# Salicylic Acid Modulates Volatile Organic Compound Profiles During CEVd Infection in Tomato Plants
## 3 Results  
Impact of Salicylic Acid Accumulation on Symptom Development and Metabolic Responses in CEVd-Infected Tomato Plants  
To better understand the changes in plants during vivoid pathogenesis, various approaches have focused on the differential expression of genes related to defensive compounds [2]. Recent studies have highlighted the critical role of salicylic acid (SA) in plant defence and its impact on vivoid pathogenesis, where SA concentration determines plant resistance. Particularly, _NahG_ tomato plants, unable to accumulate SA, display increased susceptibility to CEVd [15], whereas _RNAi_55H_ transgenic lines with higher SA levels show enhanced resistance to this pathogen [9]. To study the metabolic changes associated with CEVd tolerance and susceptibility, plants corresponding to the three genotypes (WT, _NahG_, and _RNAi_55H_) were infected with the vivoid. As previously reported, _NahG_ plants exhibited enhanced susceptibility to CEVd, whereas transgenic _RNAi_55H_ lines showed increased resistance, confirming the essential role of SA in vivoid pathogenesis. The heightened susceptibility of _NahG_ plants was evident both in the development of symptoms such as epitaxy, roughness, and chlorosis (Figure 1A), as well as in compromised growth, reflected by reduced plant height (Figure 1B). In contrast, _RNAi_55H_ plants showed no vivoid symptoms or significant height reduction. Among the secondary metabolites involved in plant immunity, VOCs have attracted significant interest due to their role in activating defence responses in neighbouring plants. Metabolomics studies have been performed to identify VOCs differentially accumulated in response to different biotic stresses [26; 33]. However, the volatilome of tomato plants in response to CEVd infection remains unexplored, and the potential role of these volatiles in vivoid pathogenesis is yet to be elucidated. To investigate the volatiles involved in the tomato-CEVd interaction, an untargeted metabolomics analysis using GC-MS was conducted on leaves of _NahG_, _RNAi_SSH_, and the corresponding genetic background 'MoneyMaker' (WT) plants, either infected or not with CEVd. The Principal Component Analysis (PCA) of the VOC profiles from both CEVd-infected and mock-treated _NahG_, _RNAi_SSH_, and wild-type (WT) tomato plants revealed distinct clustering patterns (Figure 2). The first two principal components accounted for most of the variance, contributing 44.4% (PC1) and 20.5% (PC2), respectively. Figure 1: Disease development of representative MoneyMaker (WT), _NahG_, and _RNAi_SSH_ tomato plants 21 days after CEVd infection. (**A**) Phenotype of CEVd-infected tomato plants. (**B**) Height of mock-treated and CEVd-infected tomato plants. Error bars represent the standard error of the mean. The letters show the grouping information using [PERSON]’s range test (one-way ANOVA method) with a significance level of 5% (_p_-value < 0.05). Figure 2: Principal Component Analysis (PCA) score plot based on the whole array of the mass spectra within an \(m/z\) range from 35 to 250 based on Pareto scaling. Samples included _NahG_, RNAi_SSH_, and the genetic background ‘MoneyMaker’ (WT) plants, either infected with CEVd, including CEVd_NahG_ (full red circles), _CEVd_SSH_ (full orange triangles), or CEVd_WT (full dark-green diamonds), or mock non-inoculated with Mock_NahG_ (open pink circles), Mock_SSH (open blue triangles), or Mock_WT (open green diamonds). The CEVd-infected _NahG_ plants (full red circles in Figure 2) formed a distinct cluster on the negative side of the PC1 axis, indicating that this SA-deficient genotype exhibited a specific volatilome profile during CEVd infection. In contrast, CEVd-infected _RNAi_SSH_ plants (full orange triangles), which accumulate higher levels of SA, clustered separately along the negative side of the PC2 axis.