Proteomic Analyzes of Plants with BiP Overexpression Reveal Mechanisms Related to Defense Against Moniliophthora perniciosa

Moniliophthora perniciosa is one of the main pathogens affecting the cocoa crop. The control of this phytopathogen is generally carried out by means of resistant genotypes accompanied by phytosanitary pruning. The identification of plant genes related to the plant defense mechanism is important to unravel the molecular basis of plant-pathogen interaction. One potential gene studied is the binding protein (BiP), a molecular chaperone located in the lumen of the endoplasmic reticulum that binds to newly synthesized proteins as they are translocated into the ER and maintains it in a competent state for subsequent folding and oligomerisation. BiP is an abundant protein under all growth conditions, but its synthesis is markedly induced under conditions that lead to accumulation of unbound polypeptides in the ER, such as when plants are under pathogen attack. In this study, we overexpressed the SoyBiPD gene in Solanum lycorperscum plants and inoculated them with M. perniciosa. The control (untransformed) plants showed severe symptoms of witches' broom disease, while the symptoms of the transgenic lineages varied from severe to mild according to the level of BiP in transgenic lineages. We further applied a gel-free proteomics technique to investigate the molecular mechanisms induced by BiP overexpression that confers M. perniciosa resistance. We inoculated BiP-plant (tomato superexpressing BiP) and control plants (Non-transgenic) with a mix of M. perniciosa spores. Proteins were extracted and analyzed by mass spectrometry to identify up-regulated and differentially abundant proteins between treatments associated with gene overexpression. The uninoculated NT plants showed 196 proteins, 15 of which were up-regulated, while the uninoculated BiP plants showed 214 proteins, 33 of which were up-regulated. After inoculation, the total number of proteins detected decreased to 178 in the NT group, with 37 up-regulated, and to 171 in the BiP plants, with 30 up-regulated. The identification of these proteins showed that plants overexpressing SoyBiPD had a higher abundance of resistance-related proteins such as PR2, PR3 and PR10, proteins that are part of the plant's immune system and contribute to resistance against pathogens. Transgenic lines showed higher activity of glucanase, superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX). In addition, plants overexpressing BiP, both inoculated and non-inoculated, accumulated less hydrogen peroxide (H₂O₂) compared to NT plants, suggesting a more efficient control in the production or detoxification of ROS. Interaction network analysis revealed the activation of clusters associated with defense and the interconnection between different metabolic and signaling pathways, reinforcing the defensive readiness and metabolic capacity of plants superexpressing BiP. Taken together, our results indicate that overexpression of BiP in plants increases the abundance of defense proteins, improves antioxidant capacity, modulates the accumulation of ROS and increases the activity of fungal cell wall degradation enzymes, even in the absent of M. perniciosa attack, contributing to a state of defensive readiness and greater tolerance to biotic stress. This study deepens our understanding of the molecular mechanisms of BiP-mediated induced resistance to biotic stress. Further, it highlights the biotechnological potential of the BiP gene for developing crops that are more resistant to witches' broom and other economically relevant diseases. Its application could be a promising approach to reducing losses caused by pathogens in species of economic interest, such as cocoa.