Dynamic ubiquitination determines transcriptional activity of the plant immune coactivator NPR1
Immune responses must be tightly controlled to provide appropriate , efficient and timely resistance to pathogenic threats . A major hallmark of eukaryotic immune responses is dramatic reprogramming of the transcriptome to prioritise defences over other cellular functions . In plants transcriptional reprogramming is largely orchestrated by the immune hormone salicylic acid ( SA ) that accumulates upon recognition of biotrophic pathogens . SA not only induces resistance in infected local tissues , it is also required for establishment of systemic acquired resistance ( SAR ) , a form of induced resistance with broad-spectrum effectiveness that is long-lasting and protects the entire plant from future pathogen attack ( Spoel and Dong , 2012 ) . Establishment of SAR and associated transcriptome reprogramming are mediated by the transcriptional coactivator NPR1 ( nonexpressor of pathogenesis-related ( PR ) genes 1 ) . The majority of SA-induced genes are NPR1 dependent , indicating NPR1 is a master regulator of plant immunity ( Wang et al . , 2006 ) . Consequently , loss of NPR1 function results in severely immune-compromised plants unable to activate SAR . Since NPR1 exerts its activity in the nucleus ( Kinkema et al . , 2000 ) , controlling its nuclear entry provides a means to prevent spurious activation of immune responses . Indeed , in resting cells NPR1 is sequestered in the cytoplasm as a large redox-sensitive oligomer that is formed by intermolecular disulphide linkages between conserved cysteine residues ( Mou et al . , 2003 ) . NPR1 monomers that escape oligomerization and enter the nucleus are ubiquitinated by a Cullin-RING Ligase 3 ( CRL3 ) , a modular E3 ubiquitin ligase , resulting in their degradation by the 26S proteasome ( Spoel et al . , 2009 ) . Importantly , constitutive clearance of NPR1 from nuclei of resting cells by concerted action of CRL3 and the proteasome is necessary to prevent untimely activation of its target genes and associated autoimmunity . Upon activation of SAR , NPR1 is subject to an array of post-translational modifications . A combination of alterations in redox-based modifications , phosphorylation and SUMOylation of NPR1 result in the formation of a transactivation complex that induces the transcription of immune-responsive target genes ( Skelly et al . , 2016; Withers and Dong , 2016 ) . Subsequent to these post-translational control points , NPR1 becomes phosphorylated at Ser11 and Ser15 , which surprisingly results in recruitment of CRL3 followed by its degradation ( Spoel et al . , 2009 ) . Pharmacological inhibition of the proteasome , genetic mutation of CRL3 , and mutation of Ser11/15 all stabilised NPR1 protein , yet impaired the expression of SA-induced NPR1 target genes ( Spoel et al . , 2009 ) . These findings indicate that paradoxically , ubiquitination and degradation of NPR1 are required for the full expression of its target genes . We previously proposed a proteolysis-coupled transcription model in which activation of target gene transcription results in NPR1 being marked as ‘spent’ by Ser11/15 phosphorylation ( Spoel et al . , 2009 ) . SUMOylation of NPR1 was required for Ser11/15 phosphorylation and facilitates its interaction with other transcriptional activators ( Saleh et al . , 2015 ) , suggesting that NPR1 becomes inactivated only after it has initiated gene transcription . Removal of inactive NPR1 from target promoters may be necessary to allow binding of new active NPR1 protein that can reinitiate transcription , thereby correlating the rate of NPR1 turnover to the level of target gene expression ( Spoel et al . , 2009 ) . This type of transcriptional control by unstable ( co ) activators has also been reported in other eukaryotes , including for key transcriptional regulators such as the nutrient sensor GCN4 in yeast and the estrogen receptor ERα as well as oncogenic cMyc and SRC-3 activators in humans ( Kim et al . , 2003; Lipford et al . , 2005; Métivier et al . , 2003; Reid et al . , 2003; von der Lehr et al . , 2003; Wu et al . , 2007 ) . This suggests that the use of unstable transcriptional ( co ) activators may be an evolutionary conserved mechanism for fine-tuning gene expression ( Geng et al . , 2012; Kodadek et al . , 2006 ) . While transcription-coupled degradation of unstable ( co ) activators is an attractive model for controlling transcriptional outputs in eukaryotes , it is potentially also a costly sacrificial process . Therefore we explored the alternative possibility that prior to degradation , ubiquitination itself might act as a transcriptional signal . As chains of four or more ubiquitin molecules are thought to be necessary for recruitment of most substrates to the proteasome ( Thrower et al . , 2000 ) , it is plausible that processive ubiquitination could provide a window of opportunity for NPR1 to activate its target genes . In this study we demonstrate that the transcriptional activity of NPR1 is controlled by several ubiquitin chain modifying enzymes . Both stepwise ubiquitin chain extension and trimming activities contribute to the regulation of NPR1 target genes and establishment of plant immunity . Our findings imply that in eukaryotes transcriptional outputs of unstable ( co ) activators may not be fine-tuned by their proteasomal turnover per se but rather by conjugated ubiquitin chains of dynamic variable length .
To examine if stepwise ubiquitination of NPR1 plays a role in plant immune responses we examined a potential role for E4 ligases . Unlike E3 ligases , the E4 class do not contribute towards initial ubiquitination of substrates but rather extend pre-existing ubiquitin chains ( Hoppe , 2005; Koegl et al . , 1999 ) . In Arabidopsis the E4 ligase UBE4/MUSE3 has been implicated in the degradation of NLR ( nucleotide binding and leucine-rich repeat ) immune receptors . Mutant ube4/muse3 plants exhibited enhanced disease resistance but this phenotype could only be explained in part by the increased stability of an NLR receptor ( Huang et al . , 2014 ) . Therefore we investigated if UBE4 is involved in downstream NPR1-dependent immune signalling by acquiring a loss-of-function T-DNA insertion mutant ( Figure 1—figure supplement 1 ) . Like mutants in CRL3 ligase that fail to degrade NPR1 ( Spoel et al . , 2009 ) , adult ube4 plants displayed enhanced expression of immune genes in absence of pathogen challenge ( Figure 1A ) . In agreement with this , when the potential for enhanced disease resistance was examined by using a high inoculum of Psm ES4326 , adult ube4 mutants showed autoimmunity ( Figure 1B ) . To establish if these phenotypes were dependent on SA signalling , ube4 mutant plants were crossed with SA-deficient ics1 mutants ( Wildermuth et al . , 2001 ) . The constitutive immune gene expression observed in ube4 was abolished in ube4 ics1 double mutant plants ( Figure 1C ) . Furthermore , a low inoculum dosage of Psm ES4326 that does not cause disease in wild-type ( WT ) and mutant ube4 plants , did result in bacterial proliferation in mutant ics1 plants . In agreement with the gene expression data , enhanced susceptibility was maintained in ube4 ics1 double mutants ( Figure 1D ) , indicating the autoimmune phenotype of adult ube4 plants is completely dependent on SA . Because SA-dependent immunity is largely regulated by the transcription coactivator NPR1 ( Cao et al . , 1997 ) , we crossed ube4 with npr1-1 mutant plants . Constitutive immune gene expression in ube4 plants was abolished in ube4 npr1 plants ( Figure 1E ) and this double mutant was equally susceptible to a low Psm ES4326 inoculum as npr1 single mutants ( Figure 1F ) . Collectively , these data suggest that in unchallenged plants UBE4 suppresses the expression of SA-mediated NPR1 target genes and prevents autoimmunity , conceivably by altering the stability of upstream NLR immune receptors as well as the downstream NPR1 coactivator . Because ube4 mutant phenotypes resemble those of mutants in CRL3 ligase ( Spoel et al . , 2009 ) , we investigated if UBE4 also controls NPR1 stability in the nucleus . Expression of a YFP-UBE4 fusion protein in Arabidopsis protoplasts confirmed it is indeed partly localised to the nucleus ( Figure 2—figure supplement 1A ) . We used the protein synthesis inhibitor cycloheximide to examine if UBE4 controls the stability of SA-induced NPR1 . Both SA-induced constitutively expressed NPR1-GFP ( Figure 2—figure supplement 1B ) ( Kinkema et al . , 2000 ) and endogenous NPR1 from WT plants were degraded within a few hours after exposure to cycloheximide ( Figure 2A and B ) . By contrast , both proteins were considerably more stable in the ube4 mutant genetic background . These findings were further confirmed by quantifying the amount of NPR1-GFP or endogenous NPR1 protein remaining after exposure to cycloheximide ( Figure 2—figure supplement 1C and D ) . Stabilisation of endogenous NPR1 also occurred in the previously described muse3 mutant allele of UBE4 ( Figure 2—figure supplement 1E ) ( Huang et al . , 2014 ) . Taken together these results indicate that UBE4 promotes NPR1 degradation . Recruitment of NPR1 to CRL3 for ubiquitination and subsequent degradation requires phosphorylation at residues Ser11 and Ser15 ( Spoel et al . , 2009 ) . Therefore we examined if ube4 mutants were impaired in NPR1 Ser11/15 phosphorylation . However , Ser11/15 phosphorylation of NPR1-GFP was unaffected by the ube4 mutation ( Figure 2C ) , indicating UBE4 mediates NPR1 turnover downstream of CRL3-mediated ubiquitination . We then investigated if UBE4 is involved in polyubiquitination of NPR1 . Pulldown of polyubiquitinated proteins using tandem-repeated ubiquitin-binding entities ( TUBE ) ( Hjerpe et al . , 2009 ) followed by detection of NPR1-GFP , revealed that SA stimulated polyubiquitination of NPR1-GFP ( Figure 2D ) . By contrast , SA-induced polyubiquitination of NPR1-GFP was compromised in ube4 mutants ( Figure 2D ) , but ubiquitinated NPR1 was still detected at high-molecular weight . Therefore we sought to distinguish if in ube4 mutants , NPR1 was modified by long ubiquitin chains or multiple shorter chains , both of which yield high-molecular weights on SDS-PAGE . Thus , we performed pull down experiments with recombinant S5a ubiquitin interacting motifs ( S5aUIM ) that preferentially bind chains of four or more ubiquitin molecules ( Deveraux et al . , 1994; Young et al . , 1998 ) . Compared to plants carrying wild-type UBE4 alleles , the amount of SA-induced polyubiquitinated NPR1-GFP pulled down with recombinant S5aUIM was strikingly lower in ube4 mutants ( Figure 2E ) , indicating that UBE4 promotes formation of long ubiquitin chains on NPR1 leading to its proteasomal degradation . To determine if UBE4 may act directly on NPR1 , we assessed the ability of UBE4 to physically interact with ubiquitinated NPR1 . We isolated ubiquitinated NPR1-GFP from SA and MG132-treated ube4 mutants and subsequently incubated with recombinant FLAG-UBE4 . As shown in Figure 2F , NPR1-GFP specifically pulled down FLAG-UBE4 , indicating that UBE4 physically binds to polyubiquitinated NPR1 to facilitate ubiquitin chain extension . Because UBE4 enhanced polyubiquitination of NPR1 and controlled its stability ( Figure 2 ) , we investigated if similar to CRL3 ( Spoel et al . , 2009 ) , it also promotes transcriptional activity of NPR1 . In stark contrast to cul3a cul3b mutants that were compromised in SA-induced expression of NPR1 target genes , plants carrying two different ube4 mutant alleles exhibited elevated expression levels that were much higher than in WT ( Figure 3A and B , Figure 3—figure supplement 1A ) . To explore the effect of UBE4 on the NPR1-dependent transcriptome , we performed RNA Seq on SA-treated WT , ube4 and npr1 plants . Among 2612 genes whose expression changed by ≥2 fold in response to SA in WT or ube4 mutants , 75% were stringently dependent on NPR1 ( i . e . ≥1 . 5 fold difference compared to npr1 ) ( Figure 3—source data 1 ) . We separated these genes into two categories: ( 1 ) genes that were regulated by SA in both WT and mutant ube4 plants , and ( 2 ) genes that did not make the ≥2 fold change cut-off in WT but were highly regulated by SA in ube4 mutants . The majority of SA-induced genes in category 1 , including PR1 and WRKY marker genes , received a boost in expression when UBE4 was knocked out ( Figure 3C ) . This positive effect was even clearer for category two genes ( Figure 3C and D ) . Similarly , genes suppressed by SA treatment displayed further downregulation in ube4 mutants compared to WT ( Figure 3C ) . By contrast , SA-regulated genes that were not dependent on NPR1 behaved similarly in WT and mutant ube4 plants ( Figure 3—figure supplement 1B ) , suggesting UBE4 exerts its effects predominantly through NPR1 . Together these data suggest that in absence of UBE4-mediated long-chain polyubiquitination , NPR1 remains in a highly active transcriptional state . To understand the opposing effects of CRL3 and UBE4 on transcriptional activity of NPR1 , we examined endogenous NPR1 protein levels . Compared to WT plants , SA-induced NPR1 accumulated to elevated levels in both cul3a/b and ube4 mutants ( Figure 3E ) . Thus , NPR1 protein levels cannot explain differences in transcriptional output of NPR1 . We then examined if changes in polyubiquitin chain length regulate NPR1 association with its target promoters . To that end we performed chromatin immunoprecipitation experiments on plants that constitutively expressed NPR1-GFP , thereby eliminating genotype-dependent differences in NPR1 protein level . Coinciding with elevated PR1 gene expression , at 8 hr after SA treatment more NPR1-GFP was bound to the PR1 promoter in ube4 mutants compared to plants carrying wild-type UBE4 alleles ( Figure 3F ) . This indicates that in absence of long polyubiquitin chains , early occupancy by transcriptionally competent NPR1 is increased at target promoters . We also examined a later time point after SA treatment ( 24 hr ) and found that NPR1-GFP was still associated with the PR1 promoter in plants expressing wild-type UBE4 , but not in ube4 mutants ( Figure 3G ) . Nonetheless , PR1 gene expression remained at elevated levels in these mutants ( Figure 3G ) , implying that in absence of long-chain polyubiquitination NPR1 strongly switches on target genes without the need for long-term residency at their promoters . To investigate if CRL3 and UBE4 act independently or in tandem , we crossed ube4 single with cul3a cul3b double mutants and analysed the expression of NPR1 target genes in the resulting triple mutant . Strikingly , cul3a cul3b ube4 mutants showed severe developmental defects , including stunted growth and complete sterility ( Figure 3—figure supplement 1C and D ) . Very few viable homozygous plants were recovered , perhaps suggesting these two ligases work together and share substrates . Nonetheless we were able to select just enough plants to examine the behaviour of NPR1 target genes . In cul3a cul3b ube4 mutants the SA-induced expression of several genes , including PR genes , was impaired to a similar extend as in cul3a cul3b double mutants , indicating that elevated gene expression observed in ube4 plants is dependent on CRL3 ( Figure 3H and I ) . However , a subset of NPR1 target genes ( i . e . WRKY18 , WRKY38 , WRKY62 ) were dramatically upregulated in cul3a cul3b ube4 mutants to a level higher than in any of the other genotypes . This suggests that in absence of CRL3 and UBE4 , these genes were activated through another pathway or were highly responsive to elevated homeostatic levels of NPR1 protein and may therefore not be suitable readouts for this particular epistatic analysis ( Figure 3I ) . Regardless of this specific , our broader findings suggest that CRL3 and UBE4 function sequentially in the stepwise addition or extension of ubiquitin chains on NPR1 but with opposing effects on its transcriptional activity . We then examined if in ube4 mutants NPR1 lingered in a highly transcriptional active state that does not require proteasome-mediated turnover . To negate any feedback effects of loss of UBE4 activity on endogenous NPR1 expression , seedlings constitutively expressing NPR1-GFP were treated with SA plus a range of MG132 concentrations . SA-induced PR1 and WRKY gene expression was inhibited by increasing concentrations of MG132 in NPR1-GFP ( in npr1 ) plants ( Figure 3J and Figure 3—figure supplement 1E ) . By contrast , the SA-induced expression of these NPR1 target genes was largely unresponsive to MG132 in ube4 mutants , especially at lower concentrations . Thus , loss of UBE4 largely uncoupled NPR1 target gene expression from proteasome activity , demonstrating the importance of progressive ubiquitination for NPR1 activity . In summary , our findings indicate that initial CRL3-mediated ubiquitination is required for NPR1 to attain its full transcriptional activity , while the stepwise formation of long ubiquitin chains mediated by UBE4 inactivates NPR1 and promotes its degradation by the proteasome . Trimming or removal of ubiquitin chains is performed by deubiquitinases ( DUBs ) and may provide another layer of regulation of NPR1 activity . The Arabidopsis genome is predicted to encode for at least 65 DUBs ( Vierstra , 2009; Yang et al . , 2007 ) with high likelihood of redundancy among gene families . Therefore identifying candidate genes that potentially regulate NPR1 by genetically screening mutant collections was not feasible . Instead , we used a range of pharmacological broad-spectrum and selective DUB inhibitors and assessed their effect on SA-induced gene expression . The broad-spectrum inhibitors PR-619 ( Altun et al . , 2011 ) and NSC632839 ( Aleo et al . , 2006 ) strongly impaired SA-induced gene expression across all NPR1 target genes tested ( Figure 4A ) , suggesting that DUB activity is required for their optimal expression . Furthermore , while treatment with PR-619 or NSC632839 did not affect SA-induced transcription of the NPR1 gene , it depleted NPR1 protein levels ( Figure 4B ) . Thus , DUB activity may not only be required for NPR1-dependent gene expression but also for increasing NPR1 stability . Next we tested more selective inhibitors that more specifically target one or a few DUBs . First we treated WT seedlings with various DUB inhibitors and compared the cellular levels of global ubiquitin conjugates with control- , and MG132-treated seedlings . While NSC632839 and MG132 treatments dramatically enhanced accumulation of ubiquitin conjugates , especially in combination with SA treatment , all other inhibitors had little effect on cellular ubiquitination levels ( Figure 4C ) . We then examined the effect of these DUB inhibitors on SA-induced gene expression . All inhibitors strongly suppressed SA-induced expression of NPR1 target genes ( Figure 4D ) . Furthermore , most inhibitors were effective at low micromolar concentrations and suppressed NPR1 target genes in a dose-dependent manner ( Figure 4—figure supplement 1 ) . Collectively these data provide a first indication that DUB activity may be crucial for NPR1 stability and efficient activation of SA-induced NPR1 target genes . The more selective inhibitors used in experiments described above have been shown to target DUBs in mammalian cells ( Figure 5—figure supplement 1A ) ( Altun et al . , 2011; Kapuria et al . , 2010; Liu et al . , 2003 ) . To find potential homologues we used the sequences of these mammalian DUBs to search the Arabidopsis genome using BLASTp . The identified Arabidopsis DUBs included members of the ubiquitin-specific protease ( UBP ) and ubiquitin C-terminal hydrolase ( UCH ) multi-gene families ( Figure 5—figure supplement 1A ) . We then searched mutant collections to identify T-DNA knockouts for each of these DUBs . UBP14 knockouts are lethal in Arabidopsis ( Doelling et al . , 2001 ) , while no T-DNA insertions were identified for either UCH1 or UCH2 in mutant collections of the Col-0 genetic background . Therefore we did not pursue these DUBs further . The DUB inhibitor TCID is thought to target mammalian UCH-L3 for which we identified a single Arabidopsis homologue , UCH3 . We acquired a T-DNA insertion line that displayed complete knockout of UCH3 expression ( Figure 5—figure supplement 1B ) and analysed SA-induced NPR1 target gene expression . Figure 5A shows that SA-induced PR1 and WRKY gene expression was generally comparable between uch3 and WT plants , indicating UCH3 is unlikely to play a major role . Next we identified UBP12 and UBP13 as potential plant targets of both WP1130 and P22077 inhibitors ( Figure 5—figure supplement 1A ) . Previous research has suggested a role for these two proteins in plant immunity , as ubp12 ubp13 double knockdown RNAi plants exhibited elevated expression of PR1 and increased resistance to the virulent pathogen P . syringae pv . tomato ( Ewan et al . , 2011 ) . Single knockout mutants of UBP12 and UBP13 have no observable phenotype and double knockouts are seedling lethal ( Cui et al . , 2013; Ewan et al . , 2011 ) . However we acquired the ubp12-2w allele , previously described as a weak ubp12 ubp13 double mutant ( Cui et al . , 2013 ) , and analysed this mutant for SA-induced gene expression . Similar to a previous report ( Ewan et al . , 2011 ) , we observed elevated PR1 expression in ubp12-2w plants but other NPR1 target genes were activated to a slightly lesser extent as in WT ( Figure 5B ) . This phenotype does not explain the suppressive effects we observed with pharmacological DUB inhibitors . Finally , we acquired T-DNA knockout lines for the mammalian USP14 homologues , UBP6 and UPB7 that are potentially targeted by the WP1130 inhibitor ( Figure 5—figure supplement 1A and C ) . SA-induced expression of PR1 was slightly lower in these mutants but WRKY gene expression was largely comparable to WT plants ( Figure 5C ) . Since UBP6 and UBP7 are close homologues ( Figure 5—figure supplement 2A and B ) , we generated ubp6 ubp7 double knockout mutants ( Figure 5—figure supplement 1C ) that were viable and showed no observable developmental phenotypes . However , ubp6 ubp7 mutants were impaired in activation of SA-induced gene expression ( Figure 5D ) . This indicates that UBP6 and UBP7 are functionally redundant and required for NPR1 target gene expression . Human USP14 and its yeast homologue Ubp6 have both been shown to associate with the 26S proteasome , which is necessary for their activity ( Borodovsky et al . , 2001; Leggett et al . , 2002 ) . We tested if this is also the case for Arabidopsis UBP6 by constitutively expressing FLAG-tagged UBP6 in the ubp6 ubp7 double mutant background followed by co-immunoprecipitation experiments . The proteasomal subunits S5a and RPN6 both co-immunoprecipitated with FLAG-tagged UBP6 ( Figure 6A ) , indicating UBP6 is also a proteasome-associated DUB in plants . Next we examined if UBP6 exhibits typical DUB activity . We produced recombinant T7-tagged UBP6 and incubated it with HA-tagged ubiquitin vinyl sulfone ( HA-UbVS ) , an ubiquitin mimic that cannot be hydrolysed upon irreversible binding to DUB active sites ( Borodovsky et al . , 2001 ) . HA-UbVS readily labelled T7-UBP6 but only upon addition of 26S proteasomes ( Figure 6B ) , indicating UBP6 has proteasome-activated DUB activity . Moreover , addition of WP1130 inhibitor completely blocked HA-UbVS labelling ( Figure 6B ) , illustrating the effectiveness of this inhibitor on Arabidopsis UBP6 . To examine if UBP6 can cleave ubiquitin chains we incubated recombinant UBP6 with free ubiquitin chains or with di-ubiquitin of different linkage types and compared it to activity of recombinant human USP14 . Similar to human USP14 , Arabidopsis UBP6 displayed very little deubiquitination activity on free ubiquitin chains or di-ubiquitin of K48 and K63 linkage types ( Figure 6—figure supplement 1A–1C ) . Only wild-type UBP6 but not UBP6 ( C113S ) in which the catalytic cysteine residue was mutated , was weakly capable of trimming K63-linked chains in presence of 26S proteasomes , although this activity required very long incubation times ( Figure 6—figure supplement 1B ) . These findings mirror the poor in vitro activity of human USP14 on free ubiquitin chains ( Lee et al . , 2016 ) . Instead , human USP14 deubiquitinates anchored ubiquitin chains of various linkage types , including K48 linkages that target proteins for proteasome-mediated degradation ( Lee et al . , 2016 ) . Therefore we proceeded to investigate if UBP6 activity cleaves ubiquitin chains anchored to NPR1 . Indeed , incubation of purified polyubiquitinated NPR1-GFP with recombinant UBP6 and 26S proteasomes led to the release of ubiquitin conjugates of approximately hexa-ubiquitin chain length ( Figure 6C ) . This release of ubiquitin conjugates was dependent on SA treatment , suggesting that UBP6 counteracts SA-induced polyubiquitination of NPR1 ( Figure 6—figure supplement 1D ) . Together , these results demonstrate that UBP6 is an active DUB capable of removing ubiquitin chains en bloc from NPR1 . So what is the effect of UBP6- and UBP7-mediated deubiquitination on NPR1 function ? We found that treatment of NPR1-GFP ( in npr1 ) seedlings with WP1130 inhibitor increased the levels of SA-induced polyubiquitinated NPR1-GFP while reducing the unmodified amount of this protein ( Figure 6D ) . This suggests that UBP6 and UBP7 activities are required for deubiquitination of SA-induced NPR1-GFP , thereby rescuing it from degradation . To further examine this possibility , we analysed the stability of endogenous NPR1 protein in SA-treated ubp6 ubp7 double mutants . CHX chase experiments revealed that compared to WT plants , NPR1 was destabilised in ubp6 ubp7 mutants ( Figure 6E ) . Accordingly , expression of FLAG-tagged UBP6 in the ubp6/7 mutant background restored NPR1 stability ( Figure 6—figure supplement 1E ) . These results demonstrate that UBP6 and UBP7 serve to stabilise NPR1 by removing ubiquitin chains that signal for its proteasome-mediated degradation . Given the importance of progressive ubiquitination for the transcriptional activity of NPR1 , we explored how UBP6- and UBP7-mediated deubiquitination might affect NPR1 coactivator function . Because UBP6 and UBP7 were required for NPR1-dependent PR1 gene expression ( Figure 5D ) , we questioned if NPR1 was still associated with the PR1 promoter in absence of UBP6 and UBP7 activities . Surprisingly , ChIP experiments showed that SA-induced association of NPR1-GFP with the PR1 promoter was strongly enhanced in presence of WP1130 inhibitor ( Figure 6F ) . This suggests that UBP6 and UBP7 prevent the build-up of long polyubiquitin chains that block the transcriptional activity of NPR1 . It also implies that similar to their yeast homologue , UBP6 and UBP7 exhibit proteasome inhibitory activity ( Hanna et al . , 2006 ) . This activity is thought to delay degradation of proteasome substrates , thereby creating a window of opportunity for DUBs to deubiquitinate substrates and pardon them from proteolysis . Importantly , proteasome inhibitory activity does not require the catalytic active site ( Hanna et al . , 2006 ) . Thus , to investigate how deubiquitination and proteasome inhibitory activities of UBP6 contribute to the regulation of NPR1 coactivator activity , we expressed FLAG-tagged wild-type UBP6 ( FLAG-UBP6 ) and catalytically inactive UBP6 ( C113S ) ( FLAG-UBP6m ) in ubp6 ubp7 double mutants . While ubp6 ubp7 mutants were compromised in SA-induced activation of all NPR1 target genes tested , expression of FLAG-UBP6 fully restored SA-responsiveness ( Figure 6G ) . By contrast , FLAG-UBP6m restored SA-induced expression of only a subset , but not all NPR1 target genes . A distinction was observed between WRKY and PR genes , with the former requiring catalytic DUB activity of UBP6 while the latter did not ( Figure 6G ) . These data indicate that catalytic and non-catalytic activities of UBP6 regulate distinct NPR1-dependent gene sets . Finally we examined what the relevance is of UBP6- and UBP7-regulated transcriptional activity of NPR1 in context of plant immunity . We first treated plants with or without SA before challenge inoculation with virulent Psm ES4326 . SA treatment induced resistance in WT plants but did not block bacterial propagation in ubp6/7 plants ( Figure 6H ) . Collectively , these data clearly demonstrate that UBP6 and UBP7 are required for NPR1 coactivator activity and associated development of SA-dependent immunity .
The ubiquitin-mediated proteasome system plays vital roles in the regulation of eukaryotic gene expression , in large part by controlling the abundance of transcriptional regulators . Paradoxically , proteasome-dependent instability of selected potent eukaryotic transcriptional activators is necessary for the expression of their target genes . It is thought that their transcription-coupled degradation ensures the target promoter is continuously supplied with fresh activators that reinitiate transcription , thereby maximising gene expression ( Geng et al . , 2012; Kodadek et al . , 2006 ) . However , this sacrificial process is energy-expensive ( Collins and Goldberg , 2017; Peth et al . , 2013 ) , raising a dilemma of why such mechanisms evolved to regulate transcriptional activators . Our study on the immune coactivator NPR1 , however , indicates that ubiquitin chain extension and trimming activities can fine-tune transcriptional outputs of unstable eukaryotic activators without strict requirement for sacrificial turnover . We discovered that ubiquitination of NPR1 is a stepwise event , requiring the actions of CRL3 and the E4 ligase UBE4 . In resting cells , CRL3-mediated turnover of NPR1 is important for preventing autoimmunity in absence of pathogen threat ( Spoel et al . , 2009 ) . The NPR1-dependent autoimmune phenotype of ube4 mutants is reminiscent of that observed in cul3a cul3b mutants ( Figure 1 ) ( Spoel et al . , 2009 ) , suggesting that in addition to CRL3 ligase , UBE4 is required to clear NPR1 from the nucleus and prevent untimely activation of immunity . In presence of SA , however , CRL3-mediated ubiquitination induced NPR1 coactivator activity , whereas formation of polyubiquitin chains by UBE4 blocked its activity and ultimately led to proteasome-mediated turnover ( Figures 2 and 3 ) . Rather than initiating substrate ubiquitination , E4 ligases are thought to extend existing ubiquitin chains ( Crosas et al . , 2006; Koegl et al . , 1999 ) , thereby determining substrate commitment to proteasome-mediated degradation and contributing to proteasome processivity ( Aviram and Kornitzer , 2010; Koegl et al . , 1999 ) . Functionally these enzymes are emerging as important players in limiting the activity of NLR immune receptors as well as potent eukaryotic transcriptional regulators . Arabidopsis UBE4/MUSE3 works in concert with a CRL1/SCFCPR1 ligase to regulate stability of the intercellular immune receptors SNC1 and RPS2 that recognise pathogen invasion ( Cheng et al . , 2011; Gou et al . , 2012; Huang et al . , 2014 ) . Taken together with our finding that UBE4 acts in concert with CRL3 ( Figure 3 and Figure 3—figure supplement 1 ) , this suggests a single E4 enzyme may assist in diverse ubiquitin-mediated pathways controlled by different E3 ligases . The role of Arabidopsis UBE4 in ubiquitination and degradation of NPR1 are reminiscent of stepwise ubiquitination of the mammalian tumour suppressor p53 , a potent transcriptional activator of genes involved in apoptosis , cell cycle arrest and cellular senescence . The stability of p53 is regulated by amongst others the E3 ligase MDM2 ( or HDM2 in humans ) ( Pant and Lozano , 2014 ) . Although MDM2 limits p53 activity by promoting its turnover , MDM2 only catalyses multi-monoubiquitination of p53 , which is insufficient for recognition by the proteasome ( Lai et al . , 2001 ) . Progression to the polyubiquitinated form of p53 in the nucleus is carried out by the U-box E4 ligase UBE4B that interacts with both MDM2 and p53 ( Li et al . , 2003; Wu and Leng , 2011; Wu et al . , 2011 ) . Although this is similar to the proposed roles of CRL3 and UBE4 in controlling NPR1 stability , initial ubiquitination has different effects on p53 and NPR1 . While MDM2-mediated monoubiquitination controls nucleocytoplasmic trafficking of p53 ( Li et al . , 2003 ) , it probably does not have a direct effect on intrinsic p53 activator activity . Instead , initial ubiquitination boosts NPR1 transcriptional coactivator activity , at least in part by enhancing target promoter occupancy in the short term and potentially also by promoting genomic mobility of NPR1 in the longer term ( Figure 3 ) . Although it remains unclear if CRL3 adds only monoubiquitin or generates short chains shy of tetraubiquitin , the minimal signal required for proteasome recognition ( Thrower et al . , 2000 ) , progression to polyubiquitin chain formation by UBE4 results in transcriptional shut down as polyubiquitinated NPR1 still occupied target promoters but lacked transcriptional potency ( Figure 6D and F ) . This type of stepwise ubiquitination may be a general mechanism to control unstable transcriptional ( co ) activators in eukaryotes . For example , multi-monoubiquitination of the oncogenic growth coactivator SRC-3 results in its transcriptional activation , while subsequent chain extension targets it for degradation , but E4 ligases have not yet been implicated . We propose here that stepwise ubiquitination established by the sequential actions of E3 and E4 ligases may generate a transcriptional timer that controls the activity and lifetime of unstable ( co ) activators ( Figure 7 ) . The complexity of the ubiquitin-dependent transcriptional timer was further revealed by the identification of UBP6 and UBP7 that deubiquitinated NPR1 , thereby regulating its transcriptional activity and lifetime ( Figures 5 and 6 ) . Several unstable mammalian transcription activators , including p53 and the immune activator NF-κB , are also regulated by diverse DUBs ( Colleran et al . , 2013; Pant and Lozano , 2014; Schweitzer and Naumann , 2015 ) . In these cases DUBs promote transcription by stabilising p53 and NF-κB at their target promoters . For example , loss of USP7-mediated deubiquitination of NF-κB resulted in increased turnover and decreased promoter occupancy of NF-κB ( Colleran et al . , 2013 ) . Similarly , we found that knockout of UBP6 and UBP7 resulted in enhanced turnover and decreased transcriptional output of NPR1 ( Figure 6 ) . However , inhibition of UBP6/7 deubiquitination activities with WP1130 resulted in enhanced occupancy of transcriptionally inactive NPR1 at the PR1 target promoter ( Figure 6F ) . These data suggest that ( i ) like their yeast and mammalian counterparts ( Hanna et al . , 2006; Lee et al . , 2016 ) , UBP6 and UBP7 exhibit proteasome inhibitory activities that at least temporarily prolong promoter occupancy by NPR1 , and ( ii ) UBP6 and UBP7 prevent inactivation of NPR1 by opposing the formation of long ubiquitin chains . UBP6 showed a similar DUB activity as its mammalian homologue USP14 ( Lee et al . , 2016 ) , in that it appeared to deubiquitinate NPR1 by removing ubiquitin chains en bloc ( Figure 6C and D ) . Such activity places this DUB in direct opposition to UBE4-mediated chain extension activity . In yeast Ubp6 was previously reported to oppose ubiquitin chain extension activity of the E4 ligase Hul5 , thereby regulating substrate recruitment to the proteasome ( Crosas et al . , 2006 ) . Similarly , Arabidopsis UBP6 and UBP7 opposed ubiquitin ligase activities to extend the lifetime of transcriptionally active NPR1 . Although we cannot completely rule out that these DUBs function in opposition to CRL3 , their en bloc ubiquitin removal activity suggests they more likely remove longer ubiquitin chains generated by UBE4 ( Figure 7 ) . Other DUBs may also play a role in regulating SA-responsive gene expression . This is illustrated by our findings that all tested DUB inhibitors blocked NPR1 target gene expression , while mutation of exclusively UBP6 and UBP7 had a similar effect . This discrepancy is expected because the specificity of DUB inhibitors has well-recognised limitations in that they often target multiple related DUBs ( Figure 5—figure supplement 1A ) . This is likely especially true for Arabidopsis thaliana which encodes for >60 DUB enzymes , many of which cluster into related sub-groups ( Liu et al . , 2008; March and Farrona , 2017 ) . Besides UBP6/7 other DUBs may therefore also play a role in regulating SA-responsive gene expression but their precise functions remain unknown . In summary , we report that disparate ubiquitin modifying enzymes play important roles in establishment of plant immune responses . We demonstrate that the opposing actions of an E3 and E4 ligase pair and two DUBs can fine-tune transcriptional outputs of the unstable immune coactivator NPR1 without strict requirement for its sacrificial turnover . Dynamicity in conjugated ubiquitin chain length may be a powerful mechanism for controlling the activity of unstable eukaryotic ( co ) activators in general .
All Arabidopsis plants used in this study were in the Columbia genetic background , with WT referring to wild-type Col-0 throughout . Plants were grown under long day conditions ( 16 hr photoperiod ) on soil in controlled-environment growth chambers at 65% humidity and 22°C unless otherwise stated . Seeds were stratified at 4–8°C in darkness for 2 days before moving to growth chambers . Plants were grown in a soil mix composed of peat moss , vermiculite and sand at a ratio of 4:1:1 respectively , and illumination was provided by fluorescent tube lighting at an intensity of 70–100 μmol m−2sec−1 . For experiments on seedlings , seeds were sterilised by washing in 100% ethanol for 2 mins before incubating in 50% household bleach for 20 mins . After removal of bleach , seeds were washed at least 3 times with sterile H2O before use . Sterilised seeds were spotted on Murashige and Skoog agar media and stratified before placing under lighting conditions as above . All T-DNA insertion mutants used were genotyped by PCR using standard conditions with gene specific primers in combination with left-border primers specific to each mutant collection ( Supplementary file 1 ) . The coding sequences of the UBE4 ( At5g15400 ) and UBP6 ( At1g51710 ) genes were amplified using Phusion polymerase ( NEB ) from WT Arabidopsis cDNA with the addition of CACC at the 5’ end required for TOPO cloning . The PCR products were gel-purified and cloned in to the pENTR/D-TOPO vector ( Invitrogen ) according to manufacturers’ instructions . The active site residue of UBP6 was then mutagenised to serine ( C113S ) using QuikChange Site-Directed Mutagenesis Kit according to manufacturers’ instructions . Genes were then recombined into pEarleyGate 104 and 202 plasmids by LR reaction ( Invitrogen ) as described previously ( Earley et al . , 2006 ) to generate 35S::YFP-UBE4 , 35S::FLAG-UBP6 and 35S::FLAG-UBP6 ( C113S ) constructs . These plasmids were used to transform protoplasts or to transform Agrobacterium tumifaciens strain GV3101 ( pMP90 ) as described previously ( Kneeshaw et al . , 2014 ) . After selection of positive Agrobacterium clones carrying the transgenes , approximately 6 week old flowering ubp6/7 plants were transformed as previously described ( Clough and Bent , 1998 ) . Selection of transformants was performed by spraying 10 day old seedlings with 120 μg/l BASTA at least three times . Further confirmation of transformation was performed by immunoblotting . Segregation of BASTA resistance was analysed in the T2 generation to confirm plants had single transgene insertions . For SA treatments , adult plants were sprayed with , while seedlings were immersed in 0 . 5 mM SA or H2O . CHX , MG132 and DUB inhibitors were all used to treat seedlings by immersion at the concentrations stated in respective figure legends . Vehicle controls consisted of DMSO at the appropriate concentration for each chemical used . Psm ES4326 was grown in LB media supplemented with 10 mM MgCl2 and 50 μg/ml streptomycin . Cultures were grown overnight then centrifuged at 4 , 000 rpm for 10 mins . Cells were resuspended in 10 mM MgCl2 and absorbance was measured at 600 nm before necessary dilutions were made to adjust concentrations to those indicated in figure legends . Plants were infected by pressure infiltration with a syringe through the abaxial leaf surface . For measurement of bacterial growth , a single leaf disc per plant was cut from infected leaves at the stated dpi and ground in 10 mM MgCl2 . Serial dilutions were plated on LB supplemented with 10 mM MgCl2 and 50 μg/ml streptomycin and colonies were counted after 2 days incubation at 30°C . Leaf tissue or whole seedlings were frozen and ground to a fine powder in liquid nitrogen . Samples were homogenised in RNA extraction buffer ( 100 mM LiCl , 100 mM Tris pH 8 , 10 mM EDTA , 1% SDS ) before addition of an equal volume of phenol/chloroform/isoamylalcohol ( 25:24:1 ) . The homogenate was vortexed and centrifuged at 13 , 000 rpm for 5 min . The aqueous phase was transferred to an equal volume of 24:1 chloroform/isoamylalcohol , vortexed and then centrifuged at 13 , 000 rpm for 5 min . This step was repeated once before the aqueous layer was added to a 1/3 vol of 8 M LiCl and incubated overnight at 4°C . The extract was then centrifuged at 13 , 000 rpm for 5 min at 4°C . The resulting pellet was washed with ice cold 70% ethanol then rehydrated and dissolved in 400 μl H2O for 30 min on ice . Finally , 40 μl of NaAc ( pH 5 . 3 ) and 1 ml of ice cold 96% ethanol was added before incubating for 1 hr at −20°C . The precipitate was then centrifuged at 13 , 000 rpm for 5 min at 4°C , the pellet was washed with ice cold 70% ethanol and resuspended in 50 μl of H2O . Before cDNA synthesis , RNA samples were quantified using a NanoDrop spectrophotometer ( Thermo Scientific ) and appropriate dilutions were made to ensure all samples contained equal amounts of RNA . Reverse transcription was then performed using SuperScript II reverse transcriptase ( Invitrogen ) according to the manufacturers’ instructions . qPCR was carried out on 20-fold diluted cDNA using Power SYBR Green ( Life Technologies ) and gene-specific primers ( Supplementary file 1 ) on a StepOne Plus Real Time PCR machine ( Life Technologies ) . RNA was extracted from biological duplicate samples as described above and further purified using an RNeasy Mini Kit ( Qiagen ) according to the manufacturer’s instructions . qPCR was carried out to confirm appropriate induction of SA-responsive marker genes . RNA was then quantified and submitted to GATC Biotech/Eurofins ( Constance , Germany ) for RNA sequencing . The RNA-Seq reads were aligned to the Arabidopsis thaliana TAIR10 genome using Bowtie . TopHat identified potential exon-exon splice junctions of the initial alignment . Strand NGS software in RNA-Seq workflow was used to quantify transcripts . Raw counts were normalised using DESeq with baseline transformation to the median of all samples . Data were then expressed as normalised signal values ( i . e . log2[RPKM] where RPKM is read count per kilobase of exon model per million reads ) for all statistical tests and plotting . Genes were then filtered by expression ( 20%–100% ) and differentially expressed genes determined by Benjamini Hochberg FDR with 2-way ANOVA ( p=0 . 05 ) . Additionally , we required SA-induced genes to meet a ≥ 2 fold change cut-off , whereas NPR1-dependent genes required ≥1 . 5 fold change in Col-0 or ube4 plants when compared to npr1 mutants . RNA Seq data have been deposited in Array Express at EMBL-EBIunder accession code E-MTAB-7369 . Chromatin immunoprecipitation was performed on leaf tissue of 4 week-old soil-grown adult plants essentially as described ( Yamaguchi et al . , 2014 ) but with minor modifications . 500 mg tissue was crosslinked with 1% formaldehyde by vacuum infiltration for 30 mins at room temperature . Glycine was added to a final concentration of 100 mM to quench crosslinking and vacuum infiltrated for a further 10 mins . Crosslinked tissue was washed twice with ice-cold PBS before all liquid was removed and tissue was frozen in liquid nitrogen . Nuclei were isolated and lysed as described ( Yamaguchi et al . , 2014 ) while sonication was performed using a BioRuptor Plus ( Diagenode ) . Sonication consisted of 15 cycles of 30 s ON , 30 s OFF at high power . NPR1-GFP was immunoprecipitated using ChIP grade anti-GFP ( Abcam ) before capture of immune complexes with Protein A agarose ( Millipore ) . Crosslink reversal and protein removal was performed as described previously ( Nelson et al . , 2006 ) , by boiling in the presence of Chelex 100 resin ( BioRad ) before incubation at 55°C with Proteinase K . Finally , DNA was cleaned up using PCR purification columns ( Qiagen ) and analysed by qPCR using primers listed in Supplementary file 1 . For protein degradation assays and analysis of NPR1 levels , seedlings were frozen and ground to a fine powder in liquid nitrogen before homogenising in protein extraction buffer ( PEB ) ( 50 mM Tris-HCl ( pH 7 . 5 ) , 150 mM NaCl , 5 mM EDTA , 0 . 1% Triton X-100 , 0 . 2% Nonidet P-40 , and inhibitors: 50 μg/ml TPCK , 50 μg/ml TLCK , 0 . 6 mM PMSF ) ( Spoel et al . , 2009 ) . For analyses of NPR1 phosphorylation PEB buffer was supplemented with 1X phosphatase inhibitor cocktail 3 ( Sigma ) . Samples were centrifuged at 13 , 000 rpm for 15 min at 4°C to clarify extracts , and the resulting supernatant was used for SDS-PAGE and immunoblot analysis . All antibodies used are listed in the Key Resources Table . For analysis of polyubiquitination with TUBEs , seedlings were ground to a fine powder in liquid nitrogen and homogenised in 1x PBS supplemented with 1% Triton X-100 , 10 mM NEM , 40 µM MG132 , 50 μg/ml TPCK , 50 μg/ml TLCK , 0 . 6 mM PMSF , and 0 . 2 mg/ml GST-TUBE ( Hjerpe et al . , 2009 ) . Homogenates were centrifuged at 13 , 000 rpm at 4°C for 20 mins to remove cellular debris and filtered through 0 . 22 µm filters before overnight incubation with Protino Glutathione Agarose 4B ( Machery Nagel ) , at 4°C with rotation . The agarose beads were washed 5 times with 1X PBS + 1% Triton X-100 before elution by boiling in 1X SDS-PAGE sample buffer including 50 mM DTT . NPR1-GFP was detected by immunoblotting with anti-GFP ( Roche ) . For analysis of long chain polyubiquitination , seedlings were ground to a fine powder in liquid nitrogen and homogenised in 1X PBS , supplemented with 1% Triton X-100 , 10 mM NEM , 80 μM MG115 , 50 μg/ml TPCK , 50 μg/ml TLCK , 0 . 6 mM PMSF , 1X phosphatase inhibitor cocktail 3 ( Sigma ) . Homogenates were centrifuged at 13 , 000 rpm at 4°C for 20 mins to remove cellular debris and filtered through 0 . 22 µm filters before overnight incubation with 300 μg His6-V5-S5aUIM protein immobilised on agarose . Agarose beads were washed 5 times with extraction buffer before elution at 80°C for 15 mins in 1X SDS-PAGE sample buffer including 50 mM DTT . NPR1-GFP was detected by immunoblotting with anti-GFP ( Roche ) . For proteasome co-immunoprecipitation with FLAG-UBP6 , seedlings were frozen and ground to a fine powder in liquid nitrogen before homogenising in proteasome extraction buffer ( 50 mM Tris-HCl ( pH 7 . 4 ) , 25 mM NaCl , 2 mM MgCl2 , 1 mM EDTA , 10 mM ATP , 5% glycerol , and inhibitors: 50 μg/ml TPCK , 50 μg/ml TLCK , 0 . 6 mM PMSF ) . Extracts were centrifuged at 13 , 000 rpm at 4°C for 20 mins to remove cellular debris and filtered through 0 . 22 µm filters . Anti-FLAG M2 affinity gel was washed with the above buffer before incubating with samples overnight with rotation at 4°C . The resin was washed 3 times with the same buffer before immunoprecipitated proteins were eluted by boiling in 1X SDS-PAGE sample buffer including 50 mM DTT . FLAG-UBP6 was detected using rabbit anti-FLAG antibodies while co-immunoprecipitating proteins were detected with indicated antibodies . N-terminal GST-tagged TUBE was generated by cloning the coding sequence of hHR23A into pGEX-6P-1 using EcoRI and SalI restriction sites . Primers used are listed in Supplementary file 1 . GST-TUBE expression was induced in BL21 ( DE3 ) E . coli cells with the addition of 1 mM IPTG and cultures were incubated for a further 4 hr at 28°C before collecting by centrifugation . Cells were then lysed in 1X PBS supplemented with 1 mg/ml lysozyme , 25 U/ml Benzonase nuclease , 0 . 1% Triton-X-100 and a protease inhibitor cocktail before GST-TUBE was purified using Protino Glutathione Agarose 4B according to the manufacturers’ instructions . Purified GST-TUBE was dialysed against 1X PBS and stored with the addition of 10% glycerol at −80°C until use . Recombinant S5aUIM protein was generated by synthesising residues 196–309 from human S5a with codon optimisation for E . coli into pET151/D-TOPO . The resulting His6-V5-S5aUIM protein was expressed in BL21 ( DE3 ) E . coli cells by addition of 1 mM IPTG and incubation for 24 hr at 28°C before collecting by centrifugation . Cells were then lysed in lysis buffer ( 50 mM KHPO4 pH 8 , 100 mM NaCl , 10 mM Imidazole , 1X BugBuster ( Merck ) , 25 U/ml Benzonase nuclease , 50 μg/ml TPCK , 50 μg/ml TLCK and 0 . 5 mM PMSF ) . His6-UBP6 was then purified using HisPur cobalt resin ( Thermo Fisher ) according to manufacturers’ instructions . Purified His6-V5-S5aUIM was dialysed against 1X PBS and covalently coupled to NHS-activated agarose to a final concentration of approximately 10 μg/μl following the manufacturer’s instructions ( Thermo Fisher ) . N-terminal His6-T7-tagged UBP6 was generated by cloning the coding sequence of Arabidopsis UBP6 in to the expression vector pET28a using EcoRI and SalI restriction sites . Primers used are listed in Supplementary file 1 . Expression was induced in BL21 ( DE3 ) E . coli cells with the addition of 1 mM IPTG and cultures were incubated for a further 3 hr at 28°C before collecting by centrifugation . Cells were then lysed in lysis buffer ( 50 mM KHPO4 pH 8 , 300 mM NaCl , 10 mM Imidazole , 1 mg/ml lysozyme , 25 U/ml Benzonase nuclease , 0 . 1% Triton-X- 100 , 10 mM β-mercaptoethanol , 50 μg/ml TPCK , 50 μg/ml TLCK and 0 . 5 mM PMSF ) . His6-UBP6 was then purified using HisPur cobalt resin ( Thermo Fisher ) according to manufacturers’ instructions . Purified His6-UBP6 was dialysed against 50 mM Tris-HCl pH 7 . 4 , 5M NaCl and stored with the addition of 10% glycerol at −80°C until use . Recombinant FLAG-UBE4 was produced using cell-free synthesis via two-step PCR ( Nomoto and Tada , 2018 ) using the primers listed in Supplementary file 1 . The anti-NPR1 polyclonal antibody was generated by immunising rabbits with a synthetic peptide based on a region of the NPR1 protein with the sequence N’-SALAAAKKEKDSNNTAAVKL-Cys . Rabbits were subsequently bled and antibodies were enriched by affinity purification ( Proteintech , USA ) . For HA-UbVS labelling , 10 μl reactions were prepared in 50 mM Tris-HCl pH 7 . 4 , 5 mM MgCl2 , 1 mM DTT and 1 mM ATP . Before labelling , 350 nM His6-T7-UBP6 was pre-incubated with 50 μM WP1130 or DMSO control for 10 mins before addition of 10 nM Ub-VS treated 26S proteasomes ( Ubiquigent ) . Reactions were incubated for a further 20 mins before addition of 700 nM HA-UbVS and further incubation for 30 mins . All steps were carried out at room temperature . Labelling was terminated with the addition of SDS-PAGE sample buffer including 50 mM DTT . Samples were heated at 70°C for 10 mins before SDS-PAGE and immunoblot analyses . All in vitro deubiquitination assays were performed in DUB buffer ( 50 mM Tris-HCl pH 7 . 4 , 5 mM MgCl2 , 1 mM DTT , 5 mM ATP ) . Where indicated , 1 . 25 nM Ub-VS treated 26S proteasomes and 20 nM UBP6 were added . Di-ubiquitin and polyubiquitin chain substrates were included at 400 nM . Reactions were incubated at 30°C for the times indicated in figure legends before terminating with addition of SDS-PAGE sample buffer including 50 mM DTT . Samples were heated at 70°C for 10 mins before SDS-PAGE and immunoblot analyses . For in vitro deubiquitination of NPR1-GFP isolated from plants , seedlings were treated with SA and MG132 as described in figure legends . Seedlings were frozen and ground to a fine powder in liquid nitrogen before homogenising in protein extraction buffer ( PEB ) ( 50 mM Tris-HCl ( pH 7 . 5 ) , 150 mM NaCl , 5 mM EDTA , 0 . 1% Triton X-100 , 0 . 2% Nonidet P-40 , and inhibitors: 50 μg/ml TPCK , 50 μg/ml TLCK , 0 . 6 mM PMSF ) . Extracts were centrifuged at 13 , 000 rpm at 4°C for 20 mins to remove cellular debris and filtered through 0 . 22 µm filters . GFP-Trap A agarose ( Chromotek ) was incubated with extracts for 2 hr with rotation at 4°C before washing 10 times with PEB ( without inhibitors ) then twice with DUB buffer . Supernatant was completely removed before DUB reactions were set up as described above but with NPR1-GFP immobilised on GFP-Trap A as the substrate . Proteins were eluted by boiling in 1X SDS-PAGE sample buffer including 50 mM DTT , before analysis by immunoblotting . For pathogen growth experiments confidence intervals that would allow acceptance or rejection of the null hypothesis were used to estimate sample size , while no statistical methods were used to predetermine sample sizes elsewhere , nor were any methods of randomization . All experiments were repeated a minimum of two times with similar results . For quantitative immunoblotting , band intensities were acquired using Image Studio software and a LI-COR Odyssey FC imaging device ( LI-COR Biosciences ) . NPR1 signal was normalised to GAPDH signal for three replicates , each of which consisted of an independent pool of at least 50 seedlings . For qPCR and ChIP-qPCR experiments with adult plants , samples consisted of a pool of at least six different plants while for seedlings , samples consisted of a pool of at least 50 seedlings . qPCR data is shown for three technical replicates of a representative repeated experiment . For pathogen growth experiments , replicates represent eight separate plants . In all figure legends , the statistical tests applied are stated while n refers to sample size .
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 .
Plant diseases cause devastating crop losses around the world and threaten the food supply of millions of people . Over time , plants have developed various mechanisms for fighting off infections caused by pests and other pathogens such as viruses and bacteria . When plants become infected they kick their immune system into action by rapidly switching on and off certain genes . They do this by activating the protein NPR1 which regulates the plant’s immune genes . NPR1 is essential for fighting off infections and plants that do not have this protein are highly susceptible to disease . Peculiarly , once the plant has detected an infection it builds resistance by destroying the NPR1 protein . A previous study suggested that plants do this to replace old ‘inactive’ NPR1 with newer versions that can activate the genes needed to stop the disease developing . This process , however , requires a lot of energy that could be re-directed to other aspects of the immune response . Now , Skelly et al . – including one of the researchers involved in the previous study – have explored whether there may be other reasons for why plants destroy the NPR1 protein . Plant cells target NPR1 for destruction by repeatedly tagging it with molecules called ubiquitin to form ubiquitin chains . The length of these chains determines whether a protein is stable and ready for action , or whether it is ready to be destroyed . In experiments with a commonly studied plant known as Arabidopsis thaliana , Skelly et al . found that the length of ubiquitin chains attached to the NPR1 protein could fine-tune its level of activity: short ubiquitin chains activate NPR1 , while longer chains lead to its destruction and shut down the protein . This suggests that the steps leading to the destruction of NPR1 regulate the immune genes needed to fight off disease . This work has uncovered important new components of how plants defend themselves from infection . If these findings translate to crop plants they could inform future agricultural strategies for enhancing the plant’s own defences to increase crop yields , which would provide more food for a rapidly growing population .