Journal Pre-proof

UBP12/UBP13-mediated deubiquitination of salicylic acid receptor NPR3 suppresses

plant immunity

Yu Zhou, Su-Hyun Park, Nam-Hai Chua

PII: S1674-2052(22)00408-7
DOI: https://doi.org/10.1016/j.molp.2022.11.008
Reference: MOLP 1463

To appear in: MOLECULAR PLANT

Received Date: 2 May 2022

Revised Date: 14 September 2022
Accepted Date: 18 November 2022

Please cite this article as: Zhou Y., Park S.-H., and Chua N.-H. (2022). UBP12/UBP13-mediated
deubiquitination of salicylic acid receptor NPR3 suppresses plant immunity. Mol. Plant. doi: https://
doi.org/10.1016/j.molp.2022.11.008.

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2 UBP12/UBP13-mediated deubiquitination of salicylic acid receptor NPR3
3 suppresses plant immunity
4
5 Yu Zhou1, 2 Su-Hyun Park1 Nam-Hai Chua1,2,*
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7 Temasek Life Sciences Laboratory, National University of Singapore, 1 Research
8 Link, 117604, Singapore
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10 Disruptive & Sustainable Technologies for Agricultural Precision, Singapore-MIT
11 Alliance for Research and Technology, 1 CREATE Way, 138602, Singapore
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13 *Corresponding author: Nam-Hai Chua; chua@rockefeller.edu

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15 Short Summary
16 After Pathogen infection the stability of salicylic acid (SA) receptor NPR3 is

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17 enhanced by increased cellular SA levels. SA promotes the interaction between
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UBP12/UBP13 and NPR3. These two deubiquitinases remove ubiquitin from NPR3
to stabilize the latter thereby suppressing plant immunity.
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22 Abstract
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24 Salicylic acid (SA), a defence hormone produced after pathogen challenge, is critical
25 for plant immunity. Arabidopsis NONEXPRESSER OF PR GENES 1 (NPR1) and its
26 paralogs NPR3 and NPR4 have been proposed to bind SA and mediate SA signal
27 transduction. NPR1 functions as a transcriptional co-activator to promote defence
28 gene expression, whereas NPR3 and NPR4 have been proposed to function as
29 negative regulators in SA signaling pathway. Although, the mechanism relating to
30 NPR1 regulation has been well studied, how NPR3/NPR4 proteins are regulated in
31 immune responses is still unknown. Here, we show that the stability of NPR3/NPR4
32 is enhanced by SA. In the absence of pathogen challenge, NPR3/NPR4 are unstable
33 and degraded by the 26S proteasome, whereas the increase of cellular SA levels upon

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34 pathogen infection suppress NPR3/NPR4 degradation. We showed UBP12 and

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35 UBP13, two homologous deubiquitinases from a ubiquitin-specific protease
36 subfamily, negatively regulate plant immunity by promoting NPR3/NPR4 stability.

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37 Our genetic results showed that UBP12/UBP13-mediated immunity suppression is
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partially dependent on NPR3/NPR4 functions. By interacting with NPR3 in the
nucleus in an SA-dependent manner, UBP12/UBP13 remove ubiquitin from
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40 polyubiquitinated NPR3 to protect the latter from being degraded. The stabilization of
41 NPR3/NPR4 promoted by UBP12/UBP13 is essential for basal and SA-induced
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42 immunity regulation.
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44 Key words: Plant immunity, Salicylic acid, NPR3, UBP12/UBP13

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47 Introduction
48
49 During their growth cycles, plants are frequently challenged by diverse groups of
50 pathogens, including viruses, oomycetes, fungi and bacteria (Fu and Dong, 2013).
51 Even without a circulatory system and specialized immunity cells as in animals,
52 plants have evolved several layers of immune system to defend themselves against
53 pathogen infection (Spoel and Dong, 2012). The first line of defence at the cellular
54 level against invading pathogens is pathogen-associated molecular patterns
55 (PAMP)-triggered immunity (PTI), which is mediated by recognizing PAMPs from
56 offending pathogens through cell surface-localized pattern-recognition receptors
57 (PRRs) (Boller and Felix, 2009; Couto and Zipfel, 2016; Spoel and Dong, 2012; Yu et
58 al., 2017). To establish a successful infection plant pathogens can secrete effectors
59 into plant cells to suppress PTI (Jones and Dangl, 2006). On the other hand, plants

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60 have also evolved effector-triggered immunity (ETI) to counteract pathogens. In this

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61 host-pathogen arms race, many resistance (R) proteins have been identified which can
62 detect pathogen effectors and trigger a stronger immune response. This response is

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63 known as hypersensitive response (HR), which leads to programmed cell death (PCD)
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at the infected sites (Jones and Dangl, 2006).
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66 Salicylic acid (SA), whose levels increase rapidly after pathogen challenge, plays a
67 central role in local and systemic defence (Fu and Dong, 2013; Gaffney et al., 1993;
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68 Métraux et al., 1990; Malamy et al., 1990). Previous studies have demonstrated that
69 SA can be perceived by NPR1 and two of its paralogs NPR3 and NPR4 (Ding et al.,
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70 2018; Fu et al., 2012; Manohar et al., 2014; Wu et al., 2012). These three SA receptors
71 share very similar domain structures: an N-terminal BTB/POZ domain, a central
ankyrin-repeat domain and a C-terminal transcriptional regulatory domain (Cao et al.,
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73 1997; Rochon et al., 2006; Zhang et al., 2006b). NPR1 is required for induction of
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74 local resistance and establishment of systemic acquired resistance (SAR) (Spoel and
75 Dong, 2012). SA treatment or pathogen infection causes NPR1 to migrate from the
76 cytosol to the nucleus, where it interacts with TGA transcription factors and acts as a
77 co-activator for SA-induced resistance gene expression (Després et al., 2000; Mou et
78 al., 2003; Zhang et al., 1999). In addition to transcriptome reprogramming, a recent
79 study showed NPR1 promotes cell survival by targeting substrates for ubiquitination
80 and degradation through the formation of salicylic acid-induced NPR1 condensates
81 (SINCs) (Zavaliev et al., 2020). Post-translational modifications (PTM), like
82 phosphorylation, ubiquitination and SUMOylation, are known to regulate the
83 transcriptional activity and stability of NPR1 (Saleh et al., 2015; Skelly et al., 2019;
84 Spoel et al., 2009). Despite the high homology to NPR1, NPR3 and NPR4 act as
85 negative regulators in plant immunity (Ding et al., 2018; Zhang et al., 2006b).
86 Consistent with their negative roles in SA signaling pathway, previous study showed
87 NPR3 and NPR4 act as adaptors of Cullin 3 (CUL3) E3 ligase complex through their
88 BTB/POZ domain to mediate ubiquitination and subsequent nuclear degradation of
89 NPR1 in an SA-dependent manner (Fu et al., 2012; Zavaliev et al., 2020). A recent
90 study has shown that NPR3 and NPR4 might also function independently of NPR1,

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 91 acting as transcriptional co-repressors to inhibit defence gene expression in the
92 absence of any pathogen challenge (Ding et al., 2018). Hence, in order to gain a better
93 understanding how NPR3 and NPR4 function in plant immunity, it is important to
94 investigate the detailed molecular mechanisms that regulate the activity and stability
95 of these two proteins.
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97 Here, we showed that under normal condition NPR3/NPR4 proteins are unstable and
98 degraded through the ubiquitin-proteasomal pathway. SA treatment or pathogen
99 infection results in elevated NPR3/NPR4 protein levels by increasing their stability.
100 Two ubiquitin-specific proteases, UBP12 and UBP13, that are involved in many
101 processes of plants development and defence (An et al., 2018; Cui et al., 2013;
102 Derkacheva et al., 2016; Ewan et al., 2011; Jeong et al., 2017; Lee et al., 2019; Liu et
103 al., 2022; Luo et al., 2022; Park et al., 2019; Park et al., 2022; Zhou et al., 2021),

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104 interact with NPR3 in an SA-dependent manner and deubiquitinate NPR3 to protect it

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105 from destruction. Our results suggest that in addition to repressing defence gene
106 expression in the absence of pathogen challenge, NPR3/NPR4 continue to play

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107 important roles after SA perception. UBP12/UBP13-mediated stabilization of NPR3 is
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required for NPR1 turnover and the full induction of defence genes.
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110 Results
111
112 SA perception increases NPR3/NPR4 stability
113
114 Modulation of protein stability plays a critical role in regulating cellular functions of
115 eukaryotes (Finley, 2009; Swatek and Komander, 2016). A large number of proteins
116 have been shown to be unstable and their turnover enables plants to rapidly respond
117 and adapt to changing cellular and environmental conditions (Trujillo and Shirasu,
118 2010; Xu and Xue, 2019). Previous studies have demonstrated that SA-triggered
119 proteasomal degradation of NPR1 in the nucleus is essential for full induction of its
120 target genes and establishment of systemic acquired resistance (Fu et al., 2012; Spoel
121 et al., 2009). In contrast to NPR1, post-translational modifications of NPR3/NPR4 are
122 still unknown. To examine whether NPR3/NPR4 stability is also regulated by SA, we

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123 analyzed NPR3/NPR4 protein levels in 35S::NPR3-MYC or 35S::NPR4-MYC

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124 transgenic plants after SA treatment. As negative regulators in SA signaling pathway,
125 NPR3/NPR4 are inactivated after SA perception (Ding et al., 2018); therefore, we

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126 hypothesized NPR3/NPR4 levels should be decreased by SA treatment. Contrary to
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our initial hypothesis, we found that NPR3 and NPR4 protein levels were
significantly elevated by high concentrations of SA (Figure 1A and Figure S1A).
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129 Additionally, NPR3 and NPR4 protein levels gradually increased with increasing SA
130 concentrations suggesting SA-induced NPR3/NPR4 accumulation was dosage
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131 dependent (Figure 1A and Figure S1A).

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133 Because a 35S promoter was used to express NPR3-MYC or NPR4-MYC, which
134 contains an as-1 element that is responsive to SA (Jupin and Chua, 1996; Qin et al.,
1994), we checked NPR3-MYC and NPR4-MYC transcript levels upon SA treatment.
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136 We found NPR3-MYC and NPR4-MYC transcript levels could indeed be induced by
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137 high SA concentrations (Figure 1B and Figure S1B). However, the fold-induction at
138 the transcript level was much lower than that at the protein level (Figure 1A and 1B,
139 Figure S1A and S1B). These results suggested SA can induce the accumulation of
140 NPR3/NPR4-MYC protein levels, which is partially caused by the increased
141 expression of the 35S::NPR3/NPR4-MYC transgene by SA. To further confirm the
142 effect of SA on the increase of NPR3/NPR4 protein levels, we transiently expressed in
143 Nicotiana benthamiana NPR3-FLAG transcribed from pUBQ10, a promoter which is
144 non-responsive to SA. pUBQ10::YFP was co-expressed with pUBQ10::NPR3-FLAG
145 and the YFP protein level was used as a control for expression. Upon SA treatment,
146 NPR3-FLAG protein levels were significantly increased; by contrast, YFP levels were
147 not altered by SA (Figure S1C). Moreover, we also analyzed the effect of
148 Pseudomonas syringae pv. tomato (Pst) DC3000 on NPR3 protein levels. Although
149 NPR3-MYC transcript levels were slightly induced, NPR3 protein levels were
150 significantly increased 6 hours after inoculation with Pst DC3000 (Figure 1C and 1D).
151 Taken together, our data indicated SA and pathogen infection can increase NPR3
152 stability. These results suggested NPR3/NPR4 may have important functions in plant
153 immunity after activation of SA signaling pathway. These findings are consistent with

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154 previous suggestion that NPR3/NPR4 act as adaptors of Cullin3 E3 ligase to mediate
155 nuclear NPR1 degradation in response to SA (Fu et al., 2012).
156
157 To investigate cellular mechanisms responsible for regulation of NPR3 stability, we
158 analyzed NPR3 protein levels after treatment with several protease inhibitors specific
159 for different proteolytic pathways. The 26S proteasome inhibitor MG132 prevented
160 NPR3 degradation especially in the absence of SA (Figure 1E). By contrast, a
161 protease inhibitor cocktail and the autophagosome inhibitor E64d had negligible
162 effect on NPR3 degradation (Figure S2). These results suggested that the 26S
163 proteasome activity is specifically required for NPR3 degradation. The instability of
164 NPR3 was further confirmed using a time course degradation assay in which
165 cycloheximide (CHX) was added to prevent new protein synthesis. NPR3 degradation
166 rate was faster without SA than with SA and the degradation was prevented by

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167 MG132 (Figure 1F and 1G). Taken together, our results indicated that under normal

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168 condition, NPR3 is unstable and degraded by a proteasome-dependent pathway but
169 after pathogen challenge, the increased cellular SA level suppresses NPR3

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170 degradation.
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NPR3 interacts with UBP12/UBP13 in an SA-dependent manner.
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174 Protein ubiquitination is a conserved type of PTM in eukaryotes to regulate multiple
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175 cellular processes and signaling pathways. Poly-ubiquitination is necessary for protein
176 degradation through the 26S proteasome (Moon et al., 2004; Swatek and Komander,
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177 2016). The steady state level of a poly-ubiquitinated protein is determined by the
178 opposing activities of specific ubiquitin E3 ligases and deubiquitinating enzymes
(DUBs) (Finley, 2009; Nijman et al., 2005; Swatek and Komander, 2016). In a search
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180 for E3 ligases or DUBs that may specifically regulate NPR3/NPR4 stability we
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181 considered UBP12 and UBP13 as possible candidates, because UBP12/UBP13-RNAi

182 transgenic plants exhibit enhanced disease resistance, a phenotype reminiscent of the
183 npr3npr4 double mutants (Ewan et al., 2011; Fu et al., 2012). To determine whether
184 UBP12/UBP13 are indeed involved in ubiquitin-proteasomal degradation of
185 NPR3/NPR4, we first performed yeast-two-hybrid assays to detect possible
186 interactions between NPR3/NPR4 and UBP12/UBP13. Figure 2A shows that NPR4
187 interacted with UBP12/UBP13 either in the absence or presence of SA. This
188 SA-independent association was confirmed by the observation that interactions
189 between UBP12/UBP13 and NPR4 were not affected by an NPR4 mutation (mNPR4)
190 which comprises its binding to SA (Ding et al., 2018) (Figure 2A). By contrast, NPR3
191 interacted with UBP12/UBP13 only in the presence of SA and the interactions
192 between UBP12/UBP13 and NPR3 were impaired by an NPR3 mutation (mNPR3)
193 that abolishes SA binding (Figure 2A). It is possible that the interaction between
194 NPR3 and UBP12 or UBP13 after SA treatment is caused by a potentially increased
195 NPR3 protein level in yeast cells. To examine this possibility we used an anti-HA
196 antibody to determine NPR3 protein levels in yeast cells after treatment with or
197 without SA. We found that NPR3-HA protein levels were not significantly affected

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198 upon SA treatment suggesting NPR3 displays a higher binding affinity to UBP12 or
199 UBP13 in presence of SA (Figure S3). On the other hand, NPR1 showed a very weak
200 binding affinity to UBP12/UBP13 in yeast two-hybrid assays (Figure 2A).
201
202 We used bimolecular fluorescence complementation (BiFC) assays in Nicotiana
203 benthamiana to further confirm the interactions between NPR3/NPR4 and
204 UBP12/UBP13. Indeed, fluorescence signals were observed in nuclei of cells
205 co-expressing NPR3/NPR4-nYFP and UBP12/UBP13-cYFP (Figure 2B). We also
206 performed co-immunoprecipitation (co-IP) assays using extracts derived from
207 transgenic plants co-expressing NPR3-MYC with UBP12-HA or UBP13-HA. NPR3
208 showed a very weak interaction with UBP12/UBP13 in the absence of SA (Figure 2C
209 and 2D). Upon SA treatment, however, the association between NPR3 and
210 UBP12/UBP13 was greatly enhanced (Figure 2C and 2D), consistent with the results

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211 from yeast-two-hybrid. These results suggested NPR3 formed a complex with

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212 UBP12/UBP13 in an SA-dependent manner .
213

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214 UBP12 and UBP13 act as negative regulators in plant immunity.
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A previous study using UBP12/UBP13-RNAi transgenic plants showed that
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217 UBP12/UBP13 are redundantly required for immunity in Arabidopsis (Ewan et al.,
218 2011). Because of possible off-target effects of RNAi approach, we re-examined the
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219 functions of UBP12/UBP13 in pathogen defence by using transfer DNA (T-DNA)

220 insertion alleles of these two genes. Consistent with previous results, no significant
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221 alteration in susceptibility to Pst DC3000 was observed in ubp12-1 and ubp13-3
222 single mutants compared to WT (Col-0). As double mutants of UBP12/UBP13 null
alleles are not available (Cui et al., 2013; Ewan et al., 2011), we examined the
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224 susceptibility to Pst DC3000 of ubp12-2w, which has truncated UBP12 transcripts
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225 and reduced UBP13 transcript levels (Cui et al., 2013). We found that ubp12-2w
226 showed increased resistance to pathogen infection relative to WT (Figure 3A).
227 Moreover, ubp12-2w/13-3 double mutants, with a more severe developmental
228 phenotype showed a stronger resistance to Pst DC3000 than ubp12-2w (Figure 3A).
229 In contrast to ubp12-2w and ubp12-2w/13-3, bacterial growth was significantly
230 increased in transgenic plants with elevated UBP12 or UBP13 expression (Figure 3A
231 and S4). These data supported the notion that UBP12/UBP13 act as negative
232 regulators in plant immunity.
233
234 In addition to the susceptibility to pathogen infection, we also examined the
235 expression of defence genes in UBP12/UBP13 deficient mutants and over-expressors.
236 Compared to WT, PR1 and PR2 transcript levels were significantly induced in
237 ubp12-2w and ubp12-2w/13-3, suggesting a constitutively activated immune response
238 in these mutants (Figure 3B and 3C). By contrast, defence gene expression was
239 decreased in UBP12-OE and UBP13-OE transgenic plants (Figure 3B and 3C). These
240 results are consistent with our genetic results indicating a negative role of
241 UBP12/UBP13 in basal immunity of plants.

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243 UBP12/UBP13 suppress plant immunity in an NPR3/NPR4-dependent manner.
244
245 Results presented above showed UBP12/UBP13 are required for immunity regulation
246 and interact with NPR3 and NPR4 in vitro and in vivo (Figure 2, Figure 3A-3C).
247 Together, they suggested the possibility that UBP12/UBP13 may be involved in SA
248 signal transduction and these two DUBs may regulate plant immunity through
249 NPR3/NPR4. We therefore examined the responses of WT, ubp12-2w/13-3,
250 UBP12-OE, and UBP13-OE to SA-induced immunity by pre-treating plants with SA
251 before Pst DC3000 inoculation. In WT plants, a significant reduction in the growth of
252 Pst DC3000 was observed after pre-treatment with SA (Figure 3D). Compared to WT,
253 pathogen resistance was enhanced in ubp12-2w/13-3 with or without SA, suggesting a
254 constitutive immune response in ubp12-2w/13-3. However, SA-induced resistance to

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255 pathogen infection in ubp12-2w/13-3 was significantly compromised (Figure 3D),

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256 which is similar to that observed in the npr3 and npr3npr4 double mutants (Fu et al.,
257 2012). In contrast to ubp12-2w/13-3, UBP12-OE and UBP13-OE showed increased

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258 sensitivity to SA-induced resistance (Figure 3D). We also analyzed the expression of
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defence genes in WT and ubp12-2w/13-3 in response to SA treatment. In the absence
or presence of SA, transcript levels of defence-related genes PR1, SARD1, and
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261 WRKY70 in ubp12-2w/13-3 were higher than those in WT (Figure 3E to 3G).
262 However, the fold-induction of these defence genes by SA was significantly reduced
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263 in ubp12-2w/13-3 compared to WT (Figure 3E to 3G). These data are consistent with
264 the results of pathogen resistance (Figure 3D). In conclusion, our results suggested
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265 that although UBP12/UBP13 negatively regulate basal immunity, they act as positive
266 regulators in SA-induced immunity. These functions of UBP12/UBP13 are similar to
those of NPR3/NPR4 in plant immunity.
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269 To further examine the genetic relationship between UBP12/UBP13 and NPR3/NPR4,
270 we generated npr3-2/4-2;ubp12-2w/13-3 quadruple mutants and
271 npr3-2/4-2;UBP13-OE transgenic plants and examined their responses to pathogen
272 challenge. After pathogen infection, npr3-2/4-2;UBP13-OE showed decreased
273 bacterial growth relative to UBP13-OE, but similar to npr3-2/4-2, indicating that
274 UBP13-mediated repression of plant immunity depends on NPR3/NPR4 functions
275 (Figure 3H). On the other hand, npr3-2/4-2;ubp12-2w/13-3 showed more resistance to
276 Pst DC3000 compared to both npr3-2/4-2 and ubp12-2w/13-3 (Figure 3H). These
277 results suggested that UBP12/UBP13 regulate pathogen defence through
278 NPR3/NPR4-dependent and independent pathways. Moreover, we over-expressed
279 NPR3 in ubp12-2w/13-3 and compared the susceptibility to pathogen infection
280 between ubp12-2w/13-3;35S::NPR3-MYC and ubp12-2w/13-3. We found that NPR3
281 over-expression can repress the enhanced resistance of ubp12-2w/13-3 to Pst DC3000
282 (Figure 3I). Taken together, our genetic results showed UBP12/UBP13 suppress plant
283 immunity partially through NPR3/NPR4.
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285 Our results suggested that UBP12/UBP13 act as important regulators in SA signaling
286 pathway which led us to explore whether SA may regulate UBP12/UBP13 expression
287 and/or the stability of their encoded proteins. To this end, we examined the related
288 transcript and protein levels treated with or without SA. UBP12/UBP13 transcript
289 levels as well as UBP12/UBP13 protein abundance were not significantly altered by
290 SA treatment (Figure S5). These results suggested SA signal regulates the functions of
291 UBP12 and UBP13 through increasing their binding affinity to NPR3 (Figure 2C and
292 2D) but not their expression and stability.
293
294 Deubiquitination mediated by UBP12/UBP13 protects NPR3 from degradation.
295
296 UBP12/UBP13 have been implicated in a large number of growth and development
297 processes by protecting their target proteins from being degraded (An et al., 2018;

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298 Jeong et al., 2017; Lee et al., 2019; Liu et al., 2022; Luo et al., 2022; Park et al., 2019;

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299 Zhou et al., 2021). Here, we showed UBP12/UBP13 directly interact with
300 NPR3/NPR4 and regulate immune responses partially through NPP3/NPR4. These

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301 observations raised the possibility that UBP12/UBP13 are required to deubiquitinate
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NPR3 and prevent its degradation in response to pathogen infection. To test this
hypothesis, we examined NPR3 protein levels in WT, ubp12-2w/13-3,
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304 pUBQ10::UBP12-HA (UBP12-OE), and pUBQ10::UBP13-HA (UBP13-OE)
305 backgrounds. Under normal conditions without SA, NPR3 protein levels only slightly
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306 decreased in ubp12-2w/13-3 but increased in UBP12-OE and UBP13-OE (Figure 4A

307 and 4B). After SA treatment, NPR3 protein levels in WT were significantly induced
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308 but the induction by SA was largely blocked in ubp12-2w/13-3 (Figure 4A). On the
309 contrary, NPR3 levels in UBP12-OE and UBP13-OE were higher than those in WT in
the presence of SA (Figure 4B). By contrast, NPR3-MYC transcript levels from these
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311 different genotypes were not significantly changed (Figure S6). The important role of
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312 UBP13 in enhancing NPR3 stability was further confirmed by a time course
313 degradation analysis. In the presence of CHX and SA, NPR3 degradation in the
314 UBP13-OE background was slower relative to that in WT (Figure 4C and 4D, Figure
315 S7). In conclusion, UBP12/UBP13 act redundantly to increase NPR3 stability mainly
316 after SA perception, which is consistent with the SA-dependent interactions between
317 UBP12/UBP13 and NPR3 (Figure 2C and 2D).
318
319 The relatively low NPR3 levels in ubp12-2w/13-3 may be caused by high levels of
320 NPR3 polyubiquitination and consequent enhanced degradation by the 26S
321 proteasome. Therefore, we examined the effects of MG132 on NPR3 protein levels in
322 ubp12-2w/13-3. We found that NPR3 protein levels in ubp12-2w/13-3, especially in
323 the presence of SA, can be significantly increased after MG132 treatment (Figure 4E).
324 This observation indicates the instability of NPR3 in ubp12-2w/13-3 is a result of
325 increased degradation by the 26S proteasome. Moreover, we investigated the
326 ubiquitination status of NPR3 in WT and ubp12-2w/13-3 backgrounds. Consistent
327 with the reduced NPR3 levels in ubp12-2w/13-3, highly poly-ubiquitinated NPR3 was
328 observed in ubp12-2w/13-3 relative to WT (Figure 4F). Taken together, our data

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329 confirmed the notion that UBP12/UBP13 are involved in deubiquitinating NPR3 and
330 are required for maintaining NPR3 abundance.
331
332 Despite the important role of UBP12/UBP13 in regulating NPR3 stability in response
333 to SA signal, our data showed UBP12/UBP13 interact with NPR4 constitutively
334 which is different from NPR3. Therefore, we investigated whether NPR4 stability is
335 also regulated by UBP12/UBP13. We found that NPR4 protein levels in
336 ubp12-2w/13-3 were decreased with or without SA (Figure S8). Notably, NPR4-MYC
337 transcript levels in ubp12-2w/13-3 were not significantly altered compared to those in
338 WT (Figure S6). Consistent with the SA-independent association between NPR4 and
339 UBP12/UBP13 these observations suggested that UBP12/UBP13 are constantly
340 required to maintain NPR4 stability.
341

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342 Deubiquitination activity of UBP13 is required to prevent NPR3 destabilization.

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343
344 Previous studies showed that the deubiquitination catalytic activities of

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345 UBP12/UBP13 are critical for their roles in JA signaling, shade avoidance response,
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and circadian clock regulation (Jeong et al., 2017; Lee et al., 2019; Zhou et al., 2021).
To determine whether the deubiquitination activity of UBP13 is required for plant
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348 immunity as well, we complemented ubp12-2w/13-3 with wild type UBP13
349 (UBP13(WT)) and a catalytically inactive form of UBP13 (UBP13(C207S)), in which
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350 the conserved C207 was replaced with serine (Cui et al., 2013). We found that
351 UBP13(WT), but not the UBP13(C207S) mutant, can partially but significantly rescue
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352 the small rosette and early flowering phenotype of ubp12-2w/13-3 (Figure 5A).
353 UBP13(C207S)-HA protein from transgenic plant extracts displayed two bands,
which may be caused by a PTM of S207 (Figure 5B).
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355 ubp12-2w/13-3;UBP13(WT)-OE and ubp12-2w/13-3;UBP13(C207S)-OE#20
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356 transgenic plants with equivalent UBP13(WT) and UBP13(C207S) protein levels
357 were selected for analysis of their susceptibility to Pst DC3000. Figure 5C shows that
358 UBP13(WT) but not UBP13(C207S) can partially attenuate the enhanced immune
359 response of ubp12-2w/13-3 (figure 5C), indicating the requirement of UBP13
360 deubiquitination activity in repressing plant immunity.
361
362 To further confirm the direct regulatory role of UBP12/UBP13 in NPR3 stability we
363 examined whether the deubiquitination activity of UBP13 was required to protect
364 NPR3 from degradation. We expressed UBP12(WT), UBP13(WT) and
365 UBP13(C207S) in ubp12-2w/13-3;35S::NPR3-MYC background and compared
366 NPR3-MYC protein levels in various genotypes. We found that the compromised
367 SA-induced NPR3 accumulation in ubp12-2w/13-3 can be partially rescued by
368 UBP12(WT) or UBP13(WT) but not by the catalytically deficient form
369 UBP13(C207S) (Figure 5D and S9). These results are consistent with our genetic
370 analysis (Figure 5C), demonstrating that the deubiquitinating activity of UBP13 is
371 required to maintain NPR3 stability.
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374 UBP12/UBP13-mediated stabilization of NPR3 is required for NPR1 turnover.
375
376 NPR3/NPR4 have been shown to act as adaptors of CUL3 E3 ligase through their
377 BTB domain to mediate NPR1 degradation thereby activating systemic resistance
378 against pathogen infection (Fu et al., 2012; Wang et al., 2020). Therefore, we
379 investigated whether NPR1 protein levels were altered by UBP12/UBP13. Consistent
380 with the previous study (Fu et al., 2012), NPR1 protein levels were significantly
381 induced after SA treatment and were higher in npr3-2/4-2 compared to WT. In the
382 presence of SA, NPR1 protein levels were increased in ubp12-2w/13-3 but decreased
383 in UBP13-OE relative to WT (Figure S8). Moreover, NPR3 loss-of-function can
384 partially rescue the decreased NPR1 protein abundance in UBP13-OE (Figure S10)
385 suggesting that the regulation of NPR1 stability by UBP13 is partially dependent on

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386 NPR3. By contrast, NPR1 transcript levels in different genotypes were not

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387 significantly changed compared to WT. These results indicated
388 UBP12/UBP13-mediated stabilization of NPR3 is necessary for NPR1 turnover.

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390 Discussion
391
392 As a master regulator in SAR, the perception of SA and subsequent signal
393 transduction is critical for plant innate immunity (Fu and Dong, 2013). Plant cells
394 contain three SA receptors, NPR1 and its paralogs NPR3 and NPR4, which have
395 different binding affinity to the ligand (Ding et al., 2018; Fu et al., 2012; Manohar et
396 al., 2014; Wu et al., 2012). NPR1 and NPR4 show a higher whereas NPR3 exhibits a
397 lower affinity to SA, indicating that these receptors are differentially responsive and
398 may be regulated at different stages of immune response.
399
400 Previous studies showed NPR3 and NPR4 act as CUL3 E3 ligase adaptors to interact
401 with and determine the stability of NPR1 (Fu et al., 2012). The accessibility of NPR3
402 and NPR4 to their substrate NPR1 is regulated by SA. NPR3/NPR4-mediated NPR1

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403 turnover is required not only to prevent activation of resistance in the absence of

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404 pathogen challenge but also to establish SAR after pathogen challenge. However, this
405 hypothesis is challenged by a recent study, which proposes a completely different

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406 working model for NPR3/NPR4: NPR3 and NPR4 function independently from
407
408
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NPR1 in pathogen defence (Ding et al., 2018). In this hypothesis NPR3 and NPR4 act
as co-repressors of TGAs to inhibit defence gene expression in the absence of SA.
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409 During pathogen infection elevated cellular SA level represses the activities of
410 NPR3/NPR4 but enhances the activity of NPR1, resulting in induced defence gene
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411 expression. These two different working models for the function of NPR3/NPR4 in
412 plant immunity illustrate the complexity of SA signaling pathway.
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413
414 Here, we showed NPR3/NPR4 proteins are unstable and undergo
ubiquitin-proteasome mediated degradation which can be rescued by SA (Figure 1).
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415
416 UBP12 and UBP13, which are involved in many aspects of plant growth and
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417 development, have direct effects on the stability of these two NPRs. ubp12-2w/13-3
418 mutants showed enhanced basal immune response but compromised SA-induced
419 resistance (Figure 3A and 3D). By contrast, UBP12/UBP13 over-expressors exhibited
420 increased disease susceptibility (Figure 3A). The phenotypes of ubp12-2w/13-3 are
421 similar to those of the npr3npr4 double mutants. Our in depth analysis showed
422 UBP12/UBP13 regulate plant immunity partially through NPR3/NPR4 (Figure 3H).
423 Moreover, UBP12/UBP13 form a complex with NPR3/NPR4 in the nucleus.
424 UBP12/UBP13 constitutively interact with NPR4 (Figure 2A) but their association
425 with NPR3 is enhanced by SA (Figure 2A, 2C, and 2D). UBP12/UBP13-NPR3
426 complex formation in the presence of SA facilitates NPR3 deubiquitination, which
427 protects NPR3 from destruction by the 26S proteasome (Figure 4).
428
429 Taken together, our data and previous studies suggest NPR3 and NPR4 may execute
430 different roles at different stages of plant immunity (Figure. 6). Under normal
431 conditions without pathogen challenge, both NPR3 and NPR4 act as co-repressors of
432 TGAs to prevent the expression of resistance genes. The constitutive binding of NPR4
433 to UBP12/UBP13 enhances its stability and suppresses basal immunity. At the early

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434 stages of pathogen infection, newly biosynthesized SA binds to the NPR proteins to
435 activate NPR1 but restrain the activity of NPR3/NPR4, thereby unleashing defence
436 responses. During pathogen infection, increasing SA levels facilitate the association
437 between UBP12/UBP13 and NPR3 thereby stabilizing the latter. Since NPR3/4 would
438 mediate the turnover of NPR1, the stabilization of NPR3/NPR4 by the two DUBs
439 leads to full induction of resistance genes and establishment of SAR.
440
441 PTMs of signaling proteins is a crucial regulatory mechanism in eukaryotes to
442 facilitate rapid responses to changing cellular and environmental conditions. Such
443 modifications play an important role in regulating plant development as well as
444 pathogen resistance. A large number of PTMs have been reported to control NPR1
445 activity and stability; by contrast, the roles of PTMs in regulating NPR3/NPR4
446 functions are still unknown. Here, we showed that in plant immunity the

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447 ubiquitination-proteasomal pathway determines the stability of NPR3 and NPR4. We

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448 have identified UBP12/UBP13 as DUBs for NPR3/4 in plant immunity. Our results
449 here, as well as previous reports (Fu et al., 2012; Wang et al., 2020), show that NPR3

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450 and NPR4 play important roles in regulating NPR1 stability; however, more studies
451
452
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are needed to help us better understand the precise underlying mechanism. Fu et al.,
have shown that NPR3 and NPR4 interact with CUL3A in vitro and NPR3 and NPR4
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453 mediate NPR1 degradation through Cullin 3 E3 ligase (Fu et al., 2012). A recent study
454 showed ubiquitination of NPR1 is a progressive event in which the initial
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455 modification by a CUL3 E3 ligase promotes its chromatin association and expression
456 of target genes. The polyubiquitination of NPR1 can be subsequently enhanced by the
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457 E4 ligase, UBE4, which mediates proteasomal degradation of NPR1 (Skelly et al.,
458 2019). In view of these findings it is possible that NPR3/NPR4 may be involved in
CUL 3 and UBE4-mediated NPR1 activation and turnover. Future work should also
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459
460 be directed toward understanding how the two opposing activities are regulated and
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461 coordinated in plant immune responses. In the majority of cases, phosphorylation

462 precedes ubiquitination and subsequent degradation which is also true for NPR1
463 (Spoel and Dong, 2012). Furthermore, both NPR3 and NPR4 share very similar
464 amino acid sequences and structures to NPR1, which raises the reasonable hypothesis
465 that NPR3 and NPR4 may also undergo phosphorylation. Investigation of their
466 possible phosphorylation and identification of potential kinases and phosphatases are
467 important future tasks in order to gain a full understanding of the regulation of
468 NPR3/NPR4 activities.
469
470 Plant immunity is activated at the cost of growth suppression. Plants with
471 constitutively activated immune responses display arrested growth and reduced yield.
472 UBP12 and UBP13 are known to act as positive regulators in plant growth. They
473 promote BR signaling and shade-induced rapid growth through stabilizing BRI1,
474 BES1, and PIF7 (Luo et al., 2022; Park et al., 2022; Zhou et al., 2021). Here, we
475 provide evidence that UBP12 and UBP13 negatively regulate pathogen resistance via
476 protecting NPR3 and NPR4 from destruction. All theses studies suggest a crucial role
477 of these two DUBs in balancing growth and immunity. Our genetic analysis showed

13
478 npr3-2/4-2;ubp12-2w/13-3 quadruple mutants exhibit increased resistance to
479 pathogen infection than both npr3-2/4-2 and ubp12-2w/13-3 (Figure 3H). This result
480 suggests that in addition to NPR3 and NPR4, UBP12/UBP13 may regulate plant
481 immunity though other pathway. PTI is the first defense layer of plant innate
482 immunity. Many components in PTI signaling pathway, including FLS2 and BIK1,
483 can undergo poly- or mono- ubiquitination (Lu et al., 2011; Ma et al., 2020; Wang et
484 al., 2018). It is conceivable that UBP12 and UBP13 might be involved in determining
485 the turnover of these important signaling components as well.
486
487
488
489

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490
491 Materials and methods
492
493 Constructs generation. Full-length coding regions of NPR1, NPR3, NPR4,
494 NPR3-FLAG and YFP were amplified by PCR using specific primers listed in
495 Supplementary Table 1. The UBP13 (C207S)-HA, mNPR3 (NPR3R428Q) and mNPR4
496 (NPR4R419Q) mutants were generated as previously described (Cui et al., 2013; Ding et
497 al., 2018) by overlapping PCR. After purification, the fragments were cloned into the
498 pDONR/zero entry vector using a gateway cloning approach (Invitrogen). After
499 sequence verification, NPR entry vectors were cloned into pDEST-GADT7 for yeast
500 two-hybrid, pEG202-nYFP for BIFC analysis, and pBA-GWR-MYC for
501 35S::NPR3/4-MYC transgenic plants generation, respectively. UBP13 (C207S)-HA,
502 NPR3-FLAG and YFP were introduced into pUBQ10::GWR vector through LR

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503 reactions.

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504
505 Arabidopsis thaliana mutants and transgenic plants. All plants used in this study

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506 are of the Col ecotype. ubp12-1 (GABI_244E11), ubp13-3 (SALK_132368), ubp12-2w
507
508
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(GABI_742C10), and ubp12-2w/13-3 double mutants were described previously and
provided by Xiaofeng Cao (Cui et al., 2013). pUBQ10::UBP12-HA (UBP12-OE) and
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509 pUBQ10::UBP13-HA (UBP13-OE) were previously described (Jeong et al., 2017).
510 npr3-2 (SALK_043055), and npr4-2 (SALK_098460) were obtained from ABRC.
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511 npr3-2/npr4-2 double mutants was obtained by crossing npr3-2 with npr4-2.
512 Homozygous plants were selected by genotyping. 35S::NPR3-MYC and
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513 35S::NPR4-MYC transgenic plants were generated by transforming GV3101 strain of

514 Agrobacterium harboring pBA-NPR3-MYC or pBA-NPR4-MYC vector into WT (Col-0)
using floral dipping method (Zhang et al., 2006a). 35S::NPR3-MYC was introduced
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515
516 into ubp12-2w/13-3, UBP12-OE, and UBP13-OE backgrounds by crossing
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517 35S::NPR3-MYC transgenic plants with ubp12-2w/13-3, pUBQ10::UBP12-HA and

518 pUBQ10::UBP13-HA, respectively. ubp12-2w/13-3;35S::NPR4-MYC plants were
519 obtained by crossing ubp12-2w/13-3 with 35S::NPR4-MYC.
520 ubp12-2w/13-3;UBP13(WT)-OE, ubp12-2w/13-3;UBP13(C207S)-OE,
521 ubp12-2w/13-3;UBP13(WT)-OE;NPR3-MYC, and
522 ubp12-2w/13-3;UBP13(C207S)-OE;NPR3-MYC were obtained by transforming
523 pUBQ10::UBP13(WT)-HA or pUBQ10::UBP13(C207S)-HA into ubp12-2w/13-3, or
524 ubp12-2w/13-3;35S::NPR3-MYC background, respectively.
525 npr3-2/npr4-2;UBP13-OE was generated by crossing npr3-2/npr4-2 with
526 pUBQ10::UBP13-HA.
527
528 Pathogen infection. Four-week-old plants grown under short day conditions (12h
529 Light/ 12h Dark) were used to test for resistance to Pst DC3000 as previously
530 described (Liu et al., 2015). Overnight bacterial culture was resuspended in 10 mM
531 MgCl2 and adjusted to a final OD600 of 0.002. Leaves number 5 and 6 were inoculated
532 with the diluted Pst DC3000 by needleless syringe infiltration. After 3 days post
533 inoculation (dpi), leaf disc was punched from each infected leaf with a 1-hole paper

15
534 puncher and used to determine bacterial growth (Colony forming units per disc).
535 Three independent biological replicates were carried out. For each replicate, 8 plants
536 from each indicated genotype were used for analysis.
537
538 Immunoblotting. Ten-day-old seedlings grown under 22℃, long-day (LD, 16h L/8h
539 D) condition were treated with or without 0.4mM SA for 4h, if not specific mentioned.
540 Then the samples were collected and ground to fine powder in liquid nitrogen. After
541 homogenization in a protein extraction buffer (100 mM Tris-HCl pH 7.8, 4 M urea, 5%
542 SDS, 15% glycerol, 5 mM DTT, 1 mM PMSF, 1 mM protease inhibitor cocktail) the
543 homogenates were centrifuged twice at 14,000 rpm, 4℃, for 10 min each. 4 × SDS
544 sample buffer was added to the supernatant and the mix was incubated at 95℃ for 10
545 min (Zhou et al., 2017). Western blot analysis was performed and protein levels were
546 detected with the indicated antibodies. Tubulin (TUB) was used as an internal loading

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547 control.

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548
549 RNA extraction and Quantitative real-time PCR. Ten-day-old plants grown under

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550 22℃, LD conditions were treated with or without 0.4 mM SA for 2 hours before
551
552
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being collected for total RNA extraction using a Plant Total RNA extraction kit
(Transgen). cDNA was synthesis using a Bio-rad reverse transcriptase kit. Expression
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553 of immunity response genes were analyzed by quantitative real-time PCR using
554 SYBR Premix Ex Taq II (Bio-rad) with gene-specific primers described before and
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555 listed in supplementary Table 1 (Ding et al., 2018). Expression values were
556 normalized to the expression of ACTIN2 (ACT2). Each experiment was repeated 3
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557 times. Similar results were obtained and one set of representative results was shown.
558
Yeast two-hybrid assay. The coding regions of UBP12/13 were cloned into
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559
560 pDEST-GBKT7 (BD) and those of NPR1, NPR3, mNPR3, NPR4, mNPR4 were cloned
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561 into pDEST-GADT7 (AD) by a Gate-way method (Invitrogen). Cells of

562 Saccharomyces cerevisiae yeast strain Y2H Gold (Clontech) were co-transformed with
563 a combination of pGBKT7-UBP12, UBP13 and pGADT7-NPR1, NPR3, mNPR3,
564 NPR4, mNPR4, respectively, following the manufacturer’s transformation protocol
565 (Clontech). Interactions between UBP12/13 and NPRs were verified on selective
566 plates (SD-Leu/Trp/His + 0.5 mM 3-aminotriazole) with or without 0.1 mM SA.
567
568 Bimolecular fluorescence complementation (BiFC). GV3101 strain of
569 Agrobacterium harboring pEarley Gate201-NPR3/4-nYFP and
570 pEarley Gate202-UBP12/13-cYFP were incubated at 28°C overnight with shaking.
571 After centrifugation, the pellets were resuspended in MS liquid medium containing
572 10mM MES (PH5.7), 10 mM MgCl2 and 200 µM acetosyringone and adjusted to a
573 final OD600 of 0.6. Equal volumes of Agrobacterial re-suspensions harboring the
574 indicated constructs were mixed and incubated at room temperature for 3 hours before
575 infiltration. The NLS-mcherry was used as a nucleus marker. After 48 hours of
576 infiltration fluorescence was observed by an Olympus FV3000 confocal microscope.
577

16
578 Co-immunoprecipitation (Co-IP). 35S::NPR3-MYC/pUBQ10::UBP12-HA and
579 35S::NPR3-MYC/pUBQ10::UBP13-HA plants were used to detect possible in vivo
580 interaction between NPR3 and UBP12/UBP13. 35S::NPR3-MYC transgenic plant was
581 used as a negative control. Ten-day-old plants were treated with or without 0.4 mM
582 SA for 4 hours before being collected for subsequent analysis. Tissues were ground to
583 fine powder in liquid nitrogen and homogenized in IP buffer (50 mM Tris-HCl pH 7.5,
584 1 mM EDTA, 75 mM NaCl, 0.5% Triton X-100, 5% Glycerol, 5 mM DTT, 1 mM
585 protease inhibitor cocktail). After centrifugation twice at 14,000 rpm, 4℃ for 10 min
586 each, the supernatant was mixed with 50 μl anti-HA Affinity Matrix (SIGMA) and
587 incubated at 4°C overnight. The beads were washed 5 times with washing buffer (50
588 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Triton X-100) and bound proteins were
589 eluted and denatured by 2× SDS loading buffer at 95°C for 10 min. Immunoblots
590 were carried out to detect interactions using anti-HA and anti-MYC antibodies.

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591

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592 In vivo ubiquitination analysis. Ten-day-old WT (Col-0), 35S::NPR3-MYC, and
593 ubp12-2w/13-3;35S::NPR3-MYC plants were treated with or without 0.4 mM SA in

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594 the presence of 50 µM MG132 for 4h. After homogenized in IP buffer (50 mM
595
596
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Tris-HCl pH 7.5, 1 mM EDTA, 75 mM NaCl, 0.5% Triton X-100, 5% Glycerol, 5
mM DTT, 1 mM protease inhibitor cocktail) and centrifugation, the supernatants were
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597 incubated with anti-MYC magnetic beads (Thermo) at 4°C overnight. After washing 5
598 times with washing buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Triton
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599 X-100) ubiquitinated proteins were eluted with 2×SDS sample buffer at 95°C for 10
600 min. Ubiquitinated NPR3 proteins were detected using an anti-ubiquitin (Invitrogen)
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601 antibody.
602
Accession numbers. NPR1(AT1G64280), NPR3 (AT5G45110), NPR4 (AT4G19660),
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603
604 UBP12 (AT5G06600), UBP13 (AT3G11910), PR1 (AT2G14610), PR2 (AT3G57260),
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605 SARD1 (AT1G73805), WRKY70 (AT3G56400). Sequence information is given in

606 Arabidopsis Information Resource (http://www.arabidopsis.org/).
607
608

17
609 Author contributions
610 Y. Z. and N. -H. C. designed the experiments; Y. Z. and S. -H. P. performed the
611 experiments; Y. Z. and N. -H. C. analyzed the data and wrote the manuscript.
612
613 Acknowledgments
614 We are grateful to Dr. Xiaofeng Cao of the Institute of Genetics and Developmental
615 Biology, Chinese Academy of Sciences, China, for providing ubp12-2w, ubp13-3, and
616 ubp12-2w/13-3 double mutant.
617
618 This research was supported by the National Research Foundation (NRF), Prime
619 Minister’s Office, Singapore under its Campus for Research Excellence and
620 Technological Enterprise (CREATE) program. The Disruptive and Sustainable
621 Technologies for Agricultural Precision (DiSTAP) is an interdisciplinary research

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622 group (IRG) of the Singapore-MIT Alliance for Research and Technology Centre

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623 (SMART) supported by the National Research Foundation (NRF), Prime
624 Minister’s Office, Singapore under its Campus for Research Excellence and

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625 Technological Enterprise (CREATE) program.
626
627 Competing interests
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628 The authors declare no competing interests.
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736 coactivator NPR1. eLife 8, e47005.

737 Spoel, S.H., and Dong, X. (2012). How do plants achieve immunity? Defence
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738 without specialized immune cells. Nature Reviews Immunology 12, 89-100.
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742 Swatek, K.N., and Komander, D. (2016). Ubiquitin modifications. Cell research 26,
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750 Hinds, T.R., Guttman, M., et al. (2020). Structural basis of salicylic acid perception
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755 Xu, F.-Q., and Xue, H.-W. (2019). The ubiquitin-proteasome system in plant
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773 Zhou, Y., Park, S.-H., Soh, M.Y., and Chua, N.-H. (2021). Ubiquitin-specific
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778 Expression of Both PHYTOCHROME INTERACTING FACTORs and Auxin

779 Biosynthetic Genes. Plant physiology 176, 1850-1861.
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781 Figure Legends
782
783 Figure 1. SA promotes the stability of NPR3.
784 (A and B) Response of NPR3-MYC protein levels and NPR3-MYC transcript
785 abundance to SA treatment. Ten-day-old 35S::NPR3-MYC plants were treated with
786 indicated concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.6, and 0.8 mM) of SA for 4 hours.
787 Seedlings were then harvested for protein and RNA extraction. Tubulin (TUB) and
788 ACT2 were used as internal control for proteins and transcripts, respectively. In (B),
789 data shown are average values ± SD (n=3). Asterisks indicate significant differences
790 compared with that without SA (*p< 0.05, **p < 0.01, Student’s t test).
791 (C and D) NPR3-MYC protein levels and NPR3-MYC transcript abundance from
792 leaves after inoculation with Pst DC3000 for indicated times. The 5th and 6th leaves
793 from four-week-old 35S::NPR3-MYC plants grown under SD (12h L/12h D)

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794 condition were used for Pst DC3000 (OD600=0.002) infiltration. Leaves from 3

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795 individual plants were collected at each time point for protein and RNA extraction.
796 hpi (hours post inoculation). Tubulin (TUB) and ACT2 were used as internal control

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797 for proteins and transcripts, respectively. In (D), data shown are average values ± SD
798
799
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(n=3). Asterisks indicate significant differences relative to that inoculated with Pst
DC3000 at 0 h (*p< 0.05, **p < 0.01, Student’s t test).
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800 (E) Responses of NPR3 protein levels to proteasome inhibitor MG132. Ten-day-old
801 seedlings were treated with MG132 (50 µM) for 4h in the absence or presence of SA
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802 (0.4 mM) before being collected for immunoblot analysis. Tubulin (TUB) was used as
803 a loading control.
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804 (F and G) Time course degradation of NPR3 under indicated conditions. Ten-day-old
805 35S::NPR3-MYC seedlings were pre-treated with 0.4mM SA for 3h to increase NPR3
protein amount to a high level. After washing with ddH2O plants were transferred to
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806
807 1/2 MS liquid medium containing 200 µM cycloheximide (CHX). SA (0.4 mM),
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808 MG132 (50 µM) or a combination of both chemicals were added to the buffer.
809 Samples were collected at the indicated time points. Tubulin (TUB) was used as a
810 loading control.
811 For (A), (C), and (E)-(G), numbers below the lanes refer to relative protein levels.
812
813 Figure 2. NPR3 interacts with UBP12/UBP13 in an SA-dependent manner.
814 (A) Interactions between NPR1/NPR3/mNPR3/NPR4/mNPR4 and UBP12/UBP13 in
815 yeast two-hybrid assays with or without SA (0.1 mM). AD (pGADT7); BD (pGBKT7).
816 mNPR3 and mNPR4 indicate NPR3R428Q and NPR4R419Q, respectively. These two
817 sites, R428 in NPR3 and R419 in NPR4, which are critical for SA binding were
818 mutated to glutamine (Q).
819 (B) BIFC analysis of interactions between NPR3/NPR4 and UBP12/UBP13 in
820 Nicotiana benthamiana leaves. YN (nYFP); YC (cYFP). NLS-mcherry was used as a
821 nuclear marker. Scale bar = 40 µm.
822 (C and D) In vivo interactions between NPR3 and UBP12 (C) or UBP13 (D)
823 analyzed by co-immunoprecipitation. Ten-day-old plants were treated with or without
824 0.4 mM SA for 4h before being collected for co-immunopricipitation analysis. * in (C)

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825 indicates the NPR3-MYC bands.
826
827 Figure 3. Regulation of immune response by UBP12/UBP13 is dependent on
828 NPR3/NPR4 functions.
829 (A) Growth of Pst DC3000 on WT (Col-0), ubp12-1, ubp13-3, ubp12-2w/13-3,
830 UBP12-OE, and UBP13-OE, (n=8). Data shown are average values ± SD. Asterisks
831 indicate significant differences compared with wild type (*p< 0.05, **p < 0.01,
832 Student’s t test; ns, not significant (p ≥ 0.05)).
833 (B and C) Expression of defence genes PR1 (B), and PR2 (C) in WT (Col-0),
834 ubp12-2w, ubp12-2w/13-3, UBP12-OE, and UBP13-OE. Data shown are average
835 values ± SD (n=3). Asterisks indicate significant differences compared with wild type
836 (*p< 0.05, **p < 0.01, Student’s t test).
837 (D) Growth of Pst DC3000 on WT, ubp12-2w/13-3, UBP12-OE, and UBP13-OE

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838 (n=8), which were pre-treated with or without 0.5 mM SA for 1 day before

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839 inoculation. Data shown are average values ± SD. Asterisks indicate significant
840 differences of SA induced pathogen resistance in various genotypes compared to WT

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841 (*p< 0.05, **p < 0.01, Student’s t test).
842
843
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(E to G) Expression of defence genes PR1 (E), SARD1 (F), and WRKY70 (G) in WT
(Col-0) and ubp12-2w/13-3 treated with or without 0.2 mM SA for 2h. Data shown
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844 are average values ± SD (n=3). Numbers indicate SA-induced fold change of gene
845 expression.
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846 (H) Growth of Pst DC3000 on WT, npr3-3/4-2, ubp12-2w/13-3,

847 npr3-3/4-2;ubp12-2w/13-3, UBP13-OE, and npr3-2/4-2;UBP13-OE, (n=8). Data
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848 shown are average values ± SD. Asterisks indicate significant differences between the
849 indicated genotypes (*p< 0.05, **p < 0.01, Student’s t test).
(I) Growth of Pst DC3000 on WT, ubp12-2w/13-3, 35S::NPR3-MYC,
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850
851 ubp12-2w/13-3;35S::NPR3-MYC, (n=8). Data shown are average values ± SD. The
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852 asterisks indicate significant difference between the two indicated genotypes (*p<
853 0.05, **p < 0.01, Student’s t test).
854
855 Figure 4. UBP12/UBP13 protect NPR3 from degradation through
856 deubiquitinating NPR3.
857 (A) NPR3-MYC protein levels in WT and ubp12-2w/13-3 backgrounds after
858 treatment with or without 0.4 mM SA for 4 h. Anti-UBP12 and anti-MYC antibodies
859 were used to detect UBP12 and NPR3-MYC, respectively. The smaller fragment of
860 UBP12 protein in the ubp12-2w/13-3 background is the truncated form of UBP12.
861 Tubulin (TUB) was used as a loading control.
862 (B) Changes of NPR3-MYC protein levels in WT, UBP12-OE and UBP13-OE
863 backgrounds upon SA treatment. Anti-HA and anti-MYC antibodies were used to
864 detect UBP12-HA, UBP13-HA and NPR3-MYC, respectively.
865 (C) Time course degradation of NPR3 in WT or UBP13-OE background in the
866 presence of CHX and SA. Ten-day-old seedlings were pre-treated with 0.4mM SA for
867 3h to increase NPR3 protein levels. After washing with double distilled H2O plants
868 were incubated in 1/2 MS liquid medium containing 200 µM CHX in combination

24
869 with 0.4 mM SA. Samples were harvested at indicated time points to determine
870 NPR3-MYC levels. Tubulin (TUB) protein levels were used as a loading control.
871 (D) Quantitative results of relative NPR3-MYC levels as indicated in (C). Protein
872 levels were measured by Image J software. After normalization to TUB control,
873 relative NPR3-MYC levels were presented as average values ± SD (n=3). Asterisks
874 indicate significant differences compared with the 35S::NPR3-MYC (*p< 0.05, **p <
875 0.01, Student’s t test).
876 (E) NPR3-MYC levels in WT and ubp12-2w/13-3 background after treatment with 50
877 µM MG132 or a combination with 0.4 mM SA for 4 h.
878 (F) Poly-ubiquitination status of NPR3 in WT and ubp12-2w/13-3 background.
879 Ten-day-old seedlings were treated with or without 0.4 mM SA in the presence of 50
880 µM MG132 for 4h. Anti-MYC magnetic beads was used for immunoprecipitation of
881 NPR3-MYC proteins. An anti-Ubiquitin antibody was used to detect

f
882 poly-ubiquitinated NPR3.

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883
884 Figur 5. Deubiquitination activity of UBP13 is required for suppression of

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885 immune response.
886
887
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(A) Phenotype of 4-week-old WT, ubp12-2w/13-3, ubp12-2w/13-3;UBP13(WT)-OE,
and ubp12-2w/13-3;UBP13(C207S)-OE plants grown under 22℃, SD (12h light/12h
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888 dark) condition. Scale bar = 1 cm.
889 (B) UBP13 (WT/C207S)-HA protein levels in extracts from
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890 ubp12-2w/13-3;UBP13(WT)-OE and ubp12-2w/13-3;UBP13(C207S)-OE.

891 (C) Growth of Pst DC3000 on various genotypes indicated in (A). Data shown are
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892 average values ± SD (n=8). Asterisks indicate significant differences between the
893 indicated genotypes (*p< 0.05 and **p < 0.01; ns, not significant (p≥0.05); based on
Student’s t test).
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894
895 (D) NPR3-MYC levels in WT, ubp12-2w/13-3, ubp12-2w/13-3;UBP13(WT)-OE and
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896 ubp12-2w/13-3;UBP13(C207S)-OE backgrounds. Ten-day-old plants were treated

897 with or without 0.4mM SA for 4h.
898
899 Figure 6. A working model for the function of UBP12/13 in regulating plant
900 immunity.
901 (A) In the absence of pathogen challenge, UBP12/UBP13 interact with NPR4 to
902 maintain NPR4 protein at a basal level, which is necessary to block defence gene
903 expression and suppress immune response.
904 (B) After pathogen infection, as SA levels increase, the hormone binds to
905 NPR3/NPR4. SA binding promotes the association of UBP12/UBP13 with NPR3,
906 which protects the latter from being degraded. By contrast, UBP12/1UBP13
907 constitutively bind to NPR4 to enhance the stability of the latter. The stabilized
908 NPR3/NPR4 mediate the turnover of NPR1, which is essential for full induction of
909 defence gene expression and the establishment of SAR.

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