Seeing is believing: Understanding functions of NPR1 and its paralogs in plant immunity through cellular and structural analyses

Abstract

In the past 30 years, our knowledge of how nonexpressor of pathogenesis-related genes 1 (NPR1) serves as a master regulator of salicylic acid (SA)-mediated immune responses in plants has been informed largely by molecular genetic studies. Despite extensive efforts, the biochemical functions of this protein in promoting plant survival against a wide range of pathogens and abiotic stresses are not completely understood. Recent breakthroughs in cellular and structural analyses of NPR1 and its paralogs have provided a molecular framework for reinterpreting decades of genetic observations and have revealed new functions of these proteins. Besides NPR1's well-known nuclear activity in inducing stress-responsive genes, it has also been shown to control stress protein homeostasis in the cytoplasm. Structurally, NPR4's direct binding to SA has been visualized at the molecular level. Analysis of the cryo-EM and crystal structures of NPR1 reveals a bird-shaped homodimer containing a unique zinc finger. Furthermore, the TGA3₂-NPR1₂-TGA3₂ complex has been imaged, uncovering a dimeric NPR1 bridging two TGA3 transcription factor dimers as part of an enhanceosome complex to induce defense gene expression. These new findings will shape future research directions for deciphering NPR functions in plant immunity.

Keywords: Cryo-EM; Crystal structure; NPR1; NPR2; NPR3; NPR4; Plant immunity; SA-binding domain (SBD); SA-induced NPR1 condensate (SINC); Salicylic acid (SA); Structural analysis; Systemic acquired resistance.

Figure 1:

Origin of NPR gene family across land plants. A phylogenetic tree was constructed and whole-genome duplication events were annotated according to the 1KP study [46]. Full-length protein sequences from 62 representative algae and land plants (excluding Gymnosperms) with high-quality genome sequence data were downloaded from Phytozome v12, all-against-all BLASTP [47] was performed, and ortholog groups (OGs) were identified using OrthoMCL [48] (Supplemental Table 1). From the analysis, AtNPR1, AtNPR2, and their 69 orthologs from 46 species were classified into one orthologous group (named here as the NPR1/2 clade); AtNPR3, AtNPR4, and their 121 orthologs from 47 species were classified into another orthologous group (named here as the NPR3/4 clade); AtNPR5, AtNPR6, and their 120 orthologs from 53 species were classified into the third orthologous group (named here as the NPR5/6 clade) (Supplemental Table 2). The number of rectangles represent the number of gene copies. Gene copies with the same color belong to the same orthologous group. The NPR genes not classified as orthologs are not shown.

Figure 2.

The enhanceosome model of the NPR1 dimer bridging two dimeric TGA transcription factors which recognize the as-1 (activation sequence 1) cis-element in the promoters of defense genes. SA-induced SBD–ANK docking creates a new interface to facilitate post-translational modifications and/or recruitment of transcriptional regulators (X) for the activation of defense genes. The position of key residues in NPR1 (left) and the corresponding activation steps (right) are shown.

Figure 3.

Subcellular localization npr1^sim3-GFP during ETI. The npr1^sim3-GFP/npr1–2 transgenic plant was infected at the tip with Psm ES4326/AvrRpt2. At 24 hpi, tissue was sampled from the cell death-survival boundary (diagram). GFP signal was collected across the boundary between dead and surviving regions (dashed line). Enlargements from regions adjacent to and distant from the cell death zone (dashed rectangles) are shown in bottom panels. Scale bar = 100 μm (top panel); 20 μm (bottom panels). Modified from [30].

Figure 4.. Proposed model for the hormone signaling network in plants.

Based on the recent studies of NPR functions highlighted in this review, together with reports on other plant hormones, it is conceivable that a myriad of signaling diversity can be generated through combinatorial effects of interactions at the levels of hormone (H) production [21]; hormone receptor (R) cross reactions [21,45]; and cross-binding of hormone receptors with TFs (TF) [35,36].