize
the action of CSL-gel in Figure 7 . Therefore, the low-cost and
easily synthesized CSL-gel with a novel mode of action triggeringCML30 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 (Figures 1 and
2 ). Polysaccharide immunity inducer, such as amino oligosaccharides,
chitosan, chitin, and lentinan, has been widely used in anti-plant virus
diseases [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[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 [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 N. benthamiana to TMV
(Figure 3 ). At present, plant immunity inducers as an
alternative agent are more effective in controlling plant virus disease[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[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 PR1 and PR3 to improve
plant resistance[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 [38-39].
Calcium ion (Ca2+), 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 [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 [40].
Ca2+, 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[41-42]. The activities of antioxidant enzymes,
such as SOD, POD, and CAT, in soybeans and apples treated with calcium
ions were significantly improved [43].
Furthermore, the application of calcium fertilizer effectively prevents
the occurrence of brown spot disease [44]. Our
results showed that the chitosan shell promotes the CSL-gel to stably
and sustainably release Ca2+ owing to the strong
electrostatic interaction between SA and chitosan (Figure 2 ).
The plants treated with CS-gel exhibit significantly enhanced growth
than those treated with the SL-gel (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 Ca2+ is perceived by
calmodulin (CaM) and calmodulin-like (CML ) proteins to
participate in physiological and biochemical reactions in plants[8-9]. As a type of plant-specific
Ca2+ receptor, 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 [22,
45-46]. For example, overexpression of Arabidopsis CML8confers enhanced resistance to Pseudomonas syringae in an
SA-dependent process[47]. ArabidopsisCML9 is rapidly and strongly induced by Pseudomonas
syringae and abiotic stress and abscisic acid (ABA), which acts as a
positive regulator in response to Pseudomonas syringae and a
negative regulator to salt stress[48-49]. Tomato
plants overexpressing CML44 exhibit higher antioxidant enzyme
activity and greater tolerance to abiotic
stresses[42]. Our results showed that CSL-gel
strongly induces the expression of CML30 and the silencing ofCML30 enhances the infection of TMV (Figure 4 ),
suggesting that 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 CML30 to adapt to the
increased calcium ions in the environment. In turn, the accelerated
expression of CML30 enhances the resistance against TMV. However,
the mechanisms underlying this observation remain unknown. BecauseCML as a sensor of Ca2+ regulates diverse plant
processes, the identification of CML30 interactors will aid us in
understanding the anti-virus activity of CML30 in the subsequent
studies.