## 3 Results
### Identification of Cucurbita pepo Differentially Expressed Proteins
In the RSPX vs. Control comparison (Figure 2C; Table 1; Supplementary Table S1) the pattern regarding the trend of up and down-regulated proteins was like that of PX vs. Control, with 17 and three proteins having above 4-fold changes in expression (increase and decrease, respectively). Interestingly, the highest expression decrease (9-fold) observed was for the glyoxalase-like domain-containing protein (Cp4.1 LG18g03400.1) also reported in the RS vs. Control comparison, followed by a GDSL-lipase protein (Cp4.1 LG19g06470.1) with a 6-fold decrease. Interestingly, nine out of the 17 highly expressed DEPs are common in the PX vs. Control comparison as well. Among the remaining eight DEPs (unique in this comparison), a 14-3-3 protein (Cp4.1 LG11g03840.1), a pyruvate kinase family protein (Cp4.1 LG19g03720.1), an ascorbate peroxidase (Cp4.1 LG20g08860.1), and a protein of unknown function (Cp4.1 LG05g00400.1) were reported. Regarding the comparisons between treated sample groups (PX, RS, and RSPX) the total numbers of DEPs with a 2-fold difference in expression were much less than those in the treated vs. the Control comparisons, as shown in Table 1. For the RS vs. PX comparison (Figure 2D, Table 1; Supplementary Table S1) most DEPs with a difference of 4-fold and above were grouped as down-regulated, including two carbonic anhydrases (Cp4.1 LG20g07260.1; Cp4.1 LG02g12540.1) and a thiamine thiazole synthase (Cp4.1 LG01g08250.1). A tollB protein-related protein (Cp4.1 LG12g06350.1) was the only highly up-regulated DEP with a 7-fold increase in expression in this comparison and was found also to be the highest up-regulated protein in the RSPX vs. PX comparison, with a 5-fold up-regulation (Figure 2E, Table 1; Supplementary Table S1). A WD-40 repeat protein MSI4 (Cp4.1 LG18g03560.1) and the previously-reported GDSL-lipase (Cp4.1 LG19g06470.1) protein were significantly down-regulated (nearly 5- and 6-fold, respectively) in RSPX vs. PX and RSPX vs. RS, respectively (Figure 2E,F, Table 1; Supplementary Table S1). The overlap and differences shared between the three main comparisons for DEPs with \(p\)-values < 0.05 at three different levels of expression magnitude (>1-, >2-, and >4-fold, respectively) are shown in Figure 3. A small number of highly expressed proteins (13, seven, and eight DEPs) are unique in each treatment (PX, RS, and RSPX respectively) and even less (below six DEPs) are shared between groups (Figure 3C). The numbers of shared and unique DEPs between all groups' comparisons are shown in Supplementary Figure S3. Figure 2: Identification of differentially expressed proteins (DEPs). Volcano plots show the DEPs in each of the six (**A**–**F**) comparisons for the four treatments. Treatment names are as described in Table 1. The horizontal axis represents the fold change in protein expression between the two sample groups (Log2 fold change), and the vertical axis represents the \(p\)-value-based significance of the DEPs between the two sample groups (\(-\)Log10 \(P\)). Threshold lines for DEPs screening criteria are indicated by dashed lines (placed as a visual aid), with the horizontal line representing the statistical significance threshold (\(p\)-value \(<\) 0.05) set for the current analysis and the vertical ones placed at fold-change equals two. Non-significant (NS) DEPs (\(p\)-value \(>\) 0.05) are shown in gray (\(<\)2-fold change in expression) and blue (\(>\)2-fold change in expression). Significant (\(p\)-value \(<\) 0.05) up-regulated and down-regulated DEPs are shown as red (\(<\)2-fold change) and green (\(>\)2-fold change) dots, respectively. Proteins with a 4-fold and above difference in expression are depicted with their corresponding IDs.