## 3 Results
### Recognition of Glu and Gly by GLRs Triggered Electrical Signaling in the Immune Response  
As Glu or Gly can be recognized by GLRs to trigger membrane depolarization [33], we determined Glu- or Gly-induced electrical signaling by monitoring the effect of Glu and Gly on membrane potential in tomato leaves. Glu and Gly treatment elicited significant changes in membrane potential in a short time, with an increase in amplitude of about 17-20 mV (Figure 4). Notably, the change in membrane potential induced by Glu and Gly was significantly weakened in _glr3.3_ mutants. These findings suggest that electrical signaling in tomato leaves, induced by Glu or Gly, is partly dependent on GLR3.3.  
Figure 2: Subcellular localization of GLR3.3 and GLR3.5. Tomato GLRs-GFP were transiently co-expressed with FLS2-mCherry (a plasma membrane localization maker) in _N. benthamiana_ leaves. The GFP and mCherry signals were visualized using confocal microscopy 48 h following the infiltration. Bar = 25 μm.  
The _glr3.3_ mutants exhibited more sensitivity to _B. cinerea_, which was consistent with the reduction in the Glu- or Gly-induced electrical signals. On the basis of these observations, we hypothesized that the recognition of Glu and Gly by GLRs is critical  
Figure 4: Glu or Gly affects the membrane potential of WT and _glr3.3_ leaves: (**a**) typical membrane potential changes in tomato WT or _glr3.3_ mutant leaves treated with Glu or Gly; (**b**) quantification of membrane potential changes of amplitude. The results in panel (**b**) are presented as mean values \(\pm\) SD; \(n=4\). Statistically significant differences between treatments (\(p<0.05\), Tukey’s test) are shown by different letters. The experiments described above were carried out three times, with the same findings each time.  
Figure 3: Effects of _GLR3.3_ and _GLR3.5_ editing on plant defense against _B. cinerea_ inoculation in tomato: (**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.  
in immunity against _B. cinerea_. To verify this hypothesis, a pharmacological assay was carried out by Glu- or Gly-exogenous treatment. Before _B. cinerea_ inoculation, the WT plants and the _glr3.3_ mutant were pretreated with Glu, Gly, or H2O (control). In the WT plants, the _B. cinerea_ inoculation-induced reduction in \(\Phi\)PSII and the increase in _B. cinerea actin_ mRNA accumulation were significantly alleviated by pretreatment with exogenous Glu or Gly at 3 dpi (Figure 5). However, disease symptoms in the _glr3.3_ mutant were more severe, and the beneficial effects of Glu and Gly on the resistance against _B. cinerea_ significantly decreased due to the knockout of _GLR3.3_. Thus, these results indicate that Glu- and Gly-induced plant immunity against _B. cinerea_ partly depends on GLR3.3.