Homeostasis of Arabidopsis R protein RPS2 is negatively regulated by the RING-type E3 ligase MUSE16

Abstract

The homeostasis of resistance (R) proteins in plants must be tightly regulated to ensure precise activation of plant immune responses upon pathogen infection, while avoiding autoimmunity and growth defects when plants are uninfected. It is known that CPR1, an F-box protein in the SCF E3 complex, functions as a negative regulator of plant immunity through targeting the resistance (R) proteins SNC1 and RPS2 for degradation. However, whether these R proteins are also targeted by other E3 ligases is unclear. Here, we isolated Arabidopsis MUSE16, which encodes a RING-type E3 ligase, from a forward genetic screen and suggest that it is a negative regulator of plant immunity. Unlike CPR1, knocking out MUSE16 alone in Arabidopsis is not enough to result in defense-related dwarfism, since only RPS2 out of the tested R proteins accumulated in the muse16 mutants. Thus, our study identifies a novel E3 ligase involved in the degradation of nucleotide-binding and leucine-rich repeat (NLR) R proteins, support the idea that ubiquitin-mediated degradation is a fine-tuned mechanism for regulating the turnover of R proteins in plants, and that the same R protein can be targeted by different E3 ligases for regulation of its homeostasis.

Introduction

Higher plants depend on their sophisticated immune systems to survive in nature. Two major types of immune receptors are responsible for microbial pathogen recognition and activation of downstream defense responses. Pathogen-associated molecular patterns (PAMPs, also known as MAMPs or microbe-associated molecular patterns) are recognized by - pattern recognition receptors (PRRs) localized in the plasma membrane to activate PAMP-triggered immunity (PTI) (Bigeard et al., 2015). However, successful pathogens are able to break through this PTI defense layer by delivering specialized effectors (also termed as avirulence/Avr proteins) into the host cell, which often perturb an array of host macro-molecules involved in PTI in order to promote pathogen infection (Khan et al., 2018). In turn, plants have evolved Resistance (R) genes, which are usually found to cluster together, to ward off pathogens (van Wersch et al., 2019). These intracellular immune receptors recognize pathogenic effectors and trigger a robust immune response termed effector-triggered immunity (ETI). This often includes accumulation of the plant hormone salicylic acid (SA), induction of pathogenesis-related (PR) defense marker genes, production of reactive oxygen species (ROS), and a localized cell-death response, referred to as the hypersensitive response (HR) (Wu et al., 2014). Recent studies have revealed that there is crosstalk between the PTI and ETI pathways, including the activation of the toll/interleukin-1-receptor-like (TIR) signal, which is an important mechanism for boosting plant immunity during PTI, and enhancement of PTI, which is an indispensable component of ETI (Tian et al., 2021; Yuan et al., 2021). Activation of PTI or ETI alone is suggested to be insufficient for mounting effective resistance against bacterial pathogens, and the interplay between the two immune pathways serves as an additional necessary component for this process (Ngou et al., 2021).

ETI is often a much stronger response when compared with PTI, and R protein-mediated plant immunity has a central role in combating invasion by adaptive pathogens that are able to deliver effectors (Peng et al., 2018). The majority of R proteins belong to the nucleotide-binding and leucine-rich repeat (NLR) class, which can be further divided into two subclasses based on the presence of a TIR or a coiled-coil (CC) domain at the N terminus (Sukarta et al., 2016). TIR-type NLR (TNL) proteins in Arabidopsis are represented by RESISTANCE TO PSEUDOMONAS SYRINGAE 4 (RPS4) (Gassmann et al., 1999) and SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 (SNC1) (Zhang et al., 2003; Hyun et al., 2016). Both RPS4 and SNC1 can mediate the transduction of ETI resistance signals by homo- or heterodimerization (Zhang et al., 2017). In addition, RESISTANCE TO PSEUDOMONAS SYRINGAE 2 (RPS2) (Igari et al., 2008) and RESISTANCE TO PSEUDOMONAS SYRINGAE PV. MACULICOLA 1 (RPM1) (El Kasmi et al., 2017) are typical CC-type NLR (CNL) proteins in Arabidopsis. When transformed into Oryza sativa, these CNLs are auto-activated and confer broad-spectrum resistance to various pathogens (Li et al., 2019).

In the absence of pathogens, constitutive activation or excessive accumulation of NLR proteins can unnecessarily turn on immune responses, at a cost to the normal growth and development of the plant (Chakraborty et al., 2018). Examples include the autoimmune mutants snc1 (Zhang et al., 2003) and ssi4 (suppressor of salicylic acid insensitivity of npr1-5, 4) (Shirano et al., 2002), both of which exhibit a dwarf phenotype, which is suggestive of autoimmunity. Hence, it is critical to properly control the homeostasis of NLRs; however, the regulatory mechanisms that control the levels of these proteins are not well understood.

The 26S ubiquitin-proteasome system (UPS) is a post-translational modification and regulatory system for protein levels that is found used in eukaryotes (Dikic, 2017). It has been suggested to be involved in plant disease resistance (Copeland et al., 2019; Kud et al., 2019; Qin et al., 2019). Ubiquitin (Ub) is a small, highly conserved protein that can modify a target protein through covalent attachment (Heap et al., 2017). Tagging a target with ubiquitin can alter its subcellular localization, affect its activity, or promote its degradation by the UPS (Meyer-Schwesinger, 2019). The process is mainly catalysed by three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3) (Rittinger et al., 2017). E1 activates a free Ub molecule by ATP and transfers it to E2 (Olsen et al., 2013), and then E3 specifically recognizes the target protein and transfers the Ub to it via E2 to complete the tagging (Zheng et al., 2017).

E3 thus serves as a key enzyme in the UPS by determining the substrate specificity, due to its role in conjugating targeted proteins with Ub (Iconomou et al., 2016). Among the four major E3 families, RING E3 is the largest and contains a RING domain that is responsible interacting with the E2–Ub intermediate (Reiter et al., 2018). The RING domain is ~40–60 amino acids in length, rich in spatially conserved cysteine (Cys) and histidine (His) residues, and coordinates two zinc ions (Zn²⁺) to form a zinc-finger structure (Metzger et al., 2012). Previous studies have suggested that the coordination with zinc ions of the RING domain can be disrupted by a mutation on its Cys or His residue, which leads to a dominant negative effect and inhibits the activity of E3 (Peng et al., 2013). As a result, the self-ubiquitination activity of E3 is also removed (Sui et al., 2015), thus stabilizing the E3 and its substrate.

The Arabidopsis genome has two genes encoding E1s and 37 encoding E2s, while the number of genes encoding E3s is over 1400 (Kraft et al., 2005). Due to the genetic redundancy present among these E3-encoding genes, dissecting the phenotype that results from a single mutant can be challenging. Since ubiquitin-mediated protein degradation can induce rapid changes in intracellular protein levels in response to stimuli (Zhu et al., 2014), it can be used to regulate the stability of NLR proteins and many of their downstream components (Zhang et al., 2019). Therefore, these E3s might participate in the regulation of NLR-mediated resistance. Indeed, previous studies have shown that CONSTITUTIVE EXPRESSOR OF PR GENES 1 (CPR1), an F-box protein component of the E3 complex, is involved in regulating the protein levels of SNC1 and RPS2 (Cheng et al., 2011). In parallel, MUTANT, SNC1-ENHANCING 1 (MUSE1) and its paralogue MUSE2 function as independent E3 ligases, exhibiting overlapping functions in regulating the turnover of SIDEKICK SNC1 (SIKIC) TNLs (Dong et al., 2018).

Here, we report the isolation, characterization, positional cloning, and functional analysis of Arabidopsis MUSE16 from a forward genetic screen (Huang et al., 2013). MUSE16 encodes a novel E3 ligase with a typical RING domain, which functions as a negative regulator of plant immunity through targeting RPS2 for degradation.

Materials and methods

Plant cultivation and mutant screening

All Arabidopsis thaliana seeds used in this experiment were surface-sterilized prior to sowing and vernalized at 4 °C for 2 d. When sowing, a pipette was used to spread the seeds evenly on either a soil: vermiculite: limestone (4:1:1) mixture or on a half-strength MS plate. At 2 weeks after germination, the seedlings were transplanted into new pots. The growth conditions were 22 °C under a 16/8-h-light/dark cycle at 120 µmol m^–2 s^–1. Screening of muse mutants using ethyl methanesulfonate (EMS) was performed as previously described (Huang et al., 2013). The muse16-1 mutant was identified in the F₂ generation of a muse16-1 mos4 snc1 × Col-0 cross. When crossing muse16-2 individually with snc1, the transgenic plants snc1-GFP, RPS2-HA, and RPS4-HA, the corresponding muse16-2 snc1 double-mutant plants, and muse16-2 overexpressing snc1-GFP, RPS2-HA, and RPS4-HA lines were isolated from the F₂ generation by resistance and specific primers. The specific primers used for mutant genotyping are listed in Supplementary Table S1. All the original seeds of plant genotypes used in this study are obtained from the laboratory of Prof. Xin Li (University of British Columbia Michael Smith Laboratories).

Gene expression analysis

After 3 weeks of growth, 50-100 mg of plant leaf tissue was collected for total RNA extraction using an EASYspin plant RNA extraction kit (Biomed, cat. no. RA106-02). Samples of 0.5 μg of total RNA were reverse-transcribed to obtain cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, cat. no. RR047A). Expression of defense-related genes was normalized to ACTIN1 by real-time (RT-) PCR using TB Green^® Premix Ex Taq^™ II (TakaRa, cat. no. RR820A). For semi-quantitative PCR, the amplified product was subjected to agarose gel electrophoresis and staining with GoldView II (Solarbio, cat. no. G8142) before imaging. ACTIN1 was used as a loading control in each sample. The primers for gene expression analysis used in this study are listed in Supplementary Table S1.

Pathogen infections

The methods used for infection with Hyaloperonospora arabidopsidis (H.a.) Noco2 and Pseudomonas syringae pv. maculicola (P.s.m.) strain ES4326 were as previously described by Li et al. (2001). Briefly, for H.a. Noco2 infection, 10-day-old seedlings were sprayed with a conidiospore suspension at a concentration of 10⁵ spores 1 ml^–1 of water. The plants were then grown in chambers under a 12/12-h light/dark cycle for one week at 18 °C with 80% humidity and then the number of conidiospores per gram of tissue was counted using a hemocytometer. For virulent P.s.m. ES4326 infection, bacterial cultures diluted to OD₆₀₀=0. 005 with 10 mM MgCl₂ were used to infiltrate leaves of 4-week-old soil-grown plants. Leaf disks from the infected areas were taken at 0 d and 3 d after infiltration to quantify the bacterial colony-forming units (cfu) on LB plates with appropriate antibiotic selection. For infection by avirulent Pseudomonas syringae pv. tomato (P.s.t.) DC3000 strains carrying avrRpt2, avrRps4, avrRpm1 or avrPphB, respectively, the experimental method was the same as P.s.m. ES4326 infection, but the concentration of bacterial infection was OD₆₀₀=0.001.

Positional cloning and whole-genome sequencing

Positional cloning of muse16-1 was performed using a previously described method (Huang et al., 2013). The mapping markers used were designed according to polymorphisms between the genomic sequences of the Col and Ler ecotypes that are available on TAIR (http://www.Arabidopsis.org). Once the mutation site was localized to a small region (~1 Mb), the genomic DNA of the mutant plants from the mapping population were sequenced using Illumina whole-genome sequencing following the NEB Next DNA Library Prep Master Mix Set for Illumina protocol. After comparison with the Col-0 genomic reference sequence (http://www.Arabidopsis.org), the mutations found within the flanking region were further analysed.

Sanger sequencing

To confirm the presence of mutations, direct Sanger sequencing was used. First, PCR amplification of the target was followed by a clean-up step to remove excess primers. The PCR products were then used in a cycle-sequencing reaction to generate chain-terminated fragments, followed by another clean-up step to remove unincorporated ddNTPs. The fragments were then separated by capillary electrophoresis, and the sequence was read using data analysis software. After comparison between the sequencing results and the original sequence, the existence of mutations could be confirmed. The primers used are listed in Supplementary Table S1.

Bioinformatic analysis

After obtaining the amino acid sequence of an identified candidate protein, AT2G37150, from NCBI, its 3D simulation structure model was established using the Phyre2 online website (http://www.sbg.bio.ic.ac.uk/phyre2). Amino acid sequence alignment of the C3H2C3 domain from multiple species obtained from NCBI was rendered using the Genedoc software, and plotted using Adobe Photoshop.

Plasmid construction and Arabidopsis transformation

The coding sequence of the candidate gene AT2G37150 was PCR-amplified using the primers AT2G37150 CDS cloning-F and -R from Col-0 cDNA. After KpnI and SalI double-digestion, the amplified fragment was cloned into pCamBia1305-35S to generate pCamBia1305-35S::MUSE16. For transgene complementation, the AT2G37150 genomic sequence plus~1 kb regions both upstream of the start codon and downstream of the stop codon was amplified using the primers AT2G37150 genomic cloning-F and -R from Col-0 gDNA. The amplified product was then cloned into the modified vector pGreen0229-GFP by KpnI and XhoI double-digestion to generate pGreen0229-pMUSE16::MUSE16. The plasmids were then transformed into the appropriate Arabidopsis genotypes using the Agrobacterium-mediated floral dip method (Clough et al., 1998).

Four expression vectors in the reconstituted bacterial system for the ubiquitination activity test (pCDFDuet-AtUBA1-S, pCDFDuet-MBP-ABI3-HA-AtUBA1-S, pET-28a-FLAG-UBQ, pACYDuet-AIP2-Myc-UBC8-S) were obtained from the research team of Dr Dongping Lu (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). To study the ubiquitination activity of MUSE16 and its target protein, we modified pACYDuet-AIP2-Myc-UBC8-S and pCDFDuet-MBP-ABI3-HA-AtUBA1-S into pACYDuet-MUSE16-Myc-UBC8-S and pCDFDuet-MBP-RPS2-HA-AtUBA1-S. Briefly, we removed the original E3 ligase gene AIP2 and its target protein gene ABI3 in the original vectors by BamHI and StuI double-digestion, and replaced them with the amplified genes MUSE16 and RPS2. Using wild type cDNA as the template, MUSE16 was amplified using the primers MUSE16 CDS-F and -R, while RPS2 was amplified using the primers RPS2 CDS-F and R. The dominant negative point-mutation vector construction was based on the site-directed mutagenesis overlapping extension PCR method. Using the constructed plasmid pACYDuet-MUSE16-Myc-UBC8-S described above as a template, we mutated the conserved Cys530 to a Ser residue in the MUSE16 RING domain using the primers MUSE16 DN-F and R. Once treated with DpnI enzyme, the non-mutated (methylated) plasmid template was digested and the remaining product was transformed into DH5α cells. Finally, pACYDuet-MUSE16^C530S-Myc-UBC8-S was confirmed by Sanger sequencing.

For construction of transient expression vectors, MUSE16 was amplified by the primers MUSE16 TE-F and -R, while RPS2 was amplified by the primers RPS2 TE-F and -R using pACYDuet-MUSE16-Myc-UBC8-S and pCDFDuet-MBP-RPS2-HA-AtUBA1-S as the templates, respectively. Using HindIII to digest pEAQ-GFP and BamHI to digest pHB-FLAG, MUSE16 and RPS2 were cloned into the vectors to generate pEAQ-MUSE16-GFP and pHB-RPS2-FLAG, respectively.

For vector construction for yeast two-hybrid assays, again using pACYDuet-MUSE16-Myc-UBC8-S and pCDFDuet-MBP-RPS2-HA-AtUBA1-S as the templates, respectively, MUSE16 was amplified by the primers MUSE16 YH2-F and -R, while RPS2 was amplified by the primers RPS2 YH2-F and -R. Then, using NdeI to digest pGADT7 and SalI to digest pGBKT, MUSE16 and RPS2 were cloned into the vectors to generate pGADT7-MUSE16 and pGBKT7-RPS2, respectively.

All the primers used in this section are listed in Supplementary Table S1.

Total protein extraction and western blotting

Samples of 50-100 mg of plant leaves were placed in 2-ml Eppendorf tubes and ground into powder in liquid nitrogen. The samples were mixed thoroughly with 1.5× (v:w) protein extraction buffer (100 mM Tris-HCl, pH 8.0; 0.1% SDS, and 2% β-mercaptoethanol). After 5 min, the samples were centrifuged at 16 000 g, and then the supernatant was transferred to a new tube with 4× Laemmli loading buffer (60 mM Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.01% Bromophenol Blue). The solution was boiled at 95 °C for 5 min before loading onto an SDS-PAGE gel. Western blot analysis was performed afterwards using specific antibodies.

Ubiquitination assays in E. coli

Escherichia coli strain DE3 (BL21) containing different combinations of expression vectors was inoculated into 2 ml LB liquid medium together with the corresponding antibiotics and cultured at 37 °C until OD₆₀₀= 0.4-0.6. IPTG was added to a final concentration of 0.5 mM and the bacteria were further grown at 28 °C at a shaker speed of 250 rpm for 8–10 h to induce protein expression. The culture was then stored at 4 °C overnight for the ubiquitination reaction in E. coli. Finally, the bacteria solution was centrifuged at 900 g and the pellet was washed twice with 1× PBS. Ultrasound was used to break up the cells to release the proteins. The crude protein extracts were then mixed with 1× Laemmli loading buffer, boiled at 95 °C for 5 min, separated by SDS-PAGE, and then analysed by western blotting with the corresponding antibodies.

Transient expression in N. benthamiana for MG132 treatment and co-immunoprecipitation assays

Agrobacterium strains expressing pHB-RPS2-FLAG or pEAQ-MUSE16-GFP were cultured in LB medium at 28 °C for 18 h before being transferred into a new culture medium [10.5 g l^–1 K₂HPO4, 4.5 g l^–1 KH₂PO4, 1.0 g l^–1 (NH₄)₂SO₄, 0.5 g l^–1 sodium citrate, 1 mM MgSO4, 0.2% Glc, 0.5% glycerol, 50 µM acetosyringone, 10 mM MES, pH 5.6, and 50 µg ml^–1 kanamycin] at 1:50 dilution and incubated for another 10 h. The bacteria pellets were collected by centrifuging at 3000 g for 10 min and resuspended in MS buffer (4.4 g l^–1 MS medium, 10 mM MES, and 150 µM acetosyringone), and then one or two mixed strains were infiltrated into 4-week-old Nicotiana benthamiana leaves at a concentration of OD₆₀₀=0.5. Leaves expressing RPS2-FLAG alone and MUSE16-GFP/RPS2-FLAG were further treated with the proteasome inhibitor MG132 (50 μM, dissolved in DMSO) for 12 h before sample collection. At 3 d post-infiltration, total protein was extracted from the leaves using extraction buffer containing 20 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 300 mM NaCl, 0.5% NP-40, 5 mM dithiothreitol (DTT), 100 μM PMSF, and EDTA-free Protease Inhibitor Cocktail (Roche), and examined by western blotting with the corresponding antibodies.

For co-immunoprecipitation (co-IP) assays, transient expression in N. benthamiana and total protein extraction were performed as described above. Agrobacterium GV3101 strains containing the pEAQ-E3-GFP or pHB-RPS2-FLAG vectors were syringe-infiltrated into N. benthamiana leaves. The protein extracts were centrifuged at 4000 g at 4 °C for 15 min, and the supernatant was subjected to immunoprecipitation by adding 20 µl anti-GFP magnetic beads (Shanghai Epizyme Biomedical Technology Co., Ltd) and rotating with an oscillator for 4 h at 4 °C. The beads were then washed five times with TBST before 5× sample buffer was added to release the proteins. Finally, the samples were subjected to western blot analysis with anti-FLAG or anti-GFP antibodies (both HUABIO).

Yeast two-hybrid assays

Yeast two-hybrid (Y2H) assays were performed using the Matchmaker system (Clontech) with strain AH109. Co-transformants containing pGAD and pGBK were selected on plates lacking leucine and threonine (–LW). Single colonies were re-streaked on plates that further lacked histidine and adenine (–LWHA) to monitor reporter activation. Yeast growth was recorded after incubation at 30 °C for 2–5 d.

Results

The muse16-1 mutation enhances SNC1-mediated autoimmunity in the mos4 snc1 background

In Arabidopsis, the dominant mutant snc1 exhibits constitutive activation of plant defense responses, including elevated expression of defense marker genes and enhanced resistance against both bacterial and oomycete pathogens (Li et al., 2001). The modifier of snc1 4 (mos4) mutant, which was originally identified as a genetic suppressor of snc1, reverts snc1 to a wild-type-like phenotype in the snc1 background through affecting the proper splicing of SNC1 (Palma et al., 2007; Xu et al., 2012). Here, we isolated the triple mutant muse16-1 mos4 snc1 from a forward genetic screen of mutant, snc1-enhancing (muse) in the mos4 snc1 background, which was designed to isolate negative regulators of R protein-mediated immunity (Huang et al., 2013). As shown in Fig. 1A, muse16-1 mos4 snc1 reverted to a snc1-like morphology despite the presence of the mos4 allele, exhibiting more severe dwarfism than snc1. When the triple-mutant was backcrossed to mos4 snc1, wild-type-like morphology was observed in all F₁ progeny, suggesting that the muse16-1 mutation is recessive (Supplementary Fig. S1).

To confirm that other defense-related phenotypes were enhanced in the muse16-1 mos4 snc1 triple-mutant, the expression levels of the marker genes PR-1 and PR-2 were examined using RT-PCR. As shown in Fig. 1B, expression of both PR-1 and PR-2 was increased to snc1-like levels in the triple-mutant. The snc1-like enhanced resistance against the virulent pathogen H.a. Noco2 was consistently restored in the muse16-1 mos4 snc1 seedlings (Fig. 1C). Taken together, these results indicated that muse16-1 enhanced all snc1-associated defense phenotypes in the mos4 snc1 background.

Positional cloning of muse16-1

We used a positional cloning approach to identify muse16-1. The triple mutant muse16-1 mos4 snc1 (in the ecotype Col-0 background) was crossed with the Ler ecotype and the F₁ plants were allowed to self-fertilize to generate a segregating F₂ population. Using 24 F₂ plants with enhanced snc1 morphology as a crude mapping population, the muse16-1 mutation was mapped to the bottom arm of chromosome 2 through linkage analysis. The mutation was further determined to be flanked by the markers T16B12 and F18O19 using an additional 96 snc1-enhanced F₂ plants (Fig. 2A). To confirm the initial mapping and to further localize the mutation region, a larger fine-mapping population was generated using progeny from several F₂ lines that were heterozygous for the muse16-1 mutation but homozygous at both SNC1 (snc1) and MOS4 (mos4), aiming to avoid interference from those two loci. With 549 F₃ plants from the fine-mapping population, 61 recombinants were found between T16B12 and F18O19. The muse16-1 mutation was eventually found to lie between markers F13M22 and T6A23 and was located on BAC clone F16M14 (Fig. 2A).

To identify potential molecular lesions in muse16-1, Illumina whole-genome sequencing was performed on nuclear DNA isolated from muse16-1 mos4 snc1 plants. After comparison between the triple-mutant and the Arabidopsis wild-type reference genome sequences, several candidate genes with mutations were identified. Some of the mutations could be excluded as candidates after detailed sequence analysis, because they were either false-positives or silent mutations that did not cause amino acid changes (Supplementary Table S2). Using genomic DNA from muse16-1 mos4 snc1, direct Sanger sequencing confirmed the most likely single candidate gene mutation, which exhibited a G-to-A transition in the junction between 6th intron and 7th exon of AT2G37150 (Fig. 2B; Supplementary Fig. S2). To further test the effect of the point-mutation, Sanger sequencing using the cDNA from muse16-1 mos4 snc1 and wild-type plants was carried out and confirmed an aberrant splicing pattern of AT2G37150 in muse16-1, resulting in the deletion of the first G nucleotide in the 7th exon (Fig. 2C). As a consequence of the shift in the reading frame, an early stop codon could occur, which truncated the predicted RING domain of the protein (Fig. 2D).

Through further bioinformatic analysis, it was found that the protein encoded by AT2G37150 belongs to the RING E3 family, more specifically, to the C3H2C3 type (Supplementary Fig. S3). A 3D simulation of the structure of this protein was obtained using Phyre² and it showed that the C3H2C3 domain was located at the end of the peptide chain (carbon end; Supplementary Fig. S4), and consisted of a mosaic of three Cys, two His, and three Cys conserved amino acid sequences. A BLAST analysis (http://blast.ncbi.nlm.nih.gov/) showed that this sequence was homologous in various dicotyledons including Brassica napus, Raphanus sativus, Tarenaya hassleriana, Juglans regia, and Prunus persica (Supplementary Fig. S5).

MUSE16 is identified as AT2G37150

To test whether the mutation found in AT2G37150 was responsible for the muse16 mutant phenotypes, a full-length genomic clone of AT2G37150 under the control of its native promoter was transformed into muse16-1 mos4 snc1 plants. In the T₁ generation, six independent transgenic plants exhibited a similar phenotype to mos4 snc1 (Fig. 3A). Two representative lines were selected for further examination and, consistent with the morphological phenotypes, they were both able to complement the enhanced expression levels of the PR genes, and elevated resistance of muse16-1 mos4 snc1 against the virulent pathogen H.a. Noco2 (Fig. 3B, C). Taken together, these results indicated that MUSE16 is AT2G37150, and it encodes a putative RING-type E3.

MUSE16 encodes a RING-type E3 ligase with auto-ubiquitination activity

In vitro ubiquitination experiments are often used to study modifications in plant protein ubiquitination, and can detect the biochemical functions of putative E3 ligases (O’Connor et al., 2015). Traditional ubiquitination assays require the purification of each recombinant protein from prokaryotic expression cells and then the maintenance of the activity of the proteins before mixing and incubating them together to allow the ubiquitination to occur (Peralta et al., 2013; Neklesa et al., 2017). However, the purification procedure is not always easy, especially for proteins with low solubility or low expression levels. Synthetic biological methods can help solve this problem by reconstituting the ubiquitination cascade in E. coli (Han et al., 2017).

It has been demonstrated that RING-type E3s can undergo auto-ubiquitination when their specific substrates are absent. Therefore, we tested the auto-ubiquitination activity of MUSE16 to determine its functionality in a reconstituted bacterial system (Han et al., 2017) (Fig. 4A). The results showed that FLAG-UBQ10 and MUSE16-Myc displayed a laddering pattern when all the ubiquitination components were present, as detected with anti-FLAG and anti-Myc antibodies, respectively (Fig. 4B). This suggested that MUSE16 undergoes auto-ubiquitination. As predicted, the absence of any one of the individual components resulted in an inability to achieve such laddering.

Because the RING domain contains cysteines for chelating two zinc atoms, mutagenesis of these cysteine residues in an E3 can result in the loss of its function. To further confirm the occurrence of MUSE16 auto-ubiquitination in the bacterial system, we also assayed an E3 mutant version in which the Cys230 residue of the RING domain was replaced with a Ser residue (C230S). As shown in Fig. 4B, the auto-ubiquitination activity in the mutant C230S was abolished, indicating that the cysteine residue at position 230 is critical for the E3 activity of MUSE16.

MUSE16 plays a negative role in plant basal defense

Our results for muse16-1 mos4 snc1 implied that MUSE16 might play a negative role in the plant defense response. To determine the general role of MUSE16 in plant immunity, we generated the muse16-1 single-mutant by crossing the triple-mutant with the wild type, followed by allele-specific genotyping in the F₂ population. We also obtained MUSE16-overexpressing lines by Agrobacterium-mediated transformation (Supplementary Fig. S6A, B). The SALK_021643 T-DNA insertion allele of MUSE16, renamed as muse16-2, was obtained from ABRC (https://abrc.osu.edu/), and it carries a T-DNA insertion in the 11th exon of AT2G37150 (Fig. 2B). It was also utilized for characterization of the single-mutant. Even though both the muse16-1 and muse16-2 mutants showed significantly higher relative expression levels of PR-1 and PR-2 (Fig. 5A) and enhanced disease resistance against P.s.m. ES4326 and H.a. Noco2 (Fig. 5B, C) compared to Col-0, they appeared to resemble the wild-type plants in morphology (Fig. 5D), indicating that the increased disease resistance associated with the MUSE16 mutation had not reached or exceeded the level to exhibit a defense-related dwarfism phenotype. Consistent with this, transgenic plants overexpressing MUSE16 showed increased disease susceptibility against P.s.m. ES4326 compared to Col-0, but they were not as susceptible as eds1-2 mutants which blocks TNL signaling because of lacking the nucleocytoplasmic lipase-like protein termed as ENHANCED DISEASE SUSCEPTIBILITY 1 (Supplementary Fig. S6C). Taken together, these results indicated that MUSE16 functions as a negative regulator involved in plant basal defense.

Constitutive defense responses in snc1 are promoted by muse16-2

Because it acts as a ubiquitin E3 ligase that is negatively involved in plant immune responses, it can be predicted that MUSE16 probably exercises its function by targeting some positive components of immunity for degradation. Since muse16 was originally found in the mos4 snc1 background, we first tested the genetic relationship between SNC1 and MUSE16. The muse16-2 snc1 double-mutant was obtained by crossing muse16-2 with snc1 and identifying the homologous plant in the F₂ generation using gene-specific primers. This showed that muse16-2 enhanced the defense-related dwarfism in the snc1 background (Fig. 6A, B). Consistent with its morphology, the expression levels of PR-1/PR-2 and consequent disease resistance against H.a. Noco2 in muse16-2 snc1 were much higher than in snc1 (Fig. 6C, D). These results therefore all suggested that the absence of MUSE16 promoted the SNC1-mediated immunity.

According to our characterization of muse16-2 snc1, two hypotheses could be proposed. First, that MUSE16 acts directly on SNC1, and the accumulation of SNC1 contributes to enhanced immunity; and second, that MUSE16 acts on immune components other than SNC1, and this is superimposed on the influence of snc1, causing plant immunity to be enhanced. Since the morphological phenotype of muse16 was exactly like that of the wild type, and it is different from cpr1 harboring snc1-like dwarfism (Cheng et al., 2011), the first hypothesis is probably invalid. To verify whether MUSE16 affects the stability of the TIR-type SNC1, we crossed muse16-2 into previously constructed snc1-GFP transgenic plants (Cheng et al., 2009). As expected, the levels of SNC1 did not change (Fig. 6E), suggesting that it is not targeted by MUSE16 for degradation.

MUSE16 directly targets RPS2 for degradation

In order to determine whether other R proteins could be the targets of MUSE16, and to explore the mechanism by which MUSE16 is involved in plant immune responses, we infected muse16-2 and transgenic MUSE16-overexpressing plants with pathogens containing different Avr proteins. Unlike P.s.m. ES4326 used to test basal defense, P.s.t. DC3000 strains carrying different Avr proteins can be used to detect the immune response mediated by plant R protein corresponding to its Avr protein. Compared with Col-0, muse16-2 had an increased resistance while MUSE16-overexpressing plants showed enhanced susceptibility to P.s.t. DC3000 avrRpt2 (Fig. 7A), and both exhibited similar resistance levels to P.s.t. DC3000 avrRps4, avrRpm1, and avrPphB (Supplementary S6D–F), ­indicating that MUSE16 could function as a negative regulator involved in the RPS2-mediated defense response. Therefore, we hypothesized that MUSE16 might target RPS2 to stimulate its ubiquitination, and hence mediate its degradation through the UPS pathway.

We designed the following experiments to test this hypothesis. First, muse16-2 was crossed with either RPS2-HA (Axtell et al., 2003) or RPS4-HA (Wirthmueller et al., 2007) transgenic plants and the hybrid homozygotes were obtained in the F₂ generations. These homozygotes did not have obvious differences from the parental plants in terms of morphology (Fig. 7B), although the amount of RPS2, but not of RPS4, in the muse16-2 background was clearly increased (Fig. 7C), indicating that MUSE16 might be involved in the protein degradation process of RPS2. This genetic association between MUSE16 and RPS2 prompted us to test whether MUSE16 targets RPS2 for degradation through the UPS pathway. An in vitro ubiquitination system was used to test whether RPS2 could be ubiquitinated by MUSE16 (Fig. 8A), and we found that RPS2 indeed displayed a laddering pattern (Fig. 8B), suggestive of the ubiquitinated form of RPS2 being mediated by MUSE16.

A transient expression system in leaves of N. benthamiana was then employed to further test whether RPS2 degradation mediated by MUSE16 was dependent on the 26S proteasome, and whether MUSE16 directly interacted with RPS2 in planta. As expected, RPS2 accumulation decreased when it was co-expressed with MUSE16, and this reduction could be inhibited by treatment with the proteasome inhibitor MG132 (Fig. 8C). Furthermore, the interaction between MUSE16 and RPS2 was confirmed by both Co-IP (Fig. 8D) and Y2H assays (Supplementary Fig. S7). Taken together, these results indicated that MUSE16, as an E3 ligase, functions negatively in plant immunity through directly targeting RPS2 for degradation via the UPS pathway.

Discussion

The complex signaling network of plant innate immunity relies on the harmonious effects of positive and negative regulation. Given that auto-activation of immune responses leads to the inhibition of plant growth and development, negative regulation mechanisms of disease resistance are particularly important. Control of R protein homeostasis is critical for plant health. Excessive accumulation or activation of R proteins might cause the immune response to activate constitutively, leading to defense-related dwarfism (Chakraborty et al., 2018). Given the need to balance disease resistance and with growth and development, it is a critical to uncover the negative regulatory mechanisms to which R proteins are subjected. The purpose of the muse screen that we carried out was to find negative regulators of plant immunity (Huang et al., 2013). The advantage of this screen was that all the triple mutants had the genetic background of mos4 snc1 or mos2 npr1 snc1, so that any change in any negative regulator associated with plant immunity would be magnified in the growth phenotypes. In contrast, the morphological phenotypes of the muse single-mutants might not be evident due to the presence of genetic redundancy or complementary network regulation, and this may have caused these single-mutants to be easily overlooked in previous genetic screening.

The muse16 single-mutant that we identified from the screening had all of these characteristics. From our morphological observations, the absence or overexpression of MUSE16 seemed to have no effect on normal plant growth and development (Fig. 5D; Supplementary Fig. S6A), but clues for its negative role could be identified through more detailed analyses (Fig. 5A–C; Supplementary Fig. S6C). AT2G37150, mutation of which was responsible for the muse16 mutant phenotypes, has previously been predicted to be a putative E3 ligase, and its expression can be induced by various pathogens. Our study confirms that MUSE16 is a RING-type E3 ligase harboring ubiquitination activity (Han et al., 2017) (Fig. 4B). It had a negative regulatory effect on both plant basal defense and SNC1-mediated immunity (Figs 5A–C, 6A, C, D; Supplementary S6C). Given its biochemical and biological functions, we speculate that MUSE16 might target the positive regulatory factors of disease resistance for degradation through ubiquitination, thus displaying negative regulation of plant immunity.

Although muse16 appeared to be linked to snc1, mutation of MUSE16 had little effect on SNC1 protein accumulation (Fig. 6E), suggesting that MUSE16 might not participate in plant immunity through directly influencing SNC1 stability. The more serious dwarfism that was apparent in the muse16 snc1 double-mutant plants might have been due to additive effects caused by the presence of other unknown MUSE16 target proteins involved in disease resistance.

Infection tests using several bacterial pathogens with different avirulent proteins together with western blotting analysis indicated that RPS2 might be the target protein, or one of the target proteins, of MUSE16 (Fig. 7A, CC). Further biochemical studies determined that MUSE16 directly targeted RPS2 for degradation through the UPS pathway (Fig. 8B–D). It is worth noting that this does not mean that RPS2 is the only target protein of MUSE16, because even though there are over 1400 putative ubiquitin E3 ligases in the Arabidopsis genome (Kraft et al., 2005), there are many more proteins whose stability needs to be regulated. Unlike CPR1, which targets at least two R proteins, SNC1 and RPS2 (Cheng et al., 2011), of the R proteins that we tested for degradation, MUSE16 appeared to only target RPS2, which was consistent with the fact that the cpr1 mutant shows a defense-related dwarfism and the muse16 single-mutant exhibited a phenotype similar to the wild type (Fig. 5D).

A number of studies on R protein cellular regulation through the UPS pathway have demonstrated that their regulatory mechanisms share common attributes as well as specificity, since various E3s are involved in the turnover of the same or different R proteins (Cheng et al., 2011; Huang et al., 2014; Copeland et al., 2016; Dong et al., 2018). Given that ubiquitin-mediated protein degradation enables plant cells to make rapid changes in response to stimuli at the protein level, there is no doubt that it has great potential for regulating the stability of downstream protein components involved in disease resistance. Identification and functional analysis of more E3 ligases involved in the regulation of the intracellular stability of R protein in the future will help elucidate the mechanism of E3 selection of target proteins and reveal the negative regulatory network of NLR resistance proteins in plant immunity.

Supplementary data

The following supplementary data are available at JXB online.

Fig. S1. Morphology of 3-week-old soil-grown plants of mos4 snc1, muse16-1 mos4 snc1, and T₁ progeny of muse16-1 mos4 snc1 crossed with mos4 snc1.

Fig. S2. Results of Sanger sequencing of AT2G37150 at the point-mutation site.

Fig. S3. The amino acid sequence of AT2G37150.

Fig. S4. Simulation of the 3D structure of the protein encoded by AT2G37150.

Fig. S5. Sequence comparison of the C3H2C3 domain of different species.

Fig. S6. Analysis of resistance mediated by RPM1, RPS5, RPS4 or basal defense in Col-0, different mutants, and MUSE16-overexpressing plants.

Fig. S7. Interaction between RPS2 and MUSE16 as detected by Y2H assays.

Table S1. All the primers used in this study.

Table S2. Details of candidate genes with point-mutations identified from whole-genome sequencing in the mapping region.

Acknowledgements

We thank Yan Li and Yuelin Zhang (University of British Columbia and NIBS, China) for Illumina sequencing of muse16-1 and mutation analysis, and Brian Staskawitz (University of California, Berkeley) for providing seeds of the RPS2/RPS4-HA transgenic line.

Author contributions

YH, YC, and XL designed the experiments; YH, JoL, and TH performed most of the experiments; QL and ZH carried out the analysis related to bioinformatics; XB and JgL conducted the transgenic complementation tests; YH and TH wrote the manuscript; all authors contributed to the revision of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest in relation to this work.

Funding

This work was supported by grants from the National Natural Science Foundation of China (#31500978) and the Key Project of Sichuan Provincial Department of Education (#16ZA0030).

Data availability

All data supporting the findings of this study are available within the paper and within its supplementary materials published online.

References

Author notes