Exploring the impact of NPR3 gene silencing on grapevine defense responses and arbuscular mycorrhizal symbiosis establishment

Systemic Acquired Resistance (SAR) is a crucial component of plant immunity, enabling a broad-spectrum defense against a variety of pathogens. SAR is mediated by the signalling molecule salicylic acid (SA), which activates a cascade of defense responses. Central to the regulation of SAR is NPR3, which functions as a transcriptional co-repressor of NPR1, a key positive regulator of SAR onset. By modulating NPR3 activity, it is possible to heighten the plant immune responses and enhance resistance to several pathogens. This study aims to improve the plant's resistance to biotic stresses and explore how NPR3 knock-out (through genome editing) can affect the plant interaction with soil microorganisms, particularly considering the accommodation of arbuscular mycorrhizal fungi (AMF). To achieve this, embryogenic calli from the elite grapevine cultivar Chardonnay were transformed to generate NPR3 knock-out lines. Once obtained, edited plants were assessed against spontaneous infection of Erysiphe necator, the causal agent of powdery mildew (PM). Our results demonstrated that NPR3 knock-out lines exhibited enhanced resilience to PM compared to wild-type plants. Biometric, molecular, biochemical and physiological analyses revealed that the improved resilience is associated with higher leaf thickness and overaccumulation of stilbenes with respect to WT plants. Metabarcoding of the leaf-associated fungal community highlighted a reduction of other important grape pathogens, suggesting a potential broader impact than PM tolerance alone. Beyond limiting the plant susceptibility to pathogens, another critical aspect of our study was to investigate how editing of NPR3 influences the symbiotic relationships with AMF. AM symbiosis plays a crucial role in plant health by improving nutrient and water uptake, enhancing stress tolerance, and the overall plant fitness. To assess AMF colonization, gnotobiotic-like NPR3-edited plants were inoculated with Rhizophagus irregularis under axenic conditions. A significant increase in root colonization was observed among the edited lines, indicating that NPR3 silencing improves the plant's ability to recruit and interact with AM fungi. These findings suggest that manipulating NPR3 not only enhances plant defence responses, but also positively impacts the plant's symbiotic relationships. In conclusion, our research demonstrates the potential to enhance grapevine resilience by targeting the NPR3 gene trough genome editing. Beyond the basic research information on NPR3 functional characterization, this work paves the way for developing innovative strategies to address biotic and abiotic stresses in grapevine, ensuring healthier and more sustainable vineyard management practices. Further molecular and biochemical analyses are ongoing to dissect the recruiting mechanisms altered in NPR3 mutants.