The Arabidopsis E3 ubiquitin ligase PUB4 regulates BIK1 and is targeted by a bacterial type-III effector.

Plant immunity is tightly controlled by a complex and dynamic regulatory network, which ensures optimal activation upon detection of potential pathogens. Accordingly, each component of this network is a potential target for manipulation by pathogens. Here, we report that RipAC, a type III-secreted effector from the bacterial pathogen Ralstonia solanacearum, targets the plant E3 ubiquitin ligase PUB4 to inhibit pattern-triggered immunity (PTI). PUB4 plays a positive role in PTI by regulating the homeostasis of the central immune kinase BIK1. Before PAMP perception, PUB4 promotes the degradation of non-activated BIK1, while after PAMP perception, PUB4 contributes to the accumulation of activated BIK1. RipAC leads to BIK1 degradation, which correlates with its PTI-inhibitory activity. RipAC causes a reduction in pathogen-associated molecular pattern (PAMP)-induced PUB4 accumulation and phosphorylation. Our results shed light on the role played by PUB4 in immune regulation, and illustrate an indirect targeting of the immune signalling hub BIK1 by a bacterial effector.

Plant U-box E3 ligases PUB20 and PUB21 negatively regulate pattern-triggered immunity in Arabidopsis.

KEY MESSAGE: Plant U-box E3 ligases PUB20 and PUB21 are flg22-triggered signaling components and negatively regulate immune responses. Plant U-box proteins (PUBs) constitute a class of E3 ligases that are associated with various stress responses. Among the class IV PUBs featuring C-terminal Armadillo (ARM) repeats, PUB20 and PUB21 are closely related homologs. Here, we show that both PUB20 and PUB21 negatively regulate innate immunity in plants. Loss of PUB20 and PUB21 function leads to enhanced resistance to surface inoculation with the virulent bacterium Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). However, the resistance levels remain unaffected after infiltration inoculation, suggesting that PUB20 and PUB21 primarily function during the early defense stages. The enhanced resistance to Pst DC3000 in PUB mutant plants (pub20-1, pub21-1, and pub20-1/pub21-1) correlates with extensive flg22-triggered reactive oxygen production, strong MPK3 activation, and enhanced transcriptional activation of early immune response genes. Additionally, PUB mutant plants (except pub21-1) exhibit constitutive stomatal closure after Pst DC3000 inoculation, implying the significant role of PUB20 in stomatal immunity. Comparative analyses of flg22 responses between PUB mutants and wild-type plants reveals that the robust activation of the pattern-induced immune responses may enhance resistance against Pst DC3000. Notably, the hypersensitivity responses triggered by RPM1/avrRpm1 and RPS2/avrRpt2 are independent of PUB20 and PUB21. These results suggest that PUB20 and PUB21 knockout mutations affect bacterial invasion, likely during the early stages, acting as negative regulators of plant immunity.

The Vitis yeshanensis U-box E3 ubiquitin ligase VyPUB21 enhances resistance to powdery mildew by targeting degradation of NIM1-interacting (NIMIN) protein.

KEY MESSAGE: VyPUB21 plays a key role during the defense against powdery mildew in grapes. Ubiquitin-ligating enzyme (E3), a type of protein widely found in plants, plays a key role in their resistance to disease. Yet how E3 participates in the disease-resistant response of Chinese wild grapevine (Vitis yeshanensis) remains unclear. Here we isolated and identified a U-box type E3 ubiquitin ligase, VyPUB21, from V. yeshanensis. This gene's expression level rose rapidly after induction by exogenous salicylic acid (SA), jasmonic acid (JA), and ethylene (ETH) and powdery mildew. In vitro ubiquitination assay results revealed VyPUB21 could produce ubiquitination bands after co-incubation with ubiquitin, ubiquitin-activating enzyme (E1), and ubiquitin-conjugating enzyme (E2); further, mutation of the conserved amino acid site in the U-box can inhibit the ubiquitination. Transgenic VyPUB21 Arabidopsis had low susceptibility to powdery mildew, and significantly fewer conidiophores and spores on its leaves. Expression levels of disease resistance-related genes were also augmented in transgenic Arabidopsis, and its SA concentration also significantly increased. VyPUB21 interacts with VyNIMIN and targets VyNIMIN protein hydrolysis through the 26S proteasome system. Thus, the repressive effect of the NIMIN-NPR complex on the late systemic acquired resistance (SAR) gene was attenuated, resulting in enhanced resistance to powdery mildew. These results indicate that VyPUB21 encoding ubiquitin ligase U-box E3 activates the SA signaling pathway, and VyPUB21 promotes the expression of late SAR gene by degrading the important protein VyNIMIN of SA signaling pathway, thus enhancing grape resistance to powdery mildew.

The cysteine-rich receptor-like kinase CRK4 contributes to the different drought stress response between Columbia and Landsberg erecta.

The natural variation between Arabidopsis (Arabidopsis thaliana) ecotypes Columbia (Col) and Landsberg erecta (Ler) strongly affects abscisic acid (ABA) signalling and drought tolerance. Here, we report that the cysteine-rich receptor-like protein kinase CRK4 is involved in regulating ABA signalling, which contributes to the differences in drought stress tolerance between Col-0 and Ler-0. Loss-of-function crk4 mutants in the Col-0 background were less drought tolerant than Col-0, whereas overexpressing CRK4 in the Ler-0 background partially to completely restored the drought-sensitive phenotype of Ler-0. F1 plants derived from a cross between the crk4 mutant and Ler-0 showed an ABA-insensitive phenotype with respect to stomatal movement, along with reduced drought tolerance like Ler-0. We demonstrate that CRK4 interacts with the U-box E3 ligase PUB13 and enhances its abundance, thus promoting the degradation of ABA-INSENSITIVE 1 (ABI1), a negative regulator of ABA signalling. Together, these findings reveal an important regulatory mechanism for modulating ABI1 levels by the CRK4-PUB13 module to fine-tune drought tolerance in Arabidopsis.

Impact on sales of adding a smaller serving size of beer and cider in licensed premises: an A-B-A reversal design.

BACKGROUND: Smaller serving sizes of alcoholic drinks could reduce alcohol consumption across populations thereby lowering the risk of many diseases. The effect of modifying the available range of serving sizes of beer and cider in a real-world setting has yet to be studied. The current study assessed the impact on beer and cider sales of adding a serving size of draught beer and cider (2/3 pint) that was between the current smallest (1/2 pint) and largest (1 pint) standard serving sizes. METHODS: Twenty-two licensed premises in England consented to taking part in the study. The study used an ABA reversal design, set over three 4-weekly periods, with A representing the non-intervention periods, during which standard serving sizes were served and B the intervention period when a 2/3 pint serving size of draught beer and cider was added to the existing range, along with smaller 1/2 pint and larger 1 pint serving sizes. The primary outcome was the daily volume of beer and cider sold, extracted from sales data. RESULTS: Fourteen premises started the study, of which thirteen completed it. Twelve of those did so per protocol and were included in the primary analysis. After adjusting for pre-specified covariates, the intervention did not have a significant effect on the volume of beer and cider sold per day (3.14 ml; 95%CIs -2.29 to 8.58; p = 0.257). CONCLUSIONS: In licensed premises, there was no evidence that adding a smaller serving size for draught beer and cider (2/3 pint) when the smallest (1/2 pint) and largest (1 pint) sizes were still available, affected the volume of beer and cider sold. Studies are warranted to assess the impact of removing the largest serving size. TRIAL REGISTRATION: ISRCTN: https://doi.org/10.1186/ISRCTN33169631 (08/09/2021), OSF: https://osf.io/xkgdb/ (08/09/2021).

Mediating and spatial spillover effects of public participation in environmental pollution governance mediated via traditional and new media.

A systematic study of public participation (Pub) in environmental pollution control can facilitate collaborative governance based on multiple factors, and promote the modernization of national governance. Based on the data of 30 Chinese provinces from 2011 to 2020, this study empirically analyzed the mechanism of Pub in environmental pollution governance. Based on multiple channels, a dynamic spatial panel Durbin model and an intermediary effect model were constructed. The main results are as follows: (1) Pub based on environmental letters and visits have no significant impact on local pollution reduction, while the Baidu search index of environmental pollution has the greatest effect on emission reduction, followed by environmental protection strategies based on the National People's Congress (NPC) and microblogging. (2) Pub not only has a direct positive impact on environmental control through its positive externality but also indirectly reduces environmental treatment by enhancing the intensity of environmental regulation. (3) Pub has a significant spatial spillover effect on environmental control based on geographical attenuation. Excluding environmental legislation, the direct spatial spillover effects of Pub under the networked platform and traditional channels are only significant within 1200 km and 1000 km, respectively, which decrease with increasing geographic distance within this range. When the environmental regulations are considered, the spatial spillover effects of suggestions proposed by NPC and the Chinese People's Political Consultative Conference (CPPCC) are significant within 800 km, while the attenuation boundary of Internet complaints, Baidu index, and microblogging public opinion is 1000 km. (4) The impact of Pub on environmental governance varies significantly among regions. Compared with central and western regions, the eastern region showed more effective pollution reduction based on Pub.

Arabidopsis PUB2 and PUB4 connect signaling components of pattern-triggered immunity.

Plants use pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs) and activate pattern-triggered immunity (PTI). Precise regulation of information from PRRs to downstream signaling components is vital to mounting an appropriate immune response and requires dynamic interactions of these PTI components. We used transcriptome profiling, phenotypic analysis, molecular genetics, and protein-protein interaction analysis to understand the roles of the Arabidopsis plant U-box (PUB) proteins PUB2 and PUB4 in disease resistance and PTI signaling. Loss of function of both PUB2 and PUB4 diminishes the PAMP-triggered oxidative bursts and dampens mitogen-activated protein kinase signaling, resulting in a severe compromise in resistance to not only pathogenic but also nonpathogenic strains of Pseudomonas syringae. Within PUB4, the E3 ligase activity is dispensable, but the armadillo repeat region is essential and sufficient for its function in immunity. PUB2 and PUB4 interact with PTI signaling components, including FLS2, BIK1, PBL27, and RbohD, and enhance FLS2-BIK1 and BIK1-RbohD interactions. Our study reveals that PUB2 and PUB4 are critical components of plant immunity and connect PTI components to positively regulate defense responses.

Adaptive Quality of Service Control for MQTT-SN.

Internet of Things and wireless sensor network applications are becoming more and more popular these days, supported by new communication technologies and protocols tailored to their specific requirements. This paper focuses on improving the performance of a Wireless Sensor Network operated by the MQTT-SN protocol, one of the most popular publish/subscribe protocols for IoT applications. In particular, we propose a dynamic Quality of Service (QoS) controller for the MQTT-SN protocol, capable of evaluating the status of the underlying network in terms of end-to-end delay and packet error rate, reacting consequently by assigning the best QoS value to a node. We design and implement the QoS controller in a simulated environment based on the ns-3 network emulator, and we perform extensive experiments to prove its effectiveness compared to a non-controlled scenario. The reported results show that, by controlling the quality of service, it is possible to effectively manage the number of packets successfully received by each device and their average latency, to improve the quality of the communication of each end node.

The Role of E3 Ubiquitin Ligases in Chloroplast Function.

Chloroplasts are ancient organelles responsible for photosynthesis and various biosynthetic functions essential to most life on Earth. Many of these functions require tightly controlled regulatory processes to maintain homeostasis at the protein level. One such regulatory mechanism is the ubiquitin-proteasome system whose fundamental role is increasingly emerging in chloroplasts. In particular, the role of E3 ubiquitin ligases as determinants in the ubiquitination and degradation of specific intra-chloroplast proteins. Here, we highlight recent advances in understanding the roles of plant E3 ubiquitin ligases SP1, COP1, PUB4, CHIP, and TT3.1 as well as the ubiquitin-dependent segregase CDC48 in chloroplast function.

PUB22 and PUB23 U-box E3 ubiquitin ligases negatively regulate 26S proteasome activity under proteotoxic stress conditions.

The mechanism regulating proteasomal activity under proteotoxic stress conditions remains unclear. Here, we showed that arsenite-induced proteotoxic stress resulted in upregulation of Arabidopsis homologous PUB22 and PUB23 U-box E3 ubiquitin ligases and that pub22pub23 double mutants displayed arsenite-insensitive seed germination and root growth phenotypes. PUB22/PUB23 downregulated 26S proteasome activity by promoting the dissociation of the 19S regulatory particle from the holo-proteasome complex, resulting in intracellular accumulation of UbG76V -GFP, an artificial substrate of the proteasome complex, and insoluble poly-ubiquitinated proteins. These results suggest that PUB22/PUB23 play a critical role in arsenite-induced proteotoxic stress response via negative regulation of 26S proteasome integrity.

Sustainability in the beer and pub industry during the COVID-19 period: An emerging new normal.

The COVID-19 pandemic created significant challenges for the British pub industry, due to the uncertain conditions caused by the virus, changes in consumption patterns and government measures. Studies recommend that organisations adopt innovative and flexible business models to generate added value for customers and other stakeholders as a survival and growth strategy. However, such measures require business ecosystems which encourage co-creative engagement. This qualitative study extends the concept of value co-creation beyond its current boundary as a customer-driven experiential paradigm, reconceptualising it as a driver for societal benefits. Over the period March - December 2020, we carried out in-depth interviews with pub and brewery owners, managers, and customers, combined with netnographic and offline observations of pubs' engagement with customers. We uncovered three stages of strategies and innovation during this period, which we term 'survive', 'secure', and 'sustain'. We demonstrate how multiple stakeholders benefit from the innovations of pubs and breweries negotiating each stage, advancing current scholarship on sustainable value co-creation.

Genome-wide identification and functional analysis of U-box E3 ubiquitin ligases gene family related to drought stress response in Chinese white pear (Pyrus bretschneideri).

BACKGROUND: The plant U-box (PUB) proteins are a family of ubiquitin ligases (E3) enzymes that involved in diverse biological processes, as well as in responses to plant stress response. However, the characteristics and functional divergence of the PUB gene family have not yet been previously studied in the Chinese white pear (Pyrus bretschneideri). RESULTS: In the present study, we identified 62 PbrPUBs in Chinese white pear genome. Based on the phylogenetic relationship, 62 PUB genes were clustered into five groups. The results of conserved motif and gene structure analysis supported the classification phylogenetic tree. The PbrPUB genes were unevenly distribution on 17 pear chromosomes, chromosome 15 housed most member of PUB family, with eight PUB genes. Cis-acting element analysis indicated that PUB genes might participate in diverse biological processes, especially in the response to abiotic stresses. Based on RNA-data from 'Dangshansuli' at seven tissues, we found that PUB genes exhibited diverse of expression level in seven tissues, and qRT-PCR experiment further supported the reliable of RNA-Seq data. To identify candidate genes associated with resistance, we conducted qRT-PCR experiment the expression level of pear seed plant under four abiotic stresses, including: ABA, dehydration, salt and cold treatment. One candidate PUB gene associated with dehydration stress was selected to conduct further functional experiment. Subcellular localization revealed PbrPUB18 protein was located on cell nucleus. Furthermore, heterologous over-expression of PbrPUB18 in Arabidopsis indicated that the over-expression of PbrPUB18 could enhance resistance in drought treatment. In conclusions, we systematically identified the PUB genes in pear, and provided useful knowledge for functional identification of PUB genes in pear.

Plant U-box E3 ligases PUB25 and PUB26 control organ growth in Arabidopsis.

Plant organs often grow into a genetically determined size and shape. How organ growth is finely regulated to achieve a well defined pattern is a fascinating, but largely unresolved, question in plant research. We utilised the Arabidopsis petal to study the genetic control of plant organ growth, and identify two closely related U-box E3 ligases PUB25 and PUB26 as important growth regulators by screening the targets of the petal-specific growth-promoting transcription factor RABBIT EARS (RBE). We showed that PUB25 is directly controlled by RBE in petal development in a spatial- and temporal-specific manner and acts as a major target to mediate RBE's function in petal growth. We also showed that PUB25 and PUB26 repress petal growth by restricting the period of cell proliferation, and their regulation appears to be independent of other plant E3 ligase genes implicated in growth control. PUB25 and PUB26 are among the first U-box E3 ligases shown to function in plant growth control. Furthermore, as they were also found to play a vital role in plant stress responses, PUB25 and PUB26 may act as a key hub to integrate developmental and environmental signals for balancing growth and defence in plants.

Bonding methods for chip integration with Parylene devices.

Flexible electronics require more compact interconnects for next-generation devices. Polymer devices can be bonded to integrated circuit chips, but combining flexible and rigid substrates poses unique technical challenges. Existing technologies either cannot achieve the density required for modern chips or employ specialized equipment and complex processes to do so. Here, we adapt several approaches to achieve fine-pitch bonding between rigid and flexible substrates including epoxy, ultrasonic wire, and anisotropic conductive film bonding and also introduce a novel technique called polymer ultrasonic on bump (PUB) bonding. Using Parylene C devices and various rigid substrates as our model testbed systems, we investigate these four methods across a range of bond pad size and pitch by measuring yield and resistance and by subjecting devices to thermomechanical reliability tests. We demonstrate that all methods are capable of bonding fine pitch interconnects (100 µm) at low temperature (<100 °C). Additionally, we focus on PUB bonding and join a packaged chip and a bare die to Parylene devices.

Ubiquitylation of ABA Receptors and Protein Phosphatase 2C Coreceptors to Modulate ABA Signaling and Stress Response.

Post-translational modifications play a fundamental role in regulating protein function and stability. In particular, protein ubiquitylation is a multifaceted modification involved in numerous aspects of plant biology. Landmark studies connected the ATP-dependent ubiquitylation of substrates to their degradation by the 26S proteasome; however, nonproteolytic functions of the ubiquitin (Ub) code are also crucial to regulate protein interactions, activity, and localization. Regarding proteolytic functions of Ub, Lys-48-linked branched chains are the most common chain type for proteasomal degradation, whereas promotion of endocytosis and vacuolar degradation is triggered through monoubiquitylation or Lys63-linked chains introduced in integral or peripheral plasma membrane proteins. Hormone signaling relies on regulated protein turnover, and specifically the half-life of ABA signaling components is regulated both through the ubiquitin-26S proteasome system and the endocytic/vacuolar degradation pathway. E3 Ub ligases have been reported that target different ABA signaling core components, i.e., ABA receptors, PP2Cs, SnRK2s, and ABFs/ABI5 transcription factors. In this review, we focused specifically on the ubiquitylation of ABA receptors and PP2C coreceptors, as well as other post-translational modifications of ABA receptors (nitration and phosphorylation) that result in their ubiquitination and degradation.

Application of Skyline for Analysis of Protein-Protein Interactions In Vivo.

Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein-protein interactions (PPIs) in cells is also important since these interactions are the bases of many processes, such as the cell cycle and signaling pathways. This paper describes the application of Skyline software for the identification and quantification of the biotinylated form of the biotin acceptor peptide (BAP) tag, which is a marker of in vivo PPIs. The tag was used in the Proximity Utilizing Biotinylation (PUB) method, which is based on the co-expression of BAP-X and BirA-Y in mammalian cells, where X or Y are interacting proteins of interest. A high level of biotinylation was detected in the model experiments where X and Y were pluripotency transcription factors Sox2 and Oct4, or heterochromatin protein HP1γ. MRM data processed by Skyline were normalized and recalculated. Ratios of biotinylation levels in experiment versus controls were 86 ± 6 (3 h biotinylation time) and 71 ± 5 (9 h biotinylation time) for BAP-Sox2 + BirA-Oct4 and 32 ± 3 (4 h biotinylation time) for BAP-HP1γ + BirA-HP1γ experiments. Skyline can also be applied for the analysis and identification of PPIs from shotgun proteomics data downloaded from publicly available datasets and repositories.

An exploration of the substitutions of British pub consumers during the COVID-19 crisis.

This research draws on substitution theory to understand how British pub consumers describe their substitutions in the context of the COVID-19 pandemic crisis. A two-stage research method design is adopted, with an exploratory netnography stage followed by 13 semi-structured interviews to gain a deeper insight. Five themes emerge that are either direct substitutions for the physical closing of the pub venues (online social drinking and pub-dating alternatives), or they were indirect effects of the substituting venues (in-the-home drinking, different drinks in the home and no work-related substitutions). The findings show that drinking in pubs is a functional as well as a symbolic act. Key stakeholders are recommended to remain in contact with customers through blended online and offline activities to help keep the pubs relevant to them. This will benefit the pub sector long after the COVID-19 pandemic crisis.

Genome-wide identification and expression analysis of PUB genes in cotton.

BACKGROUND: The U-box gene encodes a ubiquitin ligase that contain U-box domain. The plant U-box gene (PUB) plays an important role in the response to stresses, but few reports about PUBs in cotton were available. Therefore research on PUBs is of great importance and a necessity when studying the mechanisms of stress- tolerance in cotton. RESULTS: In this study, we identified 93, 96, 185 and 208 PUBs from four sequenced cotton species G. raimondii (D5), G. arboreum (A2), G. hirsutum (AD1) and G. barbadense (AD2), respectively. Prediction analysis of subcellular localization showed that the PUBs in cotton were widely localized in cells, but primarily in the nucleus. The PUBs in cotton were classified into six subfamilies (A-F) on the basis of phylogenetic analysis, which was testified by the analysis of conserved motifs and exon-intron structures. Chromosomal localization analysis showed that cotton PUBs were unevenly anchored on all chromosomes, varying from 1 to 14 per chromosome. Through multiple sequence alignment analysis, 3 tandem duplications and 28 segmental duplications in cotton genome D5, 2 tandem duplications and 25 segmental duplications in A2, and 143 homologous gene pairs in A2 and D5 were found; however no tandem duplications in A2 or D5 were found. Additionally, 105, 14 and 17 homologous gene pairs were found in the intra-subgenome of At and Dt, At sub-genome and Dt sub-genome of G. hirsutum, respectively. Functional analysis of GhPUB85A and GhPUB45D showed that these genes positively responded to abiotic stresses, but the expression patterns were different. In addition, although the expression levels of these two homologous genes were similar, their contributions were different when responding to stresses, specifically showing different responses to abiotic stresses and functional differences between the two subgenomes of G. hirsutum. CONCLUSIONS: This study reported the genome-wide identification, structure, evolution and expression analysis of PUBs in cotton, and the results showed that the PUBs were highly conserved throughout the evolutionary history of cotton. All PUB genes were involved in the response to abiotic stresses (including salt, drought, hot and cold) to varying degrees.

N-phenyl ureidobenzenesulfonates, a novel class of promising human dihydroorotate dehydrogenase inhibitors.

N-phenyl ureidobenzenesulfonates (PUB-SOs) is a new class of promising anticancer agents inducing replication stresses and cell cycle arrest in S-phase. However, the pharmacological target of PUB-SOs was still unidentified. Consequently, the objective of the present study was to identify and confirm the pharmacological target of the prototypical PUB-SO named 2-ethylphenyl 4-(3-ethylureido)benzenesulfonate (SFOM-0046) leading to the cell cycle arrest in S-phase. The antiproliferative and the cytotoxic activities of SFOM-0046 were characterized using the NCI-60 screening program and its fingerprint was analyzed by COMPARE algorithm. Then, human dihydroorotate dehydrogenase (hDHODH) colorimetric assay, uridine rescuing cell proliferation and molecular docking in the brequinar-binding site were performed. As a result, SFOM-0046 exhibited a mean antiproliferative activity of 3.5 µM in the NCI-60 screening program and evidenced that leukemia and colon cancer cell panels were more sensitive to SFOM-0046. COMPARE algorithm showed that the SFOM-0046 cytotoxic profile is equivalent to the ones of brequinar and dichloroallyl lawsone, two inhibitors of hDHODH. SFOM-0046 inhibited the hDHODH in the low nanomolar range (IC50 = 72 nM) and uridine rescued the cell proliferation of HT-29, HT-1080, M21 and MCF-7 cancer cell lines in the presence of SFOM-0046. Finally, molecular docking showed a binding pose of SFOM-0046 interacting with Met43 and Phe62 present in the brequinar-binding site. In conclusion, PUB-SOs and notably SFOM-0046 are new small molecules hDHODH inhibitors triggering replication stresses and S-phase arrest.

PUB4, a CERK1-Interacting Ubiquitin Ligase, Positively Regulates MAMP-Triggered Immunity in Arabidopsis.

Lysin motif (LysM) receptor-like kinase CERK1 is a co-receptor essential for plant immune responses against carbohydrate microbe-associated molecular patterns (MAMPs). Concerning the immediate downstream signaling components of CERK1, receptor-like cytoplasmic kinases such as PBL27 and other RLCK VII members have been reported to regulate immune responses positively. In this study, we report that a novel CERK1-interacting E3 ubiquitin ligase, PUB4, is also involved in the regulation of MAMP-triggered immune responses. Knockout of PUB4 resulted in the alteration of chitin-induced defense responses, indicating that PUB4 positively regulates reactive oxygen species generation and callose deposition but negatively regulates MAPK activation and defense gene expression. On the other hand, detailed analyses of a double knockout mutant of pub4 and sid2, a mutant of salicylic acid (SA) synthesis pathway, showed that the contradictory phenotype of the pub4 mutant was actually caused by abnormal accumulation of SA in this mutant and that PUB4 is a positive regulator of immune responses. The present and recent findings on the role of PUB4 indicate that PUB4 is a unique E3 ubiquitin ligase involved in the regulation of both plant immunity and growth/development.