Expression of VvNLRs in Arabidopsis thaliana activates defense-responsive pathways to counter biotic stress

Powdery mildew, caused by Erysiphe necator, is a major fungal disease affecting grapevines, leading to significant yield and quality losses. Plants defend against pathogens through pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), with nucleotide-binding leucine-rich repeat receptors (NLRs) playing a crucial role in ETI. Hormonal signaling pathways, including salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), further regulate immune responses. This study functionally characterizes two NLR genes (XP_010660247.1 and XP_003633889.1) responsive to powdery mildew in Arabidopsis thaliana. Using transgenic plants overexpressing these genes individually and in combination, we analyzed their expression patterns in response to E. necator infection and various phytohormones. Real time analysis demonstrated varied tissue-specific expression profiles across all transgenic lines. XP_010660247.1 lines exhibited early and sustained immune response post E. necator infection, indicating robust activation of the SA pathway. XP_003633889.1 lines showed delayed but prolonged activation of SA pathway genes, suggesting nuanced temporal dynamics in fungal resistance. Stacked NLR lines showed further delay in activation of SA pathway genes possibly due to mutual cross-interference in gene activation. Enhanced JA/SA pathway activation indicates heightened defense against biotic stress response. Crosstalk between JA and abscisic acid (ABA) pathways was evidenced by MYC2 upregulation in response to methyl jasmonate and ABA treatments. The ET pathway demonstrated nuanced activation with sensitivity to ET elicitor 1-aminocyclopropane-1-carboxylic acid but the inability to further activate the ET pathway in transgenic lines. Our findings provide insights into plant immune mechanisms and offer potential strategies for engineering powdery mildew-resistant grapevine varieties.