Discussion
Plant
viruses cause a common systemic disease named plant cancers that differ
from other plant diseases (Bawden et al., 1936). 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 (Roossinck et al., 2015, Roossinck and Condit,
2013). Traditional methods depend on virus deactivators and plant
immunity inducers to minimize the infection (Ryu et al., 2017). 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 (Chen et al., 2009). 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 19 (CML19 ),
and the silencing of CML19 enhances the susceptibility of tobacco
to TMV. We summarize the action of CSL-gel in Figure 8 .
Therefore, the low-cost and easily synthesized CSL-gel with a novel mode
of action triggering CML19 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 (Zhao et al., 2007). 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 (Yin et al., 2010). 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. 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 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 (Ryu et al., 2017). 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 (Chen et al., 2009). 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 (Zhao et al., 2007). 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 (Orzali et al., 2010, Hadwiger et al.,
1981).
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. 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
(Chowdhury and Choudhuri, 2010). 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 (Jones and Lunt, 1967, Wigdorowicz-Makowerowa,
1982). The activities of antioxidant enzymes, such as SOD, POD, and CAT,
in soybeans and apples treated with calcium ions were significantly
improved (Munir et al., 2016). Furthermore, the application of calcium
fertilizer effectively prevents the occurrence of brown spot disease
(Xiaomeng et al., 2017). 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 6 ). 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
(Batistic and Kudla, 2012, Defalco et al., 2010). 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 (Min et al.,
2009). For example, overexpression of Arabidopsis CML8 confers
enhanced resistance to Pseudomonas syringae in an SA-dependent
process (Xiaoyang et al.). Arabidopsis CML9 is rapidly and
strongly induced by Pseudomonas syringae and abiotic stress and
abscisic acid (ABA), which acts as a positive regulator in response toPseudomonas syringae and a negative regulator to salt stress
(Leba et al., 2012). Tomato plants overexpressing CML44 exhibit
higher antioxidant enzyme activity and greater tolerance to abiotic
stresses (Munir et al., 2016). Our results showed that CSL-gel strongly
induces the expression of CML19 and the silencing of CML19enhances the infection of TMV (Figure 4 ). In addition,
overexpression of CML19 inhibits TMV infection, suggesting thatCML19 plays a positive role in the resistance to TMV. We further
found that CML19 localizes in the plant cytosol and nucleus
(Figure S5 ). The CSL-gel maintains a stable and cumulative
release of calcium ions into the soil, which activates plantCML19 to adapt to the increased calcium ions in the environment.
In turn, the accelerated expression of CML19 enhances the
resistance against TMV. However, the mechanisms underlying this
observation remain unknown. Because CML as a sensor of
Ca2+ regulates diverse plant processes, the
identification of CML19 interactors will aid us in understanding the
anti-virus activity of CML19 in the subsequent studies.
Taken together, 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 CML19 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 CML in plants against TMV infection and expand
our tools to control plant virus diseases as well in the future.