## 3 Results and Discussion
### Transcriptional Levels of Defense-Related Genes  
At 21 dpi of all treatments, the transcription levels of four tomato-innate defense genes (PAL, PR-1, CHS, and POD) were investigated. Beside it is the first enzyme in the phenylpropanol pathway linking the primary and secondary metabolism [61], PAL is involved in salicylic acid (SA) biosynthesis [62]. SA is an important signal that regulates systemic acquired resistance (SAR) in plants [42; 63] and its  
Figure 5: **(Left) Assay of protective and curative activities of ZnO NPs (100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) against tobacco mosaic virus (TMV) on _D. stramonium_ leaves. the half-left side of the leaves was inoculated with TMV without any treatments while the half-right side treated with ZnO NPs. (Right) A histogram showing the relative expression level of _TMV-CP_ gene in TMV-infected tomato plants at 21 days post-inoculation. Where, T1 = Mock-treated plants (Control), T2 = plants inoculated with TMV only, T3 = plants treated with ZnO NPs (24 h before TMV inoculation), T4 = plants treated with ZnO NPs (24 h after TMV inoculation), and T5 = plant treated with ZnO NPs (24 h before TMV inoculation and 24 h after TMV inoculation). Columns represent mean value from three biological replicates and bars indicate standard deviation (\(\pm\)SD). Significant differences between samples were determined by one-way ANOVA using CoStat software. Means were separated by least significant difference (LSD) test at \(p\leq 0.05\) levels and indicated by small letters. Columns with the same letter means do not differ significantly. **  
Figure 6: A photograph showing the disease symptoms on tomato leaves infected with TMV at 25 days post-inoculation. (A) mock-treated plants (T1), (B) plants inoculated with TMV only (T2), (C) plants treated with ZnO NPs (100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) 24 h before inoculation of TMV (T3), (D) plants treated with ZnO NPs (100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) 24 h after inoculation of TMV (T4), (E) plant treated with ZnO NPs (100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) 24 h before inoculation of TMV and 24 h after inoculation with TMV (T5). role in plant immunity has been investigated for decades. The activation of SA, in response to pathogens, results in the accumulation and overexpression of PR-1 (SA marker gene) [64]. Compared to mock-inoculated plants (control (T1)) in the current study, a significant up-regulation of PAL with relative expression levels of 2.448-, 1.753-, and 2.590-fold change higher than control were observed in T3, T4, and T5, respectively (Figure 7). Like PAL, PR-1 expression levels in all treatments were increased with relative transcriptional levels of 1.531-, 1.375-, and 1.802-fold change in T3, T4, and T5, respectively, compared to control (Figure 7). On the contrary, PAL and PR-1 of T2 plants showed a significant down-regulation with relative expression levels of 0.064- and 0.272-fold change lower than the control treatment, respectively (Figure 7). Consequently, foliar spraying of tomato plants with the biologically synthesized ZnO NPs, either as a single treatment (before or after inoculation with TMV) or as a double treatment (before + after inoculation with TMV) triggered the expression of PAL and PR-1 genes that may cause elevated SA accumulation levels. These results suggested that the biologically synthesized ZnO NPs via _M. spicata_ may mediate the activation of the induced systemic resistance (ISR) and can play a major role in tomato SAR against TMV infection. Many researchers reported that increased resistance against virulent pathogens was associated with an increased in PAL and PR-1 expression and elevated endogenous SA accumulation levels inside plant tissues [65; 66; 67; 68; 69]. The obtained results are in agreement with [PERSON] et al. [32] who reported that upon TMV infection, chemically prepared ZnO NPs increased the expression level of the tobacco PR-1 gene that resulted in the initiation of the SA signaling pathway.