Introduction
Inducing and activating plant immune response is one of the most effective ways to combat plant virus diseases (Hammerschmidt et al., 2001). Plant virus that has a variety of infection modes, a wide range of host and a long infection cycle causes a common systemic infection (Scholthof, 2005, M. et al., 1982). 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 inhibit the replication and transfer of the virus in the plant(Yang et al., 2016, Yamakawa and H., 1998). 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(Yamakawa and H., 1998). 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 (Xiang et al., 2013, Lin et al., 2006, Xiu et al., 2006, Tang et al., 2012). 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 (Ca2+) as an essential nutrient element, can promote plant growth, improve photosynthesis, and increase the synthesis and accumulation of organic matter (Tang et al., 2012, Kwon et al., 2009). Ca2+ as a second messenger is also involved in the signal transduction of plant to stress responses (Batistic and Kudla, 2012, Defalco et al., 2010, Dodd et al.). Previous studies showed that Ca2+ signals are perceived by four types of calcium signaling sensor proteins, including calmodulin (CaM ), calmodulin-like protein (CML ), calcium-dependent protein kinase (CDPK), and calcium-like protein Calcineurin B-like (CBL) (Braam and Davis, 1990, Mccormack et al., 2005, Boonburapong and Buaboocha, 2007). However, CML, CDPK, CBL are found only in plants and some protists. CMLs , as one of the plant-specific Ca2+ receptors, participate in plant biological stress, abiotic stress as well as many developmental processes(Scrase-Field and Knight, 2003, Bender and Snedden, 2013, Nakahara et al., 2012). 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 CMLs gene (Roberts and Harmon, 1992, Mcainsh and Pittman, 2009). Accumulated evidence showed that the up-regulation of CML gene expression is able to significantly improve the plant resistance to biotic stress (Ma et al., 2008, Bo et al., 2017). For example, CML41 was found to reduce P. syringae infection by regulating plasmodesmal closure through mediating Ca2+ signaling in response to bacterial pathogens(Bo et al., 2017). Leba et al. showed that CML9 is involved in plant resistance to pathogenic bacteria through a flagellin-dependent pathway in plants(Leba et al., 2012). 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 (Nakahara et al., 2012). CML19 , also known as arabidopsis centrin 2, was found to be regulated by abiotic stress (UV resistance) and play diverse roles in DNA damage repair (Liang et al., 2006). Therefore, 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(Jeong et al., 1997, Asamura et al., 2010). Advantages of hydrogel include a prolonged drug release and action time, increased stability of loaded drugs, and enhanced control efficiency of disease(Wasikiewicz et al., 2005, Builders et al., 2008, Ali et al., 2004). In addition, many studies showed that sustained-release hydrogel exhibits the ability to carry multiple drugs and provide 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 (Mai et al., 2020). Therefore, the development of dual functions of hydrogel that could deliver a range of drugs and provide 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) (Xiang et al., 2019). 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. According to the RNA-seq, the sustainable and controlled release of calcium ions activated the expression of CML19 that enhances the resistance of tobacco against TMV with a lasting time up to 30 days after CSL-gel treatment.