ABSTRACT
Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from two-week-old seedlings treated for only 30 minutes with flg22 contained nearly 1,000 conjugates, 150 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components, such as RECEPTOR-LIKE KINASE 1 (RKL1) shown to negatively regulate plant immunity. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified novel components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants.
One-sentence summary Proteome-wide catalogs of ubiquitylated proteins revealed a rapid engagement of the ubiquitin-proteasome system in Arabidopsis innate immunity.
INTRODUCTION
Post-translational modifications (PTMs), such as phosphorylation, methylation, acetylation, glycosylation, myristoylation, and ubiquitylation diversify protein behaviors, thus increasing the functionality and regulation of the proteome. As one of the most abundant PTMs, ubiquitylation is involved in nearly all physiological and signaling processes in plants, including hormone perception, photomorphogenesis, circadian rhythms, self-incompatibility, and defense against biotic and abiotic challenges (Guerra and Callis, 2012; Trujillo and Shirasu, 2010; Vierstra, 2009; Zhou and Zeng, 2017). Attachment of the 76-amino acid ubiquitin (Ub) moiety occurs most commonly to accessible lysines (K) within the target via an isopeptide bond that links to the carboxyl (C)-terminal glycine of Ub.
This conjugation is typically mediated through a stepwise enzymatic cascade consisting of a Ub-activating enzyme (UBA, or E1), a Ub-conjugating enzyme (UBC, or E2), and finally, a Ub-protein ligase (or E3), the latter of which has greatly expanded and diversified in plants to recognize a plethora of substrates. Additional Ubs can become attached to one or more of the seven lysine residues (K6, K11, K27, K29, K31, K48, and K63) or the amino (N)-terminal amide nitrogen within previously attached Ubs, resulting in a myriad of poly-Ub chain architectures that provide additional functional diversity (Komander and Rape, 2012; Vierstra, 2009). The most common internal linkage involves lysine 48 (K48) whose poly-Ub chains mainly target proteins for breakdown by the 26S proteasome, a multisubunit proteolytic complex that recognizes the Ub moieties (Vierstra, 2009; Zhou and Zeng, 2017). Although not as well understood in plants (Paez Valencia et al., 2016; Romero-Barrios and Vert, 2018; Zhou and Zeng, 2017), other types of poly-Ub linkages, involving K6, K11, K27, K29, K33, or K63, and monoubiquitylation in which only a single Ub is attached, have also been connected to many intracellular events, including protein trafficking, signal transduction, protein-protein interactions, and protein degradation through autophagy (Komander and Rape, 2012).
In recent years, a myriad of plant defenses against a range of pathogens has become apparent. The front line of defense is to block pathogen entry by erecting physical barriers, such as wax layers, cell walls, cuticular lipids, cutin, and callose, which can be strengthened during infection (Thordal-Christensen, 2003). As a second line of defense, plants rely on innate immune responses to fend off infections internally, which are elicited upon recognition of pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs) for pattern-triggered immunity (PTI), or pathogen-encoded effectors for effector-triggered immunity (ETI), which are encoded within the pathogen (Spoel and Dong, 2012; Zhou and Zhang, 2020). The host receptors known to detect these patterns/effectors are either receptor-like kinases (RLKs) or receptor-like proteins (RLPs) (Böhm et al., 2014; Couto and Zipfel, 2016). The best understood is the RLK receptor FLAGELLIN SENSING 2 (FLS2) and its co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) that recognize the bacterial 22-amino-acid flagellin epitope - flg22. Subsequently, PTI signaling is relayed through receptor-like cytoplasmic kinases (RLCKs), the mitogen-activated protein kinase (MAPK) cascade, and transcription factors, that trigger diverse immune responses, including the production of reactive oxygen species (ROS) and the stress hormone ethylene (Couto and Zipfel, 2016; Yu et al., 2017). Pathogen effectors are recognized directly or indirectly by NUCLEOTIDE-BINDING SITE LEUCINE-RICH REPEAT (NBS-LRR) proteins (NLRs), which often leads to localized cell death at the infection sites known as the hypersensitive response (HR) (Cui et al., 2015). The defense hormone salicylic acid (SA) is indispensable for NLR-triggered HR (Cui et al., 2015).
Protein ubiquitylation is also emerging as a dynamic process in both PTI and ETI signaling (Adams and Spoel, 2018; Cheng and Li, 2012; Zhou and Zeng, 2017). As examples, the MAMP receptor FLS2 is ubiquitylated upon ligand engagement by the E3s PLANT U-BOX 12 (PUB12) and PUB13, resulting in FLS2 degradation (Lu et al., 2011; Zhou et al., 2015), while PUB13 also directs the ubiquitylation and degradation of LYSIN MOTIF RECEPTOR KINASE 5 (LYK5), an RLK that recognizes the fungal cell wall polysaccharide chitin (Liao et al., 2017). The RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1), which provides a convergent signaling hub downstream of multiple MAMP receptors, is strongly regulated by Ub addition (Ma et al., 2020; Wang et al., 2018). Two U-box E3s, PUB25, and PUB26 polyubiquitylate BIK1 to control its steady-state levels, while the RING-type E3 ligases, RING-H2 FINGER A3A (RHA3A) and RHA3B, monoubiquitylate BIK1 to direct its endocytosis and signaling activation (Ma et al., 2020; Wang et al., 2018). Additionally, the PUB22, PUB23, and PUB24 E3s work collectively to negatively regulate PTI responses through modification of EXOCYST SUBUNIT EXO-70 FAMILY PROTEIN B2 (EXO70B2), which promotes vesicle trafficking (Stegmann et al., 2012). Levels of several NLR proteins, including RESISTANT TO PSEUDOMONAS SYRINGAE 2 (RPS2) and SUPPRESSOR OF npr1-1 CONSTITUTIVE 1 (SNC1), are also regulated by CONSTITUTIVE EXPRESSOR OF PR GENES 1 (CPR1), which is the target-recognition F-Box subunit within an SKP1-CULLIN1-F-BOX (SCF) E3 complex (Cheng et al., 2011; Gou et al., 2012). Despite these examples, a systematic investigation of ubiquitylation during plant immune activation is currently lacking.
Recent advance in proteomics has enabled high-throughput and comprehensive views of ubiquitylomes under specific physiological conditions. The first catalog was generated with yeast in which wild-type Ub was replaced by a 6His-tagged variant to enable enrichment of ubiquitylated proteins by nickel-nitrotrilotriacetic acid (Ni-NTA) affinity chromatography under denaturing conditions, followed by liquid chromatography-mass spectrometric analysis (LC-MS) of the trypsinized preparations (Peng et al., 2003). Sarraco et al. (2009) then adopted this strategy for plants through the creation of a transgenic Arabidopsis line that constitutively expresses a hexa-6His-Ub concatamer which is post-translationally processed into functional 6His-Ub monomers. Subsequently, Ub-binding domains were exploited for the additional enrichment needed for more complex proteomes (Maor et al., 2007). Further improvements included Tandem-repeated Ub-Binding Entities (TUBEs) in which natural Ub-binding domains were oligomerized to increase enrichment and yield (Hjerpe et al., 2009). By combining TUBEs with the Ni-NTA affinity chromatography, stringent purifications were possible, which enabled the confident detection of over 1,000 ubiquitylated proteins in Arabidopsis (Aguilar-Hernandez et al., 2017; Kim et al., 2013). Notably, a number of Ub targets associated with disease resistance were identified.
Here, we employed this improved two-step TUBEs purification approach coupled with deep MS analysis of Arabidopsis seedlings before and shortly after exposure to flg22. The resulting ubiquitylome catalog revealed extensive proteome-wide changes soon after MAMP perception and uncovered a large number of ubiquitylated candidates potentially connected to plant innate immunity. We further observed by in vivo ubiquitylation assays distinct ubiquitylation patterns and dynamics for several immunity components, including key regulators that were modified by multiple ubiquitylation events. One target was the previously uncharacterized receptor protein kinase RECEPTOR-LIKE KINASE 1 (RKL1) that appears to negatively impact plant immunity. Interestingly, several Ub-proteasome pathway components were also confirmed as Ub targets upon PTI immune elicitation, including the E2 UBC13 and proteasome subunit REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b). Taken together, our study identified novel connections between the Ub-proteasome system (UPS) and pathogen immunity as well as provided a valuable resource to further study this PTM during immune activation.
RESULTS
TUBEs enrichment of ubiquitylated Arabidopsis proteins upon flg22 treatment
To gain a global perspective of ubiquitylation during PTI elicitation, we employed a two-step TUBEs purification protocol to enrich for ubiquitylated proteins soon after exposure of two-week-old A. thaliana ecotype Col-0 seedlings to flg22 (Kim et al., 2013), which was based on a transgenic line harboring a synthetic UBQ gene encoding six 6His-Ub repeats expressed under the control of the Cauliflower mosaic virus (CaMV) 35S promoter (Sarraco et al., 2009). The polymer is processed by endogenous deubiquitylases (DUBs) to release tagged and fully functional Ub monomers (Isono and Nagel, 2014). In the first step, extracts from hexa-6His-UBQ seedlings either untreated or treated for only 30 min to flg22 were prepared in a non-denaturing buffer containing the non-ionic detergent Triton X-100 and a protease inhibitor cocktail to minimize DUB activities that would disassemble Ub conjugates post homogenization, and then enriched for Ub-bearing species by TUBEs chromatography (Hjerpe et al., 2009; Raasi et al., 2004), using as the ligand four copies of the Ub-Associated Domain (UBA) sequence from human hHR23A interconnected through a short flexible linker, and fused with GLUTATHIONE S-TRANSFERASE (GST) (Figure 1A). Previous studies show that this GST-4X(UBA) fusion binds effectively to native Ub-conjugates bearing various chain topologies from Arabidopsis, thus allowing us to interrogate its near complete ubiquitylome (Aguilar-Hernandez et al., 2017; Kim et al., 2013). In the second step, we enriched for poly-His-containing proteins via Ni-NTA chromatography under strong denaturing conditions using buffers containing 7 M guanidine-HCl and 8 M urea for the application and washing steps, respectively, followed by elution with a buffer containing 8 M urea and 400 mM imidazole (Figure 1B).
As seen in Figure 1C and D, eluants from the TUBEs and Ni-NTA columns prepared with control and flg22-treated hexa-6His-UBQ seedlings had substantially more ubiquitylated species than those obtained from wild-type seedlings, as detected by immunoblot analysis with rabbit anti-Ub antibodies. They appeared particularly enriched in high molecular mass Ub conjugates, with limited amounts of free Ub or the Ub dimer that were easily seen in total seedling extracts (Figure 1D). The main contaminant was RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (Rubisco), which is often recognized by antibodies generated in rabbits.
Identification of Ub-conjugates by LC-MS/MS
Ub-conjugates from the hexa-6HIS-UBQ plants were trypsinized, separated by reversed-phase nanoflow liquid chromatography (LC), and subjected to tandem MS (MS/MS) using a Velos LTQ mass spectrometer in the high energy collision mode (Figure 1B). Peptides were matched to the Arabidopsis ecotype Col-0 protein database (IPI database, version 3.85; http://www.arabidopsis.org) using SEQUEST, based on the identification of two or more different matching peptides with a ≤1% false discovery rate (FDR), or if a single matching peptide of equivalent stringency was identified that harbored a canonical Ub footprint (Kim et al., 2013). This footprint was detected by the presence of a di-Gly remnant (Gly-Gly) from Ub isopeptide linked to a lysine within the peptide, which increased the monoisotopic mass of the residue by 114 Da as well as protected the site from trypsin cleavage (Peng et al., 2003; Saracco et al., 2009). To help eliminate contaminants that bind non-specifically to the TUBEs and/or Ni-NTA columns, a background LC-MS/MS database was also generated with wild-type plants that underwent the same two-step affinity protocol; these proteins were subtracted from hexa-6HIS-UBQ datasets to obtain the final ubiquitylome catalog (Kim et al., 2013). In total, we generated MS/MS datasets from three biological replicates for control, untreated, and flg22-treated seedlings for final comparisons.
The summation of this MS analysis enabled the detection of 391 Ub conjugates in control samples (204, 210, 250 proteins for biological replicates 1, 2, 3, respectively) and 570 Ub conjugates in the flg22-treated samples (391, 400, 142 proteins for biological replicates 1, 2, 3, respectively) (Supplemental Table 1). Notably, 90 proteins in the control samples and 88 proteins in the flg22-treated samples were consistently identified in all three biological replicates, which likely represented a high abundance of ubiquitylated species. When combined, we uncovered 961 ubiquitylated proteins with high confidence; 690 were confined to either control or flg22-treated samples, whereas 271 proteins were present in both (Figure 2A). We detected 299 proteins unique to the flg22-treated samples versus 120 proteins unique to the control samples, implying that the PTI elicitation rapidly and markedly increases ubiquitylation in Arabidopsis (Figure 2A).
Gene ontology analyses of flg22-treated and -untreated ubiquitylomes
To gain a global perspective for how ubiquitylation is influenced by immune elicitation, we classified the identified targets in the control and flg22-treated ubiquitylomes based on their known or predicted functions within the Gene Ontology (GO) database using the DAVID Functional Annotation Tool (Huang da et al., 2009). As shown in Figure 2B, the total number of significantly enriched GO terms (FDR <0.05) was larger for the flg22-treated ubiquitylome than for that for the control for the three main GO domains (‘Biological Processes’: 104 vs. 82, ‘Molecular Functions’: 59 vs. 45, and ‘Cellular Components’ 74 vs. 62), and were noticeably more dedicated to pathogen-related events. Enrichments included the GO terms ‘defense response to fungus’, and ‘innate immune response’, which were found only in the flg22-treated ubiquitylome, while the related terms ‘defense response to bacterium’, ‘regulation of stomatal movement’, ‘defense response by callose deposition’, and ‘oxidation-reduction process’ were more significantly enriched (Figure 2B, C).
As compared to the total proteome, we also saw significant enrichment for GO terms associated with ‘Ub-dependent protein catabolic process’ (Biological process), and ‘Cul4-RING E3 Ub ligase’ (Cellular components) in both control and flg22-treated ubiquitylomes, as were the GO terms related to stress, such as ‘response to salt stress’ and ‘response to cold stress’ (Figure 2B). Strikingly, this enrichment in Ub-related events was more robust in the flg22-treated versus control ubiquitylomes (3.7-fold vs. 2.4-fold for ‘ubiquitin-dependent protein catabolic process’ and 5.5-fold vs. 3.2-fold for ‘Cul4-RING E3 Ub ligase’, respectively). A prevalence for UPS components in the flg22-treated samples was also evident for proteins associated with ‘proteasomes’ (11.4-fold vs. 9.2-fold, respectively; Figure 2C). Taken together, this non-targeted MS analysis directly connected rapid flg22-induced ubiquitylation to Arabidopsis proteins associated with immunity and the UPS itself.
Global ubiquitylation of immunity-related proteins
Among the identified Ub conjugates were many key immunity-related proteins (Table 1). To link these ubiquitylated species to immune signaling globally, we generated an interaction network using the Search Tool for Retrieval of Interacting Genes/Proteins (STRING) database (Szklarczyk et al., 2015), which revealed an interconnected web with defense-related proteins often situated at key hubs (Figure 3A, B). Examples included plasma membrane-resident leucine-rich repeat-RLKs (LRR-RLKs), such as RECEPTOR-LIKE KINASE 7 (RLK7) and SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1/EVR) that interact with multiple LRR-RLP receptors (Figure 3A, B). Whereas SOBIR1 serves as a common component in RLP-mediated plant immunity (Albert et al., 2015; Liebrand et al., 2014), RLK7 is a receptor for PAMP-INDUCED PEPTIDE 1 (PIP1), which acts as a damage-associated molecular pattern (DAMP) to amplify plant immune signaling (Hou et al., 2014). Additionally, LysM RLK1-INTERACTING KINASE 1 (LIK1), which was specific to the flg22-treated ubiquitylome, has been shown to interact with the fungal chitin receptor complex component CERK1 to positively regulate resistance against necrotrophic pathogens (Le et al., 2014).
Downstream of the immune receptor complexes are sequential phosphorylation events involving MAPK cascades driven by MAPK KINASE KINASES (MAPKK/MEKK), MAPK KINASES (MAPKK/MKK), and MAPKS (MPK) components (Meng and Zhang, 2013). Notably, MKK5, MPK3, and MPK11, which become phosphorylated upon flg22 perception, were detected here, with MKK5 and MPK3 only identified in the flg22-treated ubiquitylome (Figure 3B). Heterotrimeric G-proteins, consisting of Gα, Gβ, and Gγ subunits, act as molecular switches to mediate diverse signal transduction events (Urano et al., 2013). In particular, ARABIDOPSIS G PROTEIN β-SUBUNIT1 (AGB1), which associates with FLS2 and regulates BIK1 stability during plant PTI (Liang et al., 2016; Liu et al., 2013), was identified in the flg22-treated samples. Ubiquitylated forms of RECEPTOR FOR ACTIVATED C KINASE (RACK)-1B and RACK1C, which scaffolds and connects heterotrimeric G proteins with MAPK cascades to form a unique signaling pathway in plant immunity (Cheng et al., 2015), were identified in both control and flg22-treated ubiquitylomes, or only the flg22-treated ubiquitylome, respectively.
Protein trafficking, regulated by a cascade of factors such as small GTPases from Rab subfamilies, also plays important roles in plant immunity (Gu et al., 2017). RabE1D, identified here in the flg22-treated ubiquitylome, is associated with Golgi and the plasma membrane and might regulate the apoplast secretion of anti-microbial proteins, such as PATHOGENESIS-RELATED PROTEIN 1 (PR1) that induce resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 (Speth et al., 2009b). Likewise, the E3 KEEP ON GOING (KEG), which localizes to the trans-Golgi network/early endosome (TGN/EE) compartments and is essential for TGN/EE-mediated secretion of PR1 and papain-like Cys protease C14 (Gu and Innes, 2011) appears to be ubiquitylated by a flg22-induced event (Figure 3B).
ETI components were also identified as flg22-specific ubiquitylation substrates. Included were four uncharacterized NLR proteins (AT1G53350, AT1G63350, AT5G45240, and AT5G48770) along with ENHANCED DISEASE RESISTANCE 1 (EDS1), which is a nucleocytoplasmic lipase-like protein indispensable for multiple TOLL-INTERLEUKIN 1 RECEPTOR (TIR) domain NLR-mediated resistance events (Cui et al., 2015). Proteins implicated in long-distance resistance were identified in the flg22-treated ubiquitylome, such as the HEAT-repeat protein ILITHYIA (ILA) that encourages systemic acquired resistance (Monaghan et al., 2010). The peroxisome-localized glycol hydrolase PENETRATION2 (PEN2) was seen in all control and flg22-treated samples and thus considered to be a high abundance Ub target. PEN2, together with PEN1 and PEN3, actuate a cell wall-based defense against non-adapted pathogens by limiting pathogen entry (Figure 3B) (Clay et al., 2009; Collins et al., 2003; Fuchs et al., 2016). Similarly, the lignin biosynthetic enzymes, CINNAMYL ALCOHOL DEHYDROGENASE 4 (CAD4), identified in both control and flg22-treated ubiquitylomes, and CAD5 identified only in flg22-treated ubiquitylomes, likely protect against pathogen invasion by modifying the lignin content of cell walls (Tronchet et al., 2010). The callose synthase POWDERY MILDEW RESISTANT 4 (PMR4), which is required for wound- and/or pathogen-induced callose formation (Meyer et al., 2009; Wawrzynska et al., 2010), was also found in both datasets (Figure 3B). Taken together, our ubiquitylome profiles implicate Ub addition in multiple defense responses ranging from the preformed cell wall-based protection, to inducible PTI and ETI, and systemic defense (Figure 3B).
Distinct ubiquitylation patterns and dynamics of immunity-related proteins upon flg22 elicitation
To examine more closely the ubiquitylation dynamics (+/−flg22) of several immunity-related substrates, we exploited a previously established in vivo ubiquitylation system in which FLAG-tagged Ub (FLAG-Ub) is co-expressed with HA- or myc-tagged targets in wild-type Arabidopsis leaf protoplasts (Zhou et al., 2014). Ubiquitylated proteins are enriched by immunoprecipitation with anti-FLAG antibody beads and then probed by immunoblot analysis with anti-HA or anti-myc antibodies to assay for possible ubiquitylated species.
As shown in Figure 4A, we successfully confirmed the ubiquitylation of MPK3, MKK5, SOBIR1, and EDS1 using this in vivo system combined with HA or myc-tagged versions. For MPK3-HA, multiple ubiquitylated species were evident in anti-FLAG immunoprecipitates with a dominant form seen at ∼52 kDa. Being ∼8 kDa bigger than that of unmodified MPK3-HA (∼43.8 kDa), we consider it likely that it represents a monoubiquitylated form. Notably, ubiquitylation of MPK3-HA was transiently enhanced soon after flg22 treatment (10 min) but quickly returned to a basal level of modification at 30 min (Figure 4A), even though the levels of unmodified MPK3-HA remained steady for one hr of flg22 treatment (Figure 4A), suggesting that MPK3 ubiquitylation triggered by flg22 helps relay the flg22 signal. By contrast, modification of MKK5-myc, easily seen by a strong monoubiquitylated species, appeared to be constitutive, i.e., its level was unchanged by flg22 elicitation (Figure 4B). For both SOBIR1-HA and EDS1-HA, poly-ubiquitylated species accumulated in the in vivo system. While, the smear of high molecular mass conjugates for SOBIR1 was strongly enhanced upon flg22 treatment, they were diminished for EDS1 (Figure 4C and 4D). Taken together, we confirmed the ubiquitylation of candidate proteins using this cell-based assay, which revealed surprisingly distinct ubiquitylation patterns and dynamics for the candidates upon immune elicitation with flg22.
Ubiquitylation of RKL1 and its role in plant immunity
The LRR III protein RECEPTOR-LIKE KINASE 1 (RKL1), identified here as a flg22-triggered ubiquitylation substrate, was previously shown to modulate the activity of the aquaporin PLASMA MEMBRANE INTRINSIC PROTEIN (PIP) family during osmotic and oxidative stress together with its close homolog RLK902 (Bellati et al., 2016). Intriguingly, while RKL1 was not yet connected to pathogen defense, RLK902 had been shown to transmit an immune signal by phosphorylating the RLCK BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1) (Zhao et al., 2019). The RKL1 protein harbors a five-LEUCINE RICH REPEAT (LRR) extracellular domain, a transmembrane domain, and a cytosolic kinase domain (Figure 5A), and is mainly expressed in the stomata, hydathodes, and trichomes of young rosette leaves, and floral organ abscission zones based on transcriptomic analyses (Tarutani et al., 2004). When expressed in our leaf protoplast system with FLAG-Ub, we found that RKL1-HA was constitutively poly-ubiquitylated in vivo, despite our proteomic analyses of seedlings which found these species only in flg22-treated samples (Figure 5B).
To further characterize the role(s) of RKL1 in PTI, we assayed both MAMP-induced ROS production and bacteria disease spread, using a T-DNA insertion mutant compromising RKL1 function available from the SALK insertion collection (Alonso et al., 2003). The rkl1-1 mutant (SALK_099094) bears an insertion within the first exon (Tarutani et al., 2004), which was confirmed here by genomic PCR analysis using gene-specific and T-DNA border primers (Figure S1A). When compared to wild-type Arabidopsis Col-0 leaves, we found that homozygous rkl1-1 leaves displayed enhanced ROS production upon flg22 treatment based on a chemiluminescence assay quantifying luminol oxidation. We also conducted an assay for disease spread by infiltrating Arabidopsis leaves with the bacterial pathogen Pst DC3000 and then measuring bacterial numbers 2 days afterwards. As shown in Figure 5D, the rkl1-1 mutant displayed significantly less infection as compared to wild type, consistent with a robust defense (Figure 5D). Together, the data implicated for the first time that RKL1 is a negative regulator of plant immunity, which is possibly influenced by ubiquitylation.
Ubiquitylation of UPS components
As revealed above (Table 2; Figures 2 and 3), multiple UPS components were preferentially identified in flg22-treated ubiquitylome. Included were the E1 UBA1, the E2 isoforms UBC13, and UBC35 (UBC13A), the E3 components CULLIN 1 (CUL1), CUL4, and UB-PROTEIN LIGASE 5 (UPL5), components of the 26S proteasome, and the DUBs Ub-Specific Proteases (UBP)-13 and UBP24 and a predicted Ub-Carboxy-Terminal Hydrolase (UCH) (At4g24320) expected to release free Ub monomers from poly-Ub chains and poly-Ub translation products. UPL5 is part of the single polypeptide HECT E3 family. By contrast, CUL1, together with S-PHASE KINASE-ASSOCIATED PROTEIN 1 (SKP1), RING BOX 1 (RBX1), and one of possibly >700 F-box protein variants, assemble into SCF-type E3 complexes (Vierstra, 2009), while the CUL4 scaffolds DNA DAMAGE-BINDING (DDB)-type E3 complexes assembled with RBX1, and one of over 85 DWD box-containing DDB proteins (Vierstra, 2009). Previously, DDB1 was implicated in PR gene expression and resistance to Agrobacterial infection in tomato (Liu et al., 2012b), which might explain the connection between CUL4 and immune elicitation, while UPL5 is required for SA- and NONEXPRESSER OF PR GENES 1 (NPR1)-mediated plant immunity (Furniss et al., 2018c). Our discovery of the DUBs UBP13, UBP24, and a UCH in the flg22-treated ubiquitylome implies that the release of Ub from targets is also a regulatory step in immune elicitation.
A common fate for ubiquitylated proteins is turnover by the 26S proteasome. This multisubunit particle consists of a 20S cylinder-shaped core protease (CP) that houses the peptidase activities, which is capped at one or both ends by the 19S regulatory particle (RP) that assist in substrate recognition, poly-Ub chain removal, unfolding, and finally translocation of the unfolded protein into to the CP for digestion (Marshall and Vierstra, 2018; Zhou and Zeng, 2017). The CP is assembled from four stacked heptameric rings of related α (PAA-PAG) and β (PBA-PBG) subunits arranged in an αββα configuration. By contrast, RP is created by a ring of six RP ATPases (RPT1-RPT6) together with a collection of non-ATPase subunits (RPN1-3 and RPN5-14). The RPT ring provides the unfoldase activities that prepare substrates for CP import, RPN10, RPN13, and possible RPN14/SEM1 serves as Ub receptors, and RPN11 is one of the several proteasome-bound DUBs that help release the Ub moieties prior to proteolysis. Unexpectedly, we identified PAC1 (α3), PBB1 (β2), and PBE2 (β5) within the CP, RPT3, and RPT6b within the RP ATPase ring, and RPN2b, RPN3a, RPN6, and RPN8a/b in the RP cap, as part of the flg22-induced ubiquitylome (Figure 2B; Tables 1 and 2). Additional RPN subunits were identified (RPT1a, RPT4b, RPT5b, RPN1a, RPN7, and RPN11) in the ubiquitylome from untreated samples as well. While this extensive modification of proteasomes is consistent with its ubiquitylation helping remove damaged particles via autophagy more generally (Marshall et al., 2015), and possibly as a target of during pathogen attack more specifically (Ustin et al., 2017), the preferential and rapid modification of some subunits after flg22 treatment also suggested a direct host role for this modification during the immune defense. As the selective modifications of several proteasome subunits occurred within 30 min of flg22 treatment and long before any impact on 26S proteasome synthesis, we propose that they reflect early defense signaling events.
Flg22 induces UBC13 ubiquitylation and its role in plant immunity
We further connected the ubiquitylation target UBC13 (AT3G46460) to flg22 perception by studying its ubiquitylation profile and impact on pathogen defense. (It should be mentioned that UBC13 is unrelated to the UBC35 and UBC36 subfamily in Arabidopsis, which is unfortunately also named UBC13A (At1g78870) and UBC13B (At1g16890), respectively (Wen et al., 2008).) As shown in Figure 6A, we confirmed that UBC13 is ubiquitylated using a UBC13-HA variant co-expressed in Arabidopsis leaf protoplasts with FLAG-Ub. Only a monoubiquitylated form was evident in the immunoprecipitates from both untreated and flg22-treated samples; intriguingly, its abundance was slightly elevated upon flg22 exposure. Whether this addition was directed by autoubiquitylation or by a second factor (E3?) is not currently known.
To examine whether UBC13 influences immune signaling, we examined two T-DNA insertion mutants ubc13-1 (CS389049) and ubc13-2, (CS389056) available from the GABI-Kat collection (Kleinboelting et al., 2012). Both mutants were predicted to harbor a T-DNA sequence within the 6th exon of UBC13, which was confirmed by genomic PCR with gene-specific and T-DNA border primers (Figure S1B, C). When compared to wild-type Arabidopsis Col-0 leaves, homozygous ubc13-2 seedlings displayed enhanced ROS production upon flg22 treatment, based on the chemiluminescence assay, and were significantly more resistant to infection spread by the pathogen Pst DC3000 (Figure 5D). Confirmation that the ubc13 mutations were responsible was provided using complementation studies that attempted to rescue the ubc13-2 phenotypes with a transgene expressing UBC13-HA. Both ROS production and infectivity by the Pst DC3000 pathogen was returned to wild-type levels in homozygous UBC13-HA ubc13-2 plants (Figure 6B-D). Together, our results imply that UBC13 negatively regulates plant immunity possibly by increasing the ubiquitylation state of UBC13.
Flg22 induces polyubiquitylation of RPN8b to potentially impact plant immunity
Our discovery that RPN8 is rapidly ubiquitylated upon treating Arabidopsis with flg22 was particularly intriguing because of its role in RP assembly where it associates with the DUB RPN11 to form an RPN11-RPN8 hetero-dimeric subcomplex (Murata et al., 2009; Smalle and Vierstra, 2004). Two paralogs of RPN8 (RPN8a, and RPN8b) assemble into the 26S particle in Arabidopsis. While the RPNA8a isoform was more highly expressed based on mRNA levels and more commonly detected within the particle by MS (Book et al., 2010), only RPN8b was identified here in flg22-treated ubiquitylome, suggesting that its modification has a selective effect on 26S proteasome structure/function. As shown in Figure 7A, we confirmed the ubiquitylation of RPN8b by co-expressing an HA-tagged variant with FLAG-Ub in our Arabidopsis protoplast ubiquitylation system. Polyubiquitylated species of RPN8b-HA were evident in the immunoprecipitates from both untreated and flg22-treated samples, but their abundance was markedly enhanced upon flg22 treatment (Figure 7A).
To test whether RPN8b contributes to immune signaling, we analyzed two T-DNA insertion mutants available from the SALK insertion collection (rpn8b-1, SALK_128568 and rpn8b-2, SALK_023568). The rpn8b-1 allele was previously shown to harbor a T-DNA sequence within the 1st intron (Palm et al., 2019), whereas we found that the rpn8b-2 allele harbored a T-DNA sequence within the 2nd intron, both of which were confirmed here by genomic PCR with gene-specific and T-DNA border primers (Figure S1D). When compared to wild-type Arabidopsis Col-0 leaves, homozygous rpn8b-1 mutant leaves showed an increase in ROS production upon flg22 perception by the chemiluminescence assay, and enhanced disease resistance upon infiltrating leaves with Pst DC3000 (Figure 7B-D). Complementation studies then connected RPN8b to the phenotypes described here; transgenic plants expressing RPN8b-HA under the control of the CaMV 35S promoter in the rpn8b-1 background had their levels of flg22-induced ROS production and disease resistance restored to those in wild type (Figure 7B-D).
Collectively, the data implicated RPN8b as a negative regulator of plant immunity possibly through a process that enhances its ubiquitylation state. Whether the RPN8a isoform is similarly ubiquitylated and involved in disease resistance is not yet known. However, we note that suppression of ROS production in rpn8b-1 seedlings by introducing RPN8b-HA was even more striking for one of the rescued lines, suggesting that overexpression of RPN8b might provide a dominant effect possibly by swamping out the function(s) of RPN8a (Figure 7B-D).
Mapping ubiquitylation sites on Ub conjugates
One strategy to demonstrate the impact of ubiquitylation on protein function/stability is to identify the modified lysine within the target and then block this modification through lysine to arginine substitutions. Consequently, a catalog of such sites is valuable, which can be detected by MS identification of Ub footprints, i.e., trypsin-protected lysines bearing an isopeptide linked di-glycine moiety (+114 kDa) (see Figure 8A). Directed queries of all of our MS datasets identified 150 ubiquitylation sites from 120 Arabidopsis proteins (Table 3), which adds to the expanding list generated by other reports (Aguilar-Hernandez et al., 2017; Kim et al., 2013; Maor et al., 2007; Saracco et al., 2009). Notable footprints within disease-related proteins identified here, some of which were previously unmapped, included K668 from CUL4, K301 from RPT6b, K502 and K507 from ARGONAUTE 7 (AGO7), K422 and K434 within the NLR proteins At1g63350 and At5g48770 respectively, K286 from UCH family protein At4g24320, and K736 from PHOSPHATIDYLINOSITOL-4-PHOSPHATE 5-KINASE (PIP5K2) (Table 3; Supplemental Table 2). Our datasets broadened the number of known Ub-attachment sites, especially within Arabidopsis proteins involved in flg22-triggered immune responses. Interestingly, noncanonical ubiquitylation sites detected in yeast or mammals, such as those involving serine, threonine, or cysteine residues (Iwai and Tokunaga, 2009; Okumoto et al., 2011; Shimizu et al., 2010) were not identified in here. Their absence is consistent with the previous ubiquitylome studies (Kim et al., 2013) and implies that these linkages are rarely, if at all, used by plants.
Analysis of poly-Ub linkages
Owing to the widespread observations that poly-Ub chains of various linkages confer unique functions to Ub (Komander and Rape, 2012; Vierstra, 2009), we examined how flg22 treatment might affect the abundances of these polymers, using the diagnostic Ub footprint peptides for quantification based on peptide spectral matches (Peng et al., 2003). While footprints on each of the seven internal lysines (K6, K11, K27, K29, K33, K48, and K63) were identified (Figure 8B), none were seen using the N-terminal methionine, which would have signified linear Ub concatemers (Iwai and Tokunaga, 2009). K48-linked Ub-Ub connections were the most abundant and comprised ∼30% of the total detected linkages in both control and flg22-treated samples (Figure 8C). The second most abundant linkage involved K63 and was followed in abundance by those involving K11, K29, K6, K33, and K27 (Figure 8B). Interestingly, flg22 exposure altered the relative abundance of poly-Ub linkages; the percentages of K6, K11, K33, and K48-linkages increased, while the percentages of K27, K29, and K63-linkages decreased (Figure 8B). The biological relevance of such changes awaits clarification.
DISCUSSION
Protein ubiquitylation is involved in nearly all aspects of eukaryotic biology, leading to a multitude of distinct signals within a variety of cellular and physiological contexts. In line with prior studies with yeast and mammals (Bennett et al., 2007; Ordureau et al., 2020), we show here and elsewhere (Aguilar-Hernandez et al., 2017) that dynamic and multifaceted changes in the plant ubiquitylome also occur. By employing a genetically encoded hexa-6HIS-UBQ variant coupled with an improved two-step enrichment protocol for ubiquitylated proteins, followed by deep LC-MS/MS analysis, we found profound and complex protein ubiquitylation patterns upon immune elicitation in Arabidopsis.
We propose that the use of two affinity-purification steps, with the second under strongly denaturing conditions, are essential to generate reliable catalogs, which resulted here in the detection of 961 possible substrates along with 150 ubiquitylation sites mapped onto 120 individual proteins. Among the 10 candidate proteins that we subsequently tested by in vivo conjugation assays, all were confirmed as ubiquitylated targets in planta. While it is possible that the ubiquitylated species seen in protoplasts were artifacts derived from ectopic expression, the fact that different types of linkages were seen (mono-versus polyubiquitylation) and that both increased and decreased responses were seen soon after flg22 elicitation argue otherwise.
We also identified Ub-Ub linkages involving all seven lysine residues based on the discovery of diagnostic footprint peptides, with those involving K48, K63, and K11 being the most abundant. Whereas K48 polyubiquitylation has been linked to 26S proteasome-mediated turnover, and K63 ubiquitylation has been connected to the endocytic internalization of plasma membrane proteins (Romero-Barrios and Vert, 2018), the functions of the other poly-Ub linkages remain largely unknown in Arabidopsis even though the total abundance of those linkages exceeds 40% of the total. In addition, we saw an increased number of ubiquitylated proteins (391 of control vs. 570 of flg22-treated) upon immune elicitation. Although the datasets were not derived from quantitative proteomic approaches, a nearly 50% increase in the number of ubiquitylated proteins seen soon after immune elicitation, suggests a strong activation of the machinery that supports Ub transfer.
Notably, our flg22-induced datasets were obtained using seedlings treated for only 30 minutes, thus implicating ubiquitylation as an early step in immune signaling. Surprisingly, several resistance proteins known to be ubiquitylation such as FLS2, BRI1, and BIK1 were not identified here (Lu et al., 2011; Ma et al., 2020; Zhou et al., 2018), which might be explained by the nature of the tissues examined or the extraction method used, which possibly preferred cytoplasmic versus plasma membrane/organelle-associated proteins. Furthermore, we should also emphasize the TUBE affinity approach likely binds polyubiquitylated proteins better than monoubiquitylated proteins and thus reduced the latter in our catalogs. One strategy to avoid this complication is to enrich for Ub footprints directly from trypsin-digested cell lysates using an anti- K-ԑ-GG antibodies specific for the GG-remnant on ubiquitylated lysines (Udeshi et al., 2013). In fact, recent studies with such antibodies in rice discovered 1,376 possible ubiquitylated proteins and revealed strong changes in ubiquitylation after a three-hr exposure to flg22 or fungal chitin (Chen et al., 2018). The only complications were that designation of a protein as an Ub target was often based on a single Ub footprint peptide and the reliability of the anti-K-ԑ-GG antibodies. Clearly, further advancements in enrichment strategies and MS instrumentation should further expand the specificity, stringency, and depth of ubiquitylome during diverse physiological conditions.
Within the flg22-induced ubiquitylomes identified here, we confirmed several Ub targets by in vivo ubiquitylation assays in protoplasts. Surprisingly, we found distinct ubiquitylation patterns and dynamic temporal responses to flg22 treatment. For example, MKK5 and MPK3 were strongly monoubiquitylated while SOBIR1 and EDS1 were mainly polyubiquitylated. Ubiquitylation of RPN8b and MPK3 was increased by flg22, with MPK3 modification showing a transient effect. In contrast, ubiquitylation of EDS1 in the protoplasts was reduced in response to flg22 treatment. One of the more interesting substrates identified here was receptor protein kinase RKL1, which was polyubiquitylated, and found genetically to negatively regulate flg22-triggered ROS and immunity against bacterial Pst DC3000 infection. Intriguingly, RLK902, a close homolog of RKL1, was previously shown to positively regulate plant disease resistance (Zhao et al., 2019), suggesting that RKL1 and RLK902 counterbalance each other in immune signaling.
Ubiquitylation often intertwines with phosphorylation in regulating protein function (Hu and Sun, 2016; Reyes et al., 2011; Skubacz et al., 2016). A well-known example in plant disease resistance is BIK1, which is phosphorylated and subsequently monoubiquitylated upon flg22 perception (Ma et al., 2020). Here, we found that the phosphorylation (Asai et al., 2002) target MPK3 is also ubiquitylated dynamically, and like phosphorylation, this modification was enhanced ∼10 min after flg22 treatment, and then was reduced to basal levels after 30 min. The cognate E3 ligase for MPK3 is currently unknown; given that MPK3 phosphorylates the E3 ligase PUB22 and regulates its turnover (Furlan et al., 2017), it is possible that PUB22 directs MPK3 ubiquitylation thus creating an internal regulatory loop. As MPK3 remained unchanged despite the dynamics of its ubiquitylation after flg22 treatment, its ubiquitylation might not commit MPK3 to turnover but confer a reversible, non-proteolytic function. In line with some ubiquitylation events being reversible, we note the detection of several DUBs in our flg22-treated ubiquitylome datasets.
Pathway analysis of our ubiquitylome datasets upon MAMP perception revealed robust changes in the ubiquitylation status for numerous components involved in the UPS, including E1, E2, E3, and DUB family members. The in vivo ubiquitylation assays further confirmed that the E2 UBC13 in monoubiquitylated in vivo with the abundance of this modification increased upon flg22 treatment. While ubc13 mutants morphologically resembled WT plants, they displayed an enhanced flg22-induced ROS burst and increased resistance against bacterial infection. Several E3s have also been implicated in immunity, including CPR1 (Cheng et al., 2011; Gou et al., 2012; Gou et al., 2009), PUB12/13 (Liao et al., 2017; Lu et al., 2011), PUB25/26 (Wang et al., 2018), PUB22/23/24 (Stegmann et al., 2012), and RHA3A/B (Ma et al., 2020). Although it is generally accepted that E3s, not E2s, are the key determinants in substrate selection, it is possible that UBC13 preferentially works with those E3s involved in immunity.
Proteasome components, including both the core and regulatory particles, are enriched in our ubiquitylome analysis, which is consistent with the observation in yeast (Peng et al., 2003), and the known role of ubiquitylation in directing proteasome turnover through autophagy (Marshall et al., 2015). Strikingly, several of the ubiquitylated subunits were only found in the flg22-treated ubiquitylome, implying an active regulation of the proteasome machinery upon flg22 perception. Previous studies showed that several regulatory particle subunits, such as RPN1a, RPN8a, and RPT2a, but not others, play a positive role in immunity (Yao et al., 2012). Unexpectedly, we found RPN8b negatively regulates the flg22-induced ROS burst and disease resistance against Pst DC3000 infection. As Arabidopsis also encodes a second isoform of RPN8 (RPN8a) that appears more dominant in providing this subunit (Book et al., 2010), one intriguing possibility is that the RPN8b isoform becomes assembled to a unique subset of proteasomes with functions more directed toward immune perception. The immunoproteasome assembled with unique isoforms of the CP subunits β1, β2, and β5 could serve as an example of such non-redundancy (Finley, 2009). Clearly, further comparisons of mutants impacting both RPN8a and RPN8b with respect to pathogen resistance, along with assays testing the ubiquitylation status of each is now necessary to explore this notion.
Author Contributions
R.D.V., P.H., and L.S. conceived the project, designed experiments, and analyzed data. X.M., C.Z., D.K., and Y.H. performed experiments and analyzed data. X.M., C.Z., R.D.V., and L.S. wrote the manuscript with inputs from all co-authors.
Declaration of Interests
The authors declare no competing interests.
MATERIALS AND METHODS
Plant materials and growth conditions
Arabidopsis thaliana 6HIS-UBQ transgenic plants in the Col-0 background were described previously (Saracco et al., 2009). The rkl1-1 (SALK_099094), ubc13-1 (CS389049), ubc13-2 (CS389056), rpn8b-1 (SALK_128568), rpn8b-2 (SALK_023568) T-DNA insertion lines were obtained from ABRC. p35S::UBC13-HA transgenic plants in the ubc13-2 background and p35S::RPB8b-HA transgenic plants in the rpn8b-1 background were generated in this study (see below for details). All Arabidopsis plants were grown in soil (Metro Mix 366, Sunshine LP5 or Sunshine LC1) in a growth chamber at 20-23 °C, 50% relative humidity and 75 μE m− 2s−1 light with a 12-hr light/12-hr dark photoperiod for four weeks before pathogen infection assay, protoplast isolation, and ROS assays.
Plasmid construction and generation of transgenic plants
MPK3-HA, MKK5-MYC, and FLAG-UBQ in a plant gene expression vector pHBT used for protoplast assays were described previously (Asai et al., 2002; Lu et al., 2011). SOBIR1, RKL1, UBC13, RPN8b, and EDS1 tagged with HA in the pHBT vector were generated as following: the gene was PCR amplified from total Col-0 cDNA with primers containing BamHI (for SOBIR1, RKL1, UBC13, and RPN8b) or NcoI (for EDS1) at the 5’ end and StuI at the 3’ end, followed by BamHI/NcoI and StuI digestion and ligation into the pHBT vector with a sequence encoding a HA tag at the 3’ end. DNA fragments cloned into the pHBT vectors were confirmed as correct by Sanger sequencing. The UBC13-HA and RPN8b-HA transgenes were further shuttled into a pCB302 vector by insertion into BamHI and StuI digestion sites. The A. tumefaciens-mediated floral dip was used to transform the above binary vectors into ubc13-2 or rpn8b-1 plants. Transgenic plants were selected by glufosinate-ammonium (Basta, 50 μg/ml) resistance. Multiple transgenic lines were analyzed by immunoblotting for protein expression. Two lines with a 3:1 segregation ratio for Basta resistance in the T3 generation were selected to obtain homozygous seeds for further studies.
Affinity purification of ubiquitylated proteins
Affinity purification of ubiquitylated proteins was described (Kim et al., 2013) with modifications. Seeds of Arabidopsis Col-0 and 6HIS-UBQ transgenic plants were vernalized at 4 °C for three days and surface-sterilized with 75% ethanol for 10 min and 5% bleach for 10 min, and plated in 1/2-strength Murashige and Skoop (MS) medium containing 1% sucrose and 0.5% agar (100 seedlings per plate) at 23 °C, 75 μE m−2s−1 light with a 12-hr light/12-hr dark photoperiod for 14 days. Approximately 5000 seedlings (50 plates) were used for each biological replicate which generated ∼16-21 gram fresh weight of tissue. Seedlings were gently transferred into a Petri dish plate and incubated overnight in water. After removing the water by vacuum, seedlings were treated for 30 min with 100 nM flg22 or water as a control.
Seedlings were gently blot-dried on Kimwipes, frozen to liquid nitrogen temperatures, and homogenized in 0.5 g/mL of extraction buffer (EB; 50 mM Tris-HCl, pH 7.2, and 200 mM NaCl), containing 0.25% Triton X-100, 13 protease inhibitor cocktail (Roche, 1 tablet per 10 ml of EB), 2 mM phenylmethanesulfonyl fluoride, 10 mM 2-chloroacetamide, 10 mM sodium metasulfite, and 1 mM N-ethylmaleimide (freshly added). The homogenate was filtered through two layers of Miracloth and the supernatant was mixed with TUBEs beads as the ratio of 40 g of tissue to 1 mL of beads. After incubated for 6 hr at 4°C, the beads were collected in a 2 × 1-0cm chromatography column and washed three times with EB and three times with EB plus 1, 2, 4 M NaCl, and eluted in 10 mL of 7 M guanidine-HCl, 100 mM NaH2PO4, and 10 mM Tris-HCl (pH 8.0, 24 °C). The eluted conjugates were incubated with 500 µl of Ni-NTA beads for 12 hr at 4°C in the same buffer with the addition of 20 mM imidazole and 10 mM 2-chloroacetamide. The beads were collected and washed once in 6 M guanidine-HCl, 0.1% SDS, 100 mM NaH2PO4, and 10 mM Tris-HCl (pH 8.0), and once in urea buffer (UB; 8 M urea, 100 mM NaH2PO4, and 10 mM Tris-HCl (pH 8.0)) also containing 0.1% Triton X-100, twice in UB plus 20 mM imidazole, and three times in UB alone, and eluted by 1 ml of UB supplemented with 400 mM imidazole. The two-step purified conjugates were filtered by the Ultracel-10K filter (Millipore) and separated by SDS-PAGE followed by staining for protein with silver, or immunoblotting with anti-Ub (Sigma-Aldrich) or anti-His (Novagen) antibodies to detect Ub conjugates.
Mass spectrometry
Purified proteins (100 μl) or control samples were reduced with 10 mM DTT at room temperature for 1 hr, and alkylated in the dark in the presence of 30 mM 2-chloroacetamide at room temperature for a further 1 hr. Excess alkylating agent was quenched with 20 μL of 200 mM DTT for 10 min. Samples, ten-fold diluted with 25 mM ammonium bicarbonate, were digested for 12 hr at 37 °C with 2 mg of sequencing-grade trypsin (Promega), followed by a second incubation for 6 hr with an additional 2 mg of trypsin. Digestion products were desalted using a C18 solid-phase extraction pipette tip (SPEC PT C18, Varian), vacuum dried, and reconstituted in 10 μL of 0.1% formic acid and 5% acetonitrile in water for MS analysis.
Samples were analyzed by a nanoflow liquid chromatography system (nanoAcquity; Waters) combined with an electrospray ionization FT/ion-trap mass spectrophotometer (LTQ Orbitrap Velos; Thermo Fisher Scientific). The LC system employed a 100 × 365 -μm fused silica microcapillary column packed with 15 cm of 3-μm-diameter, 100-Å pore size, C18 beads (Magic C18; Bruker), with the emitter tip pulled to 2 mm using a laser puller (Sutter Instruments). Peptides were loaded onto the column for 30 min at a flow rate of 500 nL/ min and eluted over 120 min at a 200 nL/min flow rate with a gradient of 2% to 30% acetonitrile in 0.1% formic acid. MS spectra were acquired in the FT orbitrap between 300 and 1500 mass-to-charge ratios (m/z) at a resolution of 60 000, followed by 10 MS/MS HCD scans of the 10 highest intensity parent ions at 42% relative collision energy and 7500 resolution, with a mass range starting at 100 mass-to-charge ratios. Dynamic exclusion was enabled with a repeat value of two for 30 sec and an exclusion window for 120 sec.
MS data analysis
The MS/MS data were searched using SEQUEST version 1.2 (ThermoFisher Scientific) against the A. thaliana ecotype Col-0 protein database (IPI database, version 3.85, containing 39,677 entries available within the Arabidopsis Information Resource [TAIR], http://www.arabidopsis.org) databse. Masses for both precursor and fragment ions were treated as monoisotopic. Met oxidation (+15.994915 Da), Cys carbamidomethylation (+57.021464 Da), the di-Gly Ub footprint indicating Ub addition to a lysine residue (+114.043 Da) were set as variable modifications. The database search allowed for up to two missed trypsin cleavages, and the ion mass tolerances were set to 10 ppm for precursor and 0.1 Da for HCD fragments. The data were filtered using a 1% FDR, and a minimum of two peptide matches was required or at least one peptide if it included a GGK Ub footprint.
GO analysis was evaluated by DAVID Functional Annotation Tool (Huang da et al., 2009) using GO annotations in the TAIR GO database (http://www.arabidopsis.org/). Fold enrichments were calculated based on the frequency of proteins annotated to the term compared with their frequency in the proteome. The p-value combined with the FDR correction was used as criteria of significant enrichment for GO catalogs, whereas a p-value < 0.05 were considered to be enriched for GO terms. The GO annotation was classified based on the “biological processes,” “molecular functions,” and “cellular components” categories and visualized as a bubble plot by R Project 4.0.0 (https://www.r-project.org/) (Bonnot et al., 2019). Protein interaction networks were created by the STRING database version 11.0 (Szklarczyk et al., 2015), and visualized by Cytoscape version 3.8.0 (Shannon et al., 2003).
Detection of ROS production
Leaves from 4-5-week-old, soil-grown Arabidopsis plants were punched into 5-mm diameter discs. The discs were incubated in 100 μl of water with gentle shaking overnight, which was then replaced with 100 μl of reaction solution containing 50 μM luminol and 10 μg/ml horseradish peroxidase (Sigma-Aldrich) supplemented with or without 100 nM flg22. Luminescence was measured with a luminometer (GloMax®-Multi Detection System, Promega) with a setting of 1 min as the interval for 40 min. Detected values for ROS production were indicated as means of Relative Light Units (RLU).
Pathogen infection assays
Pseudomonas syringae pv. tomato (Pst) DC3000 was cultured for overnight at 28 °C in King’s B medium supplemented with rifamycin (50 μg/ml). Cells were collected by centrifugation at 3,500 rpm, washed, and re-suspended to the density of 5 × 105 cfu/ml in 10 mM MgCl2. Leaves from four-week-old Arabidopsis plants were hand-inoculated with bacterial suspension using a needleless syringe. To measure in planta bacterial growth, three to four samples, each containing two 6-mm diameter leaf discs, were homogenized in 100 μl of water, and plated in serial dilutions on medium containing 1% tryptone, 1% sucrose, 0.1% glutamic acid, 1.8% agar, and 25 μg/ml rifamycin. Plates were incubated at 28 °C and bacterial colony forming units (cfu) were counted after 2 days.
In vivo ubiquitylation assays
Protoplast isolation and transient expression assay were as described previously (Zhou et al., 2014). Protoplasts were freshly isolated from leaves of four-week-old Col-0 plants and transfected at 2 × 105 cell/ml (500 μL) with various combinations of plasmids harboring genes encoding FLAG-UBQ (25 μL at 2 µg DNA/µL), and the HA or MYC-tagged proteins of interested (25 μL at 2 µg DNA /µL). The protoplasts were incubated for overnight at room temperature followed by a 2-hr treatment with DMSO alone or 2 μM MG132 dissolved in DMSO, and then a 2-hr exposure to 100 nM flg22. After homogenization in 300 μL of Immunoprecipitation (IP) buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM Na2EDTA, 1% Triton X-100, 2 mM Na3VO4, 2 mM NaF, 1 mM DTT, and 1:100 diluted protease inhibitor cocktail (Sigma-Aldrich), the FLAG-tagged proteins were immunoprecipitated by incubation of the extracts with 5 μL of anti-FLAG antibody agarose beads (Sigma-Aldrich) for 1 hr at 4 °C with gentle shaking. The anti-FLAG antibody beads were collected and washed three times with IP buffer (without cocktail) and once with 50 mM Tris-HCl (pH 7.5), and then incubated in 30 μL of hot SDS-PAGE sample buffer for 5 min. Twenty μL of the sample before adding the beads was used as the input control. The samples were separated by SDS-PAGE followed by electrophoretic transfer onto Immun-Blotpolyvinylidene difluoride (PVDF) membranes (Bio-Rad), and immunoblotting with appropriate antibodies. Antibodies against plant Ub (van Nocker et al., 1996) and 6His (Kim et al., 2013) were as described. Anti-FLAG, anti HA, and, anti-myc antibodies were purchased from Sigma-Aldrich (A8592), Roche (12013819001), and Santa Cruz (sc-40), respectively.
Statistical analyses
Data for quantification analyses are presented as mean ± standard error of the mean (s.e.m.). The statistical analyses were performed by Student’s t-test or one-way analysis of variance (ANOVA) test (* p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001). The number of replicates is shown in the figure legends.
Acknowledgements
We thank the Arabidopsis Biological Resource Center (ABRC) for T-DNA insertion mutant seeds, and members of the laboratories of L.S., P.H. and R.D.V. for discussions and comments of the experiments. The work was supported by grants from the National Institutes of Health (NIH) (R01-GM092893) and National Science Foundation (NSF) (MCB-1906060) to P.H., NIH (R01-GM097247) and the Robert A. Welch Foundation (A-1795) grant to L.S., and a grant from the NIH (R01-GM124452) to R.D.V. C. Z. and Y.H. were partially supported by the China Scholarship Council (CSC).