Proteasomal Degradation of JAZ9 by Salt- and Drought-Induced Ring Finger 1 During Pathogen Infection

Pathogens suppress plant defense responses by promoting ubiquitination and degradation of target plant proteins during infection through the proteasome complex (Banfield 2015; Nomura et al. 2006; Singer et al. 2013; Üstün and Börnke 2014, 2015). During infection, pathogen effectors target the proteasome complex to hijack the defense response (Üstün et al. 2016). Bacteria-secreted type 3 effectors (T3Es) XopJ from Xanthomonas campestris pv. vesicatoria and HopZ4 from Pseudomonas syringae pv. lachrymans inhibit regulatory particle triphosphatase 6, a subunit of the 19S recognition particle of proteasome to suppress plant defense (Üstün and Börnke 2014, 2015). T3Es HopM1, HopAO1, HopA1, and HopG1 from P. syringae were identified as putative inhibitors of proteasomal activity (Üstün et al. 2016). Proteasomal degradation of proteins involves ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) (Ji et al. 2018). Really interesting new gene (RING)-finger proteins, involved in protein–protein interaction (Metzger et al. 2014) with E3 ligase catalytic activity, are activated by elicitor or avirulent factors and act as both positive and negative regulators of plant defense (Dreher and Callis 2007). The coronatine (COR)-insensitive protein 1 (COI1), a component of Skp1/Cullin/F-box (SCF^COI1) complex, mediates the ubiquitination and degradation of jasmonate zinc-finger inflorescence meristem (ZIM) domain (JAZ) proteins during jasmonate signaling (Delauré et al. 2008). JAZ proteins bind to different transcription factors and repress their activity in various developmental and pathogen infection conditions (Guo et al. 2018). The phytotoxin COR secreted by a pathogen modulates plant defense by acting as a structural mimic of jasmonic acid isoleucine (JA-Ile) (He et al. 2004).

We previously showed that overexpression of salt- and drought-induced ring finger 1 (SDIR1), a RING-type E3 ubiquitin ligase, in Arabidopsis, makes it hypersusceptible to hemibiotrophic pathogen P. syringae pv. tomato DC3000 and resistant to necrotrophic pathogen Erwinia carotovora (Ramu et al. 2021). In contrast, the Atsdir1 mutant plants exhibited resistance to P. syringae pv. tomato DC3000 and susceptibility to E. carotovora (Ramu et al. 2021). Overexpression of AtSDIR1 in Arabidopsis showed drought stress tolerance by interacting and partially degrading SDIR1-interacting protein 1 (SDIRIP1), which acts upstream of abscisic acid (ABA) signaling (Zhang et al. 2007; Zhang et al. 2015). SDIR1 destabilizes F-box proteins EIN3-binding F-box protein 1 (EBF1) and EBF2 to promote accumulation of EIN3 involved in ethylene responses and temperature stress (Hao et al. 2021). Similar to sdir1 mutants (Ramu et al. 2021), the coi1 ring domain ligase rglg3 and rglg4 mutants also showed pathogen resistance phenotype against hemibiotrophic bacterial pathogens (Xie et al. 1998; Zhang et al. 2012). The molecular interactors of SDIR1 during pathogen infection are not known. To establish the molecular mechanisms governed by SDIR1-mediated pathogen susceptibility, we identified several proteins that interact with SDIR1 using a yeast two-hybrid (Y2H) screen. We report that SDIR1 interacts with jasmonate ZIM-domain 9 (JAZ9) and degrades through a proteasome-mediated pathway during pathogen infection.

RESULTS

SDIR1 interacts with JAZ9 in the presence of COR or jasmonic acid.

The Arabidopsis AtSDIR1 overexpression plants are hypersusceptible to hemibiotrophic bacterial pathogens and resistant to necrotrophic bacterial pathogens (Ramu et al. 2021). To understand the molecular mechanisms of SDIR1-mediated disease susceptibility or resistance, a Y2H screening was performed using an Arabidopsis mixed elicitor-induced cDNA library (Lee et al. 2017) to identify SDIR1-interacting proteins. SDIR1 possesses N-terminal transmembrane (TM) domains (78 amino acids [aa]), a C-terminal RING domain, a metal-binding site, and a palmitoylation (PAL) site (Fig. 1). When full-length SDIR1 was used for the Y2H screen, we did not find any SDIR1-interacting plant proteins. We speculated that deletion of the RING domain would prevent the degradation of interacting proteins and, also, deleting the TM domain would help in identifying the interactors in the yeast system. The Y2H screening was performed with Δ^TMSDIRΔRING (79 to 210 aa) and identified several SDIR1-interacting proteins, including JAZ proteins (7, 18, 38, and 53 other clones) (Supplementary Table S1). Because JAZ proteins have been shown to play a role in defense against bacterial pathogens (Zhang et al. 2017), we further characterized them. Interestingly, the full-length JAZ9 protein did not interact with Δ^TMSDIR1, Δ^TMSDIRΔMetal, Δ^TMSDIRΔPAL, and Δ^TMSDIRΔRING proteins in minus leucine/tryptophan/histidine triple drop-out media (TDO) even though a partial clone of JAZ9 interacted with Δ^TMSDIRΔRING in the Y2H screening (Fig. 1B). We speculate that a structural difference in full-length and partial JAZ9 proteins could be contributing to this discrepancy. We anticipated that there may be a cofactor required for SDIR1 interaction with JAZ9. Melotto et al. (2008) previously reported that JAZ9 interacts with COI1 in Y2H when growth medium is externally supplied with cofactors COR or JA-Ile. External supplementation of COR, jasmonic acid (JA), or methyl jasmonate (MeJA) facilitated interaction of full-length JAZ9 and SDIR1 proteins and the 5-bromo-4-chloro-3-indolyl−β-d-galactoside (X-gal) staining confirmed these interactions (Fig. 1B). However, the interaction with MeJA was weak (Fig. 1B) and the interaction in the absence of cofactor was also found when proteasomal inhibitor MG132 was added to the yeast growth media (Fig. 1B). These results suggest that SDIR1–JAZ9 interaction could be occurring only in specific conditions when the pathogen secretes COR or when phytohormone JA accumulates in the plants.

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The interaction of SDIR1 with JAZ9 was further confirmed by pull-down assay. The AtSDIR1- glutathione-S-transferase (GST) protein (expressed and purified from Escherichia coli) was incubated with total protein from stable HA-JAZ9-expressing Arabidopsis plants (Lee et al. 2018). Hemagglutinin (HA) resin was used to pull down HA-JAZ9 and its interacting proteins. The SDIR1-GST pull-down proteins were detected using GST and HA tag antisera and the interaction of SDIR1 with JAZ9 was evident (Fig. 1C; Supplementary Fig. S1). These results demonstrate that SDIR1 interacts with JAZ9 both in vitro and in vivo.

To further validate in planta interaction of SDIR1 with JAZ9, we performed bimolecular fluorescence complementation (BiFC) assay in Nicotiana benthamiana. Full-length AtSDIR1 fused to the N-terminal half of the enhanced yellow fluorescent protein (eYFP) and AtJAZ9 fused to the C-terminal half of eYFP and was coexpressed in N. benthamiana. The reconstitution of yellow fluorescence suggests interaction of SDIR1-JAZ9 in endoplasmic reticulum (ER) or plasma membrane (PM) (Fig. 2A; Supplementary Fig. S2A). SDIR1 and JAZ9 are supposed to be localized to the ER membrane and nucleus, respectively (Withers et al. 2012; Zhang et al. 2007; Zhang et al. 2015). However, we did not see interaction in the nucleus. We anticipated interaction in the nucleus can happen if the TM domain is deleted in SDIR1. Surprisingly, upon deletion of the TM domain of SDIR1, interaction of nEYFP-Δ^TMSDIR1 and AtJAZ9-cEYFP was detected in the ER membrane in the background of ER-specific marker and in cytosol (Fig. 2A; Supplementary Fig. S2B). Pearson coefficient data clearly suggest the colocalization of interaction on PM or ER (Fig. 2B). As a negative control, we used the AtGCN4 protein (Kaundal et al. 2017) that showed no interaction with SDIR1 (Supplementary Fig. S2C).

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SDIR1 degrades JAZ9 via the proteasome-mediated pathway during pathogen infection.

The importance of the RING domain for E3 ligase activity of SDIR1 was shown previously (Zhang et al. 2007; Zhang et al. 2015). We speculated that JAZ9 is a target of SDIR1-mediated polyubiquitination and subsequent degradation. To confirm the polyubiquitination of JAZ9 before degradation by SDIR1, the full-length SDIR1 fused with GST was expressed in E. coli and purified. A truncated SDIR1 protein in which a C-terminus RING domain was deleted and fused with GST (Δ^TMSDIR1ΔRING-GST) was purified, to determine the role of this domain in polyubiquitination. C-terminal His-tagged JAZ9 was expressed and purified from E. coli. The ubiquitination activity was determined in the presence of ubiquitin-conjugating enzymes, wheat (Triticum aestivum) E1 and a human E2 (UBCh5b), purified GST-SDIR1 or GST-Δ^TMSDIR1ΔRING, and His-JAZ9-tagged proteins (Fig. 3A). Interestingly, the self-polyubiquitination of SDIR1 was detected by nickel–horseradish peroxidase (for detecting HA-ubiquitin) (Fig. 3A). In the absence of a RING domain, self-ubiquitination of SDIR1 was completely abolished due to loss of E3 ligase activity. These results are similar to previously reported assays on other ring finger proteins, which showed that the RING motif is essential for E3 ligase activity (Xi et al. 2002). The in vitro ubiquitination assay show an SDIR1-dependent ladder of ubiquitinated forms when JAZ9 is present (Fig. 3Aii). When probed with His antibodies, the JAZ9 also appeared to be ubiquitinated, as shown by the presence of high molecular weight JAZ9 protein (Fig. 3Aiii; Supplementary Fig. S3).

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To demonstrate the degradation of JAZ9 by SDIR1, GST-SDIR1 fusion protein was purified from E. coli. Arabidopsis transgenic plants expressing HA-JAZ9 were infected with host pathogen P. syringae pv. tomato DC3000 and, after 24 h postinoculation (hpi), total plant protein was extracted. Purified GST-SDIR1 and plant proteins were mixed and incubated at room temperature for 0, 1, 2, 3, and 4 h with or without MG132. The JAZ9 protein degradation started from 1 h and was completely degraded after 3 h of incubation (Fig. 3Bi). Proteasomal inhibitor MG132 was able to inhibit the HA-JAZ9 degradation by SDIR1, suggesting that JAZ9 is degraded via the 26S proteasome system during pathogen stress (Fig. 3Bi). In the absence of SDIR1, there was no degradation of JAZ9 (Fig. 3Bii). No degradation of JAZ9 was observed in SDIR1-treated samples in the absence of the pathogen (Fig. 3Biii; Supplementary Fig. S3). Therefore, both SDIR1 and pathogen infection are needed for degradation of JAZ9. These results suggest that SDIR1 degrades JAZ9 through a proteasome-mediated pathway.

DISCUSSION

The SDIR1 overexpression in Arabidopsis led to hypersusceptibility to hemibiotrophic pathogens and resistance to necrotrophic pathogens (Ramu et al. 2021). SDIRIP1, EBF1, and EBF2 were identified previously as targets of SDIR1 that acts upstream of ABA- and ethylene-signaling pathways (Hao et al. 2021; Zhang et al. 2007; Zhang et al. 2015). During pathogen infection, we found that SDIR1 interacts with many defense-responsive JAZ proteins. The interaction of SDIR1 with JAZ9 was observed in the presence of COR or JA. MeJA, JA-phenylalanine, 12-oxo-phytodienoic acid, and JA-Ile are known to provide active signals during pathogenesis (Melotto et al. 2008; Xin and He 2013). Similar to this scenario, the interaction of COI1 with JAZ1, JAZ3, and JAZ9 was observed only in the presence of COR and JA-Ile (Melotto et al. 2008; Thines et al. 2007). The interaction of JAZ9 with SDIR1 occurs at the PM and cytosolic fraction (Fig. 2). JAZ9 localization to the nucleus is compromised in the myc2 mutant; JAZ9 interaction with MYC2 represses the JA-dependent gene expression (Withers et al. 2012). Previously, in JAZ9 protein RKJas, two COI1-interacting residues were identified, and mutation in this region resulted in resistance to SCF^COI-mediated degradation (Browse 2009; Song et al. 2011; Withers et al. 2012).

The polyubiquitination of SDIR1 is enhanced in the presence of JAZ9 in the ubiquitination assay. The JAZ9 protein was completely degraded by SDIR1 after 3 h when coexpressed together in N. benthamiana, and proteasome inhibitor MG132 prevents the degradation. The hypersusceptibility of AtSDIR1 overexpression lines to hemibiotropohic pathogens and compromised nonhost resistance of jaz9 mutant (Ramu et al. 2021) could be attributed to the role of SDIR1 in JA or pathogen-mediated response to trigger the proteasome complex. JAZ proteins are known to be negative regulators of gene expression in JA-mediated responses (Withers et al. 2012; Zhang et al. 2015). The degradation of JAZ9 mediated by SDIR1 could be activating the MYC2 transcription factor and other proteins, leading to susceptibility against hemibiotrophic pathogen P. syringae pv. tomato DC3000. SDIR1 may also be involved in pathogen-associated molecular pattern-mediated plant defense, which depends on JA signaling components of the SCF complex such as Cullin 1, JAZs, COI1, and F-box proteins for modulating downstream transcription factors such as MYC2 (Browse 2009; Burgess et al. 2012). JAZ9 is known to act as a repressor for the MYB transcription factors under cold- and pathogen-stress conditions (Lv et al. 2017; Qi et al. 2011). The transcriptomic data from Arabidopsis SDIR1 overexpression plants showed higher transcript levels of MYB30 and MYB28 genes (Ramu et al. 2021). The higher levels of these genes could be due to removal of the repression activity of JAZ9 by SDIR1 through proteasome-mediated degradation. It is evident that JA, ABA, and ethylene signaling events are modulated by SDIR1 through JAZ, SDIRIP1, and EBF target proteins, respectively (Hao et al. 2021; Ramu et al. 2021; Zhang et al. 2015). Therefore, SDIR1 could be acting as a common regulator of phytohormone signaling in plants exposed to multiple environmental stresses.

MATERIALS AND METHODS

Y2H assays.

Y2H assays were performed using ProQuest Two-Hybrid System (Thermo Fisher Scientific). AtSDIR1 was fused to the GAL4 DNA-binding domain in pDEST32 as the bait construct. pEXP-AD502 was used to develop a cDNA library from mixed-elicitor-treated Arabidopsis for screening. AtJAZ9 was fused to the GAL4 activation domain in pDEST22 as prey protein using a Gateway cloning method. The TM (Δ^TMSDIR1), RING (Δ^TMSDIR1ΔRING), metal-binding (Δ^TMSDIR1ΔMetal), and PAL (Δ^TMSDIR1ΔPAL) domain-deleted constructs were expressed in pDEST32 vector. Bait and prey constructs were cotransformed into yeast MaV203 yeast-competent cells (Thermo Fisher Scientific). Positive clones were identified on TDO or minus leucine/tryptophan/histidine/uracil quadruple drop-out medium (QDO) synthetic-defined medium containing 20 mM 3-aminotriazole. The liquid media contained X-gal to detect interaction by development of blue color. During growth in TDO and QDO media, 1.5 and 60 μM COR, JA, and MeJA (Sigma-Aldrich, St. Louis) were included in the drop-out media. MG132 (10 μm), a proteasome degradation inhibitor, was added to the QDO.

BiFC assay.

AtSDIR1 and Δ^TMSDIR1 were fused to N-terminal eYFP in pSITE-nEYFP vector and AtGCN4 was used as negative control. The C-terminal part of eYFP in pSITE-cEYFP was fused to AtJAZ9 in pSITE-nEYFP (Kudla and Bock 2016). Agrobacterium strain GV3101 harboring these vectors along with PM or ER (RFP-PM/RFP-ER) markers were infiltrated to 3-week-old N. benthamiana leaves in 1:1 ratio at an optical density of 0.6 in morpholineethanesulfonic acid (MES) buffer. Sixteen hours prior to imaging, 10 µM MG132 was infiltrated into the leaf to avoid protein degradation. Three days after infiltration, RFP (red fluorescent protein) or YFP fluorescence was detected at 561 or 514 nm, respectively, under ×63 oil immersion using a confocal laser-scanning microscope (Leica-TCS SP5 AOBS and Lieca-SP8). To analyze the colocalization, more than 10 images from a minimum of three biological replications were processed for Pearson’s correlation coefficient analysis in Leica SP5 software (Dunn et al. 2011).

Pull-down assay.

For the pull-down assay, AtSDIR1 was expressed in pDEST15 with a GST tag, and N-terminal HA-JAZ9 was expressed in Arabidopsis stable lines. Leaf tissue from Arabidopsis Col-0 plants and HA-tagged JAZ9-overexpressing transgenic plants were homogenized in protein extraction buffer (50 mM Tris-HCL [pH 7.5], 75 mM NaCl, 0.2% Triton X-100, 5 mM EDTA, 5 mM EGTA, 1 mM dithiothreitol, 100 μM MG132, 10 mM NaF, 2 mM Na₂VO₄, and 1% protease inhibitor cocktail). AtSDIR1 was purified with GST resin (Thermo Fisher Scientific) and Pierce 660 nm of protein assay reagent (Thermo Fisher Scientific) was used for quantifying the protein content. The recombinant SDIR1 protein of 2.5 μg was added to 5 μg of total crude protein from HA-JAZ9-overexpressing Arabidopsis plants and the mix was incubated overnight at 4°C. The mixture was later incubated for 2 h at 4°C with Anti-HA Agarose conjugated resin (Thermo Fisher Scientific). Precipitated samples were resolved by sodium dodecyl sulfate (SDS) polyacrylamidegel electrophoresis by washing with Tris-buffered saline with 0.1% Tween 20, and dissolving in 2× SDS protein loading buffer. Immunoblot assays were performed with anti-GST or anti-HA (Abcam, Cambridge, MA, U.S.A.). The enhanced chemiluminescence system (GE Healthcare Bio-Sciences, Pittsburgh, PA, U.S.A.) was used for detection.

In vitro ubiquitination assay.

Crude extract containing recombinant wheat (T. aestivum) E1 (Sigma-Aldrich), E2 UBcH5b (Sigma-Aldrich), purified SDIR1 fused with GST tag, Δ^TMSDIR1ΔRING-GST, JAZ9-His tags, and HA-ubiquitin (Sigma-Aldrich) were used. Reactions were performed using the protocol described previously (Xi et al. 2002). The reaction was stopped by adding protein-loading buffer and boiling for 10 min. Later, proteins were transferred to the nitrocellulose membrane and the anti-HA antibody (Abcam) was used for detection.

Protein degradation and gel blot analyses.

AtSDIR1-GST was expressed in BL21 E. coli and HA-JAZ9 was expressed in transgenic Arabidopsis lines. SDIR1-GST was purified from E. coli. After 24 hpi in HA-JAZ9-expressing Arabidopsis with or without the host pathogen P. syringae pv. tomato DC3000, the leaf samples were ground in extraction buffer (50 mM Tris-MES [pH 7.5], 80 mM NaCl, 10 mM MgCl₂, 10% glycerol, 0.2% NP40, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail) (Sigma-Aldrich) for total protein and quantified using Pierce 660 nm protein assay reagent (Thermo Fisher Scientific). Equal concentrations were taken for the assay. Both SDIR1-GST- and HA-JAZ9-tagged protein samples were incubated for 0, 0.5, 1, 2, 3, and 4 h at room temperature. MG132 (1.6 μM) (Sigma-Aldrich) was added to one of the samples (incubated for 3 h) to inhibit the proteasome-mediated protein degradation. Later, proteins were blotted on nitrocellulose membrane, and GST antisera (Abcam) or HA antisera (Sigma-Aldrich) were used to detect the SDIR1 and JAZ9 protein levels. The GST and HA antisera were diluted to 1:10,000, followed by a secondary goat antirabbit antibody conjugated to horseradish peroxidase. The protein gel blots were visualized using ECL solution (GE Healthcare Bio-Sciences).

ACKNOWLEDGMENTS

K. S. Mysore acknowledges the Fulbright-Nehru Academic and Professional Excellence Award.