## 4 Discussion  
GLRs are cation-channel proteins that exist in bacteria, plants, and animals. In animals, GLRs are proven to be involved in neurotransmission and immune regulation, and their ligand Glu acts as a signal agent between the two systems [34; 35; 36]. In plants, GLRs play a positive role in immunity against biotic stress [10; 12; 13]. Notably, GLR-induced defense against RKNs and insect bites was shown to be associated with electrical signals [3]. However, the relationship between GLR-mediated fungal-disease resistance and electrical signals has not been fully substantiated. Here, we provide convincing evidence demonstrating that tomato GLR3.3- and GLR3.5-modulated plant immunity against the typical fungal pathogen _B. cinerea_ is related to electrical signals. In this study, the role of GLRs in tomato seedling immunity against _B. cinerea_ was unveiled for the first time. The application of the GLR inhibitor DNQX enhanced the susceptibility of tomato plants to _B. cinerea_ (Figure 1). Moreover, knocking out _GLR3.3_ or _GLR3.5_ significantly attenuated plant immunity, leading to increased disease symptoms in leaves (Figure 3). These findings support the notion that tomato plants require GLRs to mount an adequate defense against the necrotichip fungal pathogen _B. cinerea_. Although overexpression of radish _GluR_ inhibits the growth of _B. cinerea_[37], none of the Arabidopsis _glr_ mutants were found to be more sensitive to _B. cinerea_[12], suggesting that the function  
Figure 5: Glu- or Gly-induced immunity against _B. cinerea_ partly depends on GLR3.3. Four-week-old tomato plants were treated with 20 mM Glu, 20 mM Gly, or water once per day for two days before _B. cinerea_ inoculation: (**a**) pseudocolor images of tomato leaves showing \(\Phi\)PSII at 3 dpi; (**b**) quantification of \(\Phi\)PSII at 3 dpi; (**c**) transcript abundance of _B. cinerea actin_ in tomato leaves inoculated with _B. cinerea_ at 1 dpi. The results in panels (**b,c**) are presented as mean values \(\pm\) SD; \(n=4\). Statistically significant differences between treatments (\(p<0.05\), [PERSON]’s test) are shown by different letters. The experiments described above were carried out three times, with the same findings each time. of GLRs may differ among species. In the present study, the _glr3.3/glr3.5_ double-mutants showed a more sensitive phenotype compared with the single-mutant _glr3.3_ and _glr3.5_. These findings suggest that GLR3.3 and GLR3.5 function redundantly. Notably, a comparison of the phenotypes between _glr3.3_ and _glr3.5_ mutants suggests that GLR3.3 plays the main role in defense against _B. cinerea_. Electrical signals are a rapid response involved in GLR-mediated plant resistance to biotic stress. Here, the electrical signals induced by Glu or Gly were significantly weakened in _glr3.3_ mutants. Glu or Gly treatment induced the membrane potential for a short time. However, the variation in amplitude is substantially decreased in _glr3.3_ mutants (Figure 4). Similarly, in Arabidopsis, the generation and propagation of electrical signals induced by leaf damage are weakened in _glr3.3_ and _glr3.6_ mutants [3]. However, the weakening of wound-induced surface potential changes is manifested in the shortening of the duration, but the amplitude remained the same as WT in Arabidopsis _glr3.3_ mutants after wounding the leaves. These differences in results may be due to differences in species and treatments. Consistent with our results, the knockout of _GLR3.3_ inhibits Glu treatment-induced membrane depolarization in Arabidopsis roots [38]. All of these results suggest that the electrical signals induced by Glu or Gly partly depend on GLR3.3. Exogenous treatment with Glu induces immune responses to multiple diseases, such as the rice blast fungus _Magnaporthe orgza_, the heterotrophic fungus _Alternaria alternata_ in tomato fruits, and the soil-borne bacterial pathogen _Ralstonia solanacearum_[39; 40; 41]. [PERSON] et al. (2019) reported that Glu treatment enhances the resistance of tomato fruits against _B.