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.