Co-expression and function of head-to-head NLR gene pair Pik-H4

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 Pik 1 -H4 ) within P Pik-H4 emerged as core regions for expressing Pik 1 -H4 and Pik 1 -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 Pik 1 -H4 and Pik 2 -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 Pik 1 -H4 and Pik 2 -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 Pik 1 -H4 or Pik 2 -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.