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.