Co‐Expression Pattern Analysis of Head‐to‐Head NLR Gene Pair <i>Pik‐H4</i>

Nucleotide-binding leucine-rich repeat (NLR) genes play a critical role in plant effector-triggered immunity (ETI) against pathogen invasion. However, the regulatory mechanisms governing NLR expression and functional dynamics, particularly in head-to-head NLR gene pairs, remain poorly understood. In this study, we investigated the regulatory mechanisms, subcellular localization and functional pathways associated with Pik-H4 gene pair. Bidirectional Pik-H4 promoter (P_Pik-H4) strengths were found across the whole plants and exhibited co-expressed patterns in tissues and cells, and the P_Pik-H4 activity was upregulated in vascular bundles during blast fungus invasion. Additionally, altering the co-expression of Pik₁-H4 and Pik₂-H4 via overexpression in rice or Nicotiana benthamiana did not compromise the immune response. Promoter analysis identified two minimal promoter regions that are essential for bidirectional transcription, and mutagenesis of the bidirectional TATA box confirmed its role in gene regulation. This dual-function promoter coordinates Pik-H4 expression in both directions, a regulatory innovation previously unreported in NLR-mediated immunity. In planta subcellular localization revealed Pik₁-H4 relocates to vesicles, indicating its role in effector recognition, while Pik₂-H4 predominantly accumulated in the nucleus. These new discoveries of Pik protein extended the putative immune function of NLR pairs. Transcriptome analysis demonstrated that Pik-H4-mediated resistance induces significant transcriptome reprogramming between 12- and 24-h postinoculation. In summary, these findings provide novel insights into the regulatory complexity and functional divergence within NLR bidirectional gene pairs in response to pathogen invasion.