NLR gene pair Pik1-H4 andPik2-H4 co-express in a BDG manner
NLR genes can be classified into singletons and pairs, depending on the number of genes involved in plant immunity. The Arabidopsis TNL gene pair RRS1 and SOC3 was reported to be organized in a BDG manner(Zhang et al. , 2017). It is well-known that the NLR gene pairs in the Pik locus are H2H genes. Despite this, there is limited knowledge about regulating NLR gene expression. In this study, we conducted a comprehensive analysis of the expression pattern ofPik-H4 and found that under the regulatory control of PPik-H4 , Pik-H4 exhibited significantly positive co-expression across various tissues. Furthermore, the infection caused by the rice blast fungus induced its expression in leaves and leaf sheaths. Based on these findings, we hypothesize thatPik-H4 , functioning as a pair of resistance genes, maintains a basal expression level throughout the plant during the resting phase. This basal expression level acts as a surveillance system to detect the presence of effector proteins promptly, thereby initiating the disease resistance pathway upon pathogen invasion. Interestingly, whilePikh-1 maintains constitutive transcription, Pikh-2 is up-regulated in response to the rice blast fungus challenge(Zhaiet al. , 2014). Identifying ten W-boxes in PPik-H4 (Figure 1A) highlights the significance of the W-box, a binding site for WRKY TFs, in pathogen-induced expression(In et al. , 2020). Notably, the observed divergence in expression between Pik-H4 and Pikh might stem from differences in their BDP sequences (Figure S1). Furthermore, our investigation revealed robust promoter activity of Pik-H4 within the vascular bundles of roots, stems, and leaf sheaths. One possible explanation for this pattern is that the hyphae of M. oryzae find a more accessible propagation route through vascular bundles, thereby necessitating heightened expression in these regions to provide a specific defense response. As a sensor protein, Pik1-H4 exhibited an expression level approximately 0.6 times higher than that of Pik2-H4, and a noticeable divergence in expression was observed between the two at 24 hpi (Figure S4C). This finding was consistent with the results of promoter activity analyses following injection (Figure 4). Moreover, the appearance of rice blast fungus disrupted the linear expression relationship of Pik-H4 (Figure 4J, K), suggesting that M. oryzae might stimulate Pik-H4expression in invaded and peripheral cells. This phenomenon prompts further exploration into the intricate mechanisms underlying signal transduction and NLR expression, which is vital for gaining deeper insights into the plant immune system’s response to disease challenges.
It is well known that NLRs play a crucial role in triggering ROS production as part of the plant’s defense response. For H2H NLR pairs, Pikp and Pikh are essential for initiating HR when confronted with the AvrPik effector(Zhai et al. , 2014; Zdrzałek et al. , 2020b). This mechanism contrasts with non-H2H NLR pairs likePigm (Deng et al. , 2017) andRGA4 /RGA5 (Cesari, Kanzaki, et al. , 2014), which function differently regarding HR induction. We broke the expression relationship between Pik1-H4 andPik2-H4 under the control of PPik-H4 by overexpressing one of them inPik-H4 NILs and expressing different ratios of Pik-H4 in tobacco, the rice blast resistance and conductivity in ion leakage assays were not affected (Figure 5A, G). This intriguing result suggests that the expression levels of Pik-H4 do not significantly influence the functional integrity of Pik1-H4 and Pik2-H4 proteins involved in immunity. It further indicates that the coordinated action of the Pik1-H4/Pik2-H4/AvrPik-E unit is sufficient to trigger HR, indicating the robustness of the plant’s immune response machinery.
Pik1-H4 and Pik2-H4 are organized in a BDG manner, functioning together to trigger rice blast resistance. This collaborative action is facilitated by the co-regulation of Pik-H4 through bidirectional promoters, which are influenced by the presence of rice blast fungus in planta.Notably, a feedback regulatory mechanism exists betweenPik1-H4 and Pik2-H4(Figure 5E, F), further highlighting the intricate nature of their coordinated function. The gene structure of this BDG is well-suited to facilitate the controlled up-regulation of Pik-H4 expression during immune responses, while also ensuring a consistent baseline expression in the absence of pathogen infection. This inherent readiness to counteract pathogens at any moment indicates the BDG structure’s adaptive advantage in enhancing the plant’s overall immune preparedness. Through the structure of BDG, NLR gene pairs establish a direct transcriptional regulatory link, which offers a more resource-efficient way to execute immune responses.