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 distaltissues;thissystemicacquiredresistance (SAR) requires salicylic acid (SA), which activatesNONEXPRESSEROFPATHOGENESISRELATED 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-mediateddegradation,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 etal. (2015) identifya role forSNF1-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 inductionofPR1,butotherSnRK2s were suppressed (see figure). Plants overexpressingSnRK2.8 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. 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 interactionoccursinthenucleusandthecytoplasm.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 ofmutant 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 notaffectNPR1phosphorylationbySnRK2.8. 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 responsetopathogen infection, indicating that this phosphorylation does not affect the balance of monomers and oligomers. Cell fractionationassaysandexaminationofa fusionof NPR1 to GFP showed that in the wild-type background, NPR1 was in the nuclei of distal cells following pathogen infection. However, the snrk2.8-1mutants showed a lower signal in the nuclei, indicating that the SnRK2.8meditated phosphorylation of NPR1 affects its localization to the nucleus in distal cells. By contrast, in cells local to the infection, thewild 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-triggeredmonomerizationandSnRK2.8-mediatedphosphorylation. This mechanism allows the plant to activate SAR while avoiding the deleterious effects of high levels of SA in distal organs. This intriguingworksets thestage for future identification of the mechanisms activating SnRK2.8, possibly includingnitricoxide,andexaminationof the role of other mobile immune signals in activating NPR1, themaster regulator of SAR.