Abstract
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.