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 up-regulated 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 Pik 1-H4 relocates to vesicles, indicating its role in effector recognition, while Pik 2-H4 predominantly accumulated in the nucleus. These new discoveries of Pik protein extended putative immune function of NLR pairs. Transcriptome analysis demonstrated that Pik-H4-mediated resistance induces significant transcriptome reprogramming between 12- and 24-hours post-inoculation. In summary, these findings provide novel insights into the regulatory complexity and functional divergence within NLR bidirectional gene pairs in response to pathogen invasion.

Nucleotide-binding, leucine-rich repeat (NLR) genes play a pivotal role in shaping plant effector-triggered immunity in response to pathogen invasions. However, the mechanisms governing the expression and behavior of NLRs, particularly in the context of head-to-head NLR gene pairs, in the presence of pathogens, remain uncovered. In this study, we dissected the Pik-H4 promoter (P Pik-H4) at the TATA boxes and conducted an in-depth investigation into split promoter activity using Agro-infiltration assays. The segments spanning 593-1232 bp and 2016-2492 bp (starting from -1 bp of Pik1-H4) within P Pik-H4 emerged as core regions for expressing Pik1-H4 and Pik1-H4 respectively. Nevertheless, merging these two core fragments failed to recover the promoter activity in both directions. Employing Gus staining, promoter activity assays and qRT-PCR, we unveiled the co-expression of Pik1-H4 and Pik2-H4 throughout the whole plant. Additionally, in the presence of the rice blast fungus, their co-amplification was observed in leaves and leaf sheaths. Strikingly, Pik-H4 exhibited heightened expression within vascular bundles. Moreover, perturbing the Pik1-H4 and Pik2-H4 co-expression relationship via overexpression in rice or Nicotiana did not disrupt the immune response. Upon infection, the singleton Pik 1-H4 localized within vesicles, while Pik 2-H4 predominantly occupied the nucleus within leaf sheath cells. Transcriptome analysis highlighted Pik-H4-mediated resistance triggering a transcriptome reprogramming between 12 and 24 hours post-inoculation. Notably, overexpression of Pik1-H4 or Pik2-H4 enriches various pathways compared to the Pik-H4 Lijiangheituanxingu near-isogenic lines. In summary, these findings unravel the intricate dynamics of co-expression and singular functionality within NLR bidirectional gene pairs upon pathogen invasion.