Phosphorylation and Nuclear Localization of NPR1 in Systemic Acquired Resistance

When plants sense a pathogen attack, they activate defenses in the immediate area and in distal tissues; this systemic acquired resistance (SAR) requires salicylic acid (SA), which activates NONEXPRESSER OF PATHOGENESIS-RELATED GENES1 (NPR1) in Arabidopsis thaliana (reviewed in Fu and Dong, 2013). This activation involves the release of NPR1 monomers from oligomers, which is also regulated by the cell’s redox state. Monomeric NPR1 is imported into the nucleus, interacts with bZIP transcription factors of the TGA family, and induces the expression of genes encoding antimicrobial pathogenesis-related proteins. Monomeric NPR1 also undergoes proteasome-mediated degradation, mediated by the NPR1 paralogs NPR3 (in the presence of SA) and NPR4 (in the absence of SA).

As if this were not complicated enough, measurements of SA levels during the establishment of SAR indicate the existence of other mechanisms regulating NPR1: During SAR, the biosynthesis of SA increases only a little in distal tissues, leading to the question of whether other factors activate NPR1 in those tissues. Shedding light on this, Lee et al. (2015) identify a role for SNF1-RELATED PROTEIN KINASE 2.8 (SnRK2.8), a serine/threonine kinase, in plant immunity. Following infection with Pseudomonas syringae pv tomato DC3000/avrRpt2, transcript levels of SnRK2.8 rapidly increased in distal tissues, before the induction of PR1, but other SnRK2s were suppressed (see figure). Plants overexpressing SnRK2.8 showed SA-dependent induction of PR1 expression and increased disease resistance, but SnRK2.8 activation was independent of SA. Notably, snrk2.8-1 mutants showed no effects on SA accumulation, but PR1 expression and SAR were suppressed in the snrk2.8-1 mutant.

Model for NPR1 and SnRK2.8 in systemic acquired resistance. Pathogen infection induces local cell death, the hypersensitive response (HR), and production of SA. Systemic signals activate NPR1 in distal tissues. Here, Lee et al. show (bold arrows) that distal activation of NPR1 involves SnRK2.8, which acts with SA to induce SAR. (Reprinted from Lee et al. [2015], Figure 8.)

Given the involvement of SnRK2.8 in immunity, the authors next examined the interaction between SnRK2.8 and NPR1. Indeed, coimmunoprecipitation and yeast two-hybrid assays showed that SnRK2.8 and NPR1 directly and strongly interact in yeast and in planta. Also, bimolecular fluorescence complementation assays showed that this interaction occurs in the nucleus and the cytoplasm. In vitro phosphorylation assays showed that SnRK2.8 phosphorylates NPR1. Moreover, two-dimensional gel electrophoresis of plants expressing a MYC-tagged version of NPR1 showed that induction of SAR also induces phosphorylation of NPR1 by SnRK2.8. Mass spectrometry and examination of mutant versions of NPR1 showed that SnRK2.8 phosphorylates NPR1 on Ser-589 and possibly on Thr-373; mutations of two other known sites of phosphorylation, Ser-11 and Ser-15, did not affect NPR1 phosphorylation by SnRK2.8.

What effect does phosphorylation by SnRK2.8 have on NPR1? The authors found that the wild type and the snrk2.8-1 mutant showed similar levels of NPR1 monomers in response to pathogen infection, indicating that this phosphorylation does not affect the balance of monomers and oligomers. Cell fractionation assays and examination of a fusion of NPR1 to GFP showed that in the wild-type background, NPR1 was in the nuclei of distal cells following pathogen infection. However, the snrk2.8-1 mutants showed a lower signal in the nuclei, indicating that the SnRK2.8-meditated phosphorylation of NPR1 affects its localization to the nucleus in distal cells. By contrast, in cells local to the infection, the wild type and snrk2.8-1 mutants showed similar patterns of NPR1-GFP localization. Moreover, NPR1 with substitutions at Ser-589 or Thr-373 failed to localize to the nucleus. The authors suggest that NPR1 localization occurs in two steps: SA-triggered monomerization and SnRK2.8-mediated phosphorylation. This mechanism allows the plant to activate SAR while avoiding the deleterious effects of high levels of SA in distal organs. This intriguing work sets the stage for future identification of the mechanisms activating SnRK2.8, possibly including nitric oxide, and examination of the role of other mobile immune signals in activating NPR1, the master regulator of SAR.