Dynamic ubiquitination determines transcriptional activity of the plant immune coactivator NPR1

Abstract

Activation of systemic acquired resistance in plants is associated with transcriptome reprogramming induced by the unstable coactivator NPR1. Immune-induced ubiquitination and proteasomal degradation of NPR1 are thought to facilitate continuous delivery of active NPR1 to target promoters, thereby maximising gene expression. Because of this potentially costly sacrificial process, we investigated if ubiquitination of NPR1 plays transcriptional roles prior to its proteasomal turnover. Here we show ubiquitination of NPR1 is a progressive event in which initial modification by a Cullin-RING E3 ligase promotes its chromatin association and expression of target genes. Only when polyubiquitination of NPR1 is enhanced by the E4 ligase, UBE4, it is targeted for proteasomal degradation. Conversely, ubiquitin ligase activities are opposed by UBP6/7, two proteasome-associated deubiquitinases that enhance NPR1 longevity. Thus, immune-induced transcriptome reprogramming requires sequential actions of E3 and E4 ligases balanced by opposing deubiquitinases that fine-tune activity of NPR1 without strict requirement for its sacrificial turnover.

Functional switching of NPR1 between chloroplast and nucleus for adaptive response to salt stress

“…chloroplast and nuclear isolation, protein extraction, and western blotting. To extract total protein from tobacco leaves, frozen samples were ground into powder and suspended in protein extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 0.2% Nonidet P-40 (NP-40) containing 50 μg/ml of tosyl-L-phenylalaninyl-chloromethylketone, 50 μg/ml of tosyl-L-lysine-chloromethylketone, serine protease inhibitors, 0.6 mM phenylmethylsulfonyl fluoride (PMSF), 80 μM MG115, 80 μM MG132, and complete protease inhibitor cocktail tablet (Roche, USA) 20 .…”

“…Nuclear NPR1 interacts with transcription factor (TF) members of the TGA class of basic domain/leucine zipper 19 . However, there are exceptional cases in which nuclear localization of NPR1 is not a requirement for SA-dependent gene regulation 20 , indicating SA-activated cytosolic NPR1 has a novel function beyond transcriptional coactivator in nucleus 18 .…”

Proteasome-associated ubiquitin ligase relays target plant hormone-specific transcriptional activators

“…Given that UPLs are associated with the proteasome, we reasoned that they might function sequentially after CRL3 and UBE4 ligases to modify NPR1 and promote its degradation. We previously reported that in contrast to upl mutants, mutant ube4 plants accumulate highly active NPR1 that is modified by short ubiquitin chains (14). Unfortunately, we were unable to obtain homozygous upl3 ube4 double mutants, suggesting this combination was lethal.…”

“…By contrast, the plant immune hormone salicylic acid (SA) stimulates the step-wise ubiquitination of NPR1, a master TA of hundreds of immune genes and promoter of cell survival (11)(12)(13). Initial SA-induced ubiquitination of NPR1 by a Cullin-RING Ligase 3 (CRL3) activates NPR1, while subsequent ubiquitin chain elongation by Ubiquitin conjugation factor E4 (UBE4) ligase deactivates NPR1 and targets it for degradation (14). In addition, SCF HOS15 ligase targets NPR1 for degradation to limit and prevent untimely activation of immune genes (15).…”

Plant proteostasis – shaping the proteome: a research community aiming to understand molecular mechanisms that control protein abundance

“…A main focus has been the interplay between these modifications and immunity. Ubiquitin and ubiquitin-like pathways regulate every layer of immunity from New Phytologist perception to plant reprogramming (Orosa et al, 2018;Turek et al, 2018;Skelly et al, 2019). Key regulators in immunity appear to be tightly regulated by one or more PTM(s).…”