Single-Cell RNA-Sequencing of Soybean Reveals Transcriptional Changes and Antiviral Functions of GmGSTU23 and GmGSTU24 in Response to Soybean Mosaic Virus.

Soybean mosaic virus (SMV) stands as a prominent and widespread threat to soybean (Glycine max L. Merr.), the foremost legume crop globally. Attaining a thorough comprehension of the alterations in the transcriptional network of soybeans in response to SMV infection is imperative for a profound insight into the mechanisms of viral pathogenicity and host resistance. In this investigation, we isolated 50 294 protoplasts from the newly developed leaves of soybean plants subjected to both SMV infection and mock inoculation. Subsequently, we utilized single-cell RNA sequencing (scRNA-seq) to construct the transcriptional landscape at a single-cell resolution. Nineteen distinct cell clusters were identified based on the transcriptomic profiles of scRNA-seq. The annotation of three cell types-epidermal cells, mesophyll cells, and vascular cells-was established based on the expression of orthologs to reported marker genes in Arabidopsis thaliana. The differentially expressed genes between the SMV- and mock-inoculated samples were analyzed for different cell types. Our investigation delved deeper into the tau class of glutathione S-transferases (GSTUs), known for their significant contributions to plant responses against abiotic and biotic stress. A total of 57 GSTU genes were identified by a thorough genome-wide investigation in the soybean genome G. max Wm82.a4.v1. Two specific candidates, GmGSTU23 and GmGSTU24, exhibited distinct upregulation in all three cell types in response to SMV infection, prompting their selection for further research. The transient overexpression of GmGSTU23 or GmGSTU24 in Nicotiana benthamiana resulted in the inhibition of SMV infection, indicating the antiviral function of soybean GSTU proteins.

Transcriptome-wide m6A methylation profiling identifies GmAMT1;1 as a promoter of lead and cadmium tolerance in soybean nodules.

Lead (Pb) and cadmium (Cd) are common heavy metal pollutants that are often found in the soil in soybean agricultural production, adversely impacting symbiotic nitrogen fixation in soybean nodules. In this study, the exposure of soybean nodules to Pb and Cd stress was found to reduce nitrogenase activity. Shifts in the RNA methylation profiles of nodules were subsequently examined by profiling the differential expression of genes responsible for regulating m6A modifications and conducting transcriptome-wide analyses of m6A methylation profiles under Pb and Cd stress condition. Differentially methylated genes (DMGs) that were differentially expressed were closely related to reactive oxygen species activity and integral membrane components. Overall, 19 differentially expressed DMGs were ultimately determined to be responsive to both Pb and Cd stress, including Glyma.20G082450, which encodes GmAMT1;1 and was confirmed to be a positive regulator of nodules tolerance to Pb and Cd. Together, these results are the first published data corresponding to transcriptome-wide m6A methylation patterns in soybean nodules exposed to Cd and Pb stress, and provide novel molecular insight into the regulation of Pb and Cd stress responses in nodules, highlighting promising candidate genes related to heavy metal tolerance, that may also be amenable to application in agricultural production. ENVIRONMENTAL IMPLICATIONS: Lead (Pb) and cadmium (Cd) are prevalent heavy metal pollutants in soil, and pose a major threat to crop production, food security and human health. Here, MeRIP-seq approach was employed to analyze the regulatory network activated in soybean nodules under Pb and Cd stress, ultimately leading to the identification of 19 shared differentially expressed DMGs. When overexpressed, GmATM1;1 was found to enhance the Pb and Cd tolerance of soybean nodules. These results provide a theoretical basis for studies on tolerance to heavy metals in symbiotic nitrogen fixation, and provide an approach to enhancing Pb and Cd tolerance in soybean production.

Changes in the m6A RNA methylome accompany the promotion of soybean root growth by rhizobia under cadmium stress.

Cadmium (Cd) is the most widely distributed heavy metal pollutant in soil and has significant negative effects on crop yields and human health. Rhizobia can enhance soybean growth in the presence of heavy metals, and the legume-rhizobia symbiosis has been used to promote heavy-metal phytoremediation, but much remains to be learned about the molecular networks that underlie these effects. Here, we demonstrated that soybean root growth was strongly suppressed after seven days of Cd exposure but that the presence of rhizobia largely eliminated this effect, even prior to nodule development. Moreover, rhizobia did not appear to promote root growth by limiting plant Cd uptake: seedlings with and without rhizobia had similar root Cd concentrations. Previous studies have demonstrated a role for m6A RNA methylation in the response of rice and barley to Cd stress. We therefore performed transcriptome-wide m6A methylation profiling to investigate changes in the soybean RNA methylome in response to Cd with and without rhizobia. Here, we provide some of the first data on transcriptome-wide m6a RNA methylation patterns in soybean; m6A modifications were concentrated at the 3' UTR of transcripts and showed a positive relationship with transcript abundance. Transcriptome-wide m6A RNA methylation peaks increased in the presence of Cd, and the integration of m6A methylome and transcriptome results enabled us to identify 154 genes whose transcripts were both differentially methylated and differentially expressed in response to Cd stress. Annotation results suggested that these genes were associated with Ca2+ homeostasis, ROS pathways, polyamine metabolism, MAPK signaling, hormones, and biotic stress responses. There were 176 differentially methylated and expressed transcripts under Cd stress in the presence of rhizobia. In contrast to the Cd-only gene set, they were also enriched in genes related to auxin, jasmonic acid, and brassinosteroids, as well as abiotic stress tolerance. They contained fewer genes related to Ca2+ homeostasis and also included candidates with known functions in the legume-rhizobia symbiosis. These findings offer new insights into how rhizobia promote soybean root growth under Cd stress; they provide candidate genes for research on plant heavy metal responses and for the use of legumes in phytoremediation.

Effector-Dependent and -Independent Molecular Mechanisms of Soybean-Microbe Interaction.

Soybean is a pivotal staple crop worldwide, supplying the main food and feed plant proteins in some countries. In addition to interacting with mutualistic microbes, soybean also needs to protect itself against pathogens. However, to grow inside plant tissues, plant defense mechanisms ranging from passive barriers to induced defense reactions have to be overcome. Pathogenic but also symbiotic micro-organisms effectors can be delivered into the host cell by secretion systems and can interfere with the immunity system and disrupt cellular processes. This review summarizes the latest advances in our understanding of the interaction between secreted effectors and soybean feedback mechanism and uncovers the conserved and special signaling pathway induced by pathogenic soybean cyst nematode, Pseudomonas, Xanthomonas as well as by symbiotic rhizobium.

A retrospective assessment of the clinical efficacy of different internal fixation methods in the treatment of distal fibula fractures in the elderly.

An ankle fracture is a fracture of the distal tibia or fibula that forms the ankle joint, usually associated with ligament and soft tissue injury, and is a common type of lower limb fracture and one of the most common types of fracture in the elderly. Although ankle fractures are one of the most common injuries seen by orthopedic trauma surgeons, there is no uniform protocol for the diagnosis and treatment of ankle fractures in the elderly, and there are many controversial indications for surgery. The aim of this study is to assess the clinical efficacy of different internal fixation methods in the treatment of distal fibular fractures in the elderly, in an effort to improve the rational selection and application of clinical acts. A retrospective analysis was performed on 68 cases of patients who suffered an ankle fracture and were treated with different internal fixation methods according to the fracture types and individual differences in distal fibula fractures. The postoperative therapeutic effect assessment was performed in terms of clinical examination, imaging evaluation, and AOFAS ankle-hind foot function scoring. There was no unhealed bone, ankle instability and loose/fractured internal fixation. Fracture healing time was 2.7 to 4.0 months (average 3.2 months). AOFAS score was 88.3 ± 6.2, of which, 34 excellent cases, 30 good cases, and 4 fair cases. Ankle activity dorsiflexion 6º~18º, average 15º; plantar flexion 26º~47º, average 37º. A good clinical efficacy could be achieved from the most appropriate individualized internal fixation for distal fibula fractures of elderly patients.

GsRSS3L, a Candidate Gene Underlying Soybean Resistance to Seedcoat Mottling Derived from Wild Soybean (Glycine soja Sieb. and Zucc).

Soybeans are a major crop that produce the best vegetable oil and protein for use in food and beverage products worldwide. However, one of the most well-known viral infections affecting soybeans is the Soybean Mosaic Virus (SMV), a member of the Potyviridae family. A crucial method for preventing SMV damage is the breeding of resistant soybean cultivars. Adult resistance and resistance of seedcoat mottling are two types of resistance to SMV. Most studies have focused on adult-plant resistance but not on the resistance to seedcoat mottling. In this study, chromosome segment-substituted lines derived from a cross between Suinong14 (cultivated soybean) and ZYD00006 (wild soybean) were used to identify the chromosome region and candidate genes underlying soybean resistance to seed coat mottling. Herein, two quantitative trait loci (QTLs) were found on chromosome 17, and eighteen genes were found in the QTL region. RNA-seq was used to evaluate the differentially expressed genes (DEGs) among the eighteen genes located in the QTLs. According to the obtained data, variations were observed in the expression of five genes following SMV infection. Furthermore, Nicotiana benthamiana was subjected to an Agrobacterium-mediated transient expression assay to investigate the role of the five candidate genes in SMV resistance. It has also been revealed that Glyma.17g238900 encoding a RICE SALT SENSITIVE 3-like protein (RSS3L) can inhibit the multiplication of SMV in N.benthamiana. Moreover, two nonsynonymous single-nucleotide polymorphisms (SNPs) were found in the coding sequence of Glyma.17g238900 derived from the wild soybean ZYD00006 (GsRSS3L), and the two amino acid mutants may be associated with SMV resistance. Hence, it has been suggested that GsRSS3L confers seedcoat mottling resistance, shedding light on the mechanism of soybean resistance to SMV.

Regulation of osteoblast autophagy based on PI3K/AKT/mTOR signaling pathway study on the effect of ß-ecdysterone on fracture healing.

OBJECTIVES: To investigate the effects of ß-ecdysterone on fracture healing and the underlying mechanism. METHODS: MTT assay was used to detect the cell viability. AO/PI and flow cytometry assays were used to determine the apoptotic rate. The expression level of RunX2, ATG7 and LC3 was evaluated by qRT-PCR and Western blot assays. X-ray and HE staining were conducted on the fractured femur. Immunohistochemical assay was used to detect the expression level of Beclin-1 and immunofluorescence assay was used to measure the expression level of LC3 in the fractured femurs. Western blot was utilized to determine the expression level of PI3K, p-AKT1, AKT1, p-mTOR, mTOR, p-p70S6K, and p70S6K. RESULTS: The ALP activity and the expression of RunX2 in fractured osteoblasts were significantly elevated, the apoptotic rate was suppressed by rapamycin, 60, and 80 µM ß-ecdysterone. The state of autophagy both in fractured osteoblasts and femurs was facilitated by rapamycin and ß-ecdysterone. Compared to control, Garrett score was significantly promoted in rapamycin and ß-ecdysterone groups, accompanied by ameliorated pathological state. Lastly, the PI3K/AKT/mTOR pathway both in fractured osteoblasts and femurs was inhibited by rapamycin and ß-ecdysterone. CONCLUSION: ß-ecdysterone might facilitate fracture healing by activating autophagy through suppressing PI3K/AKT/mTOR signal pathway.

Icariin alleviates osteoarthritis by regulating autophagy of chondrocytes by mediating PI3K/AKT/mTOR signaling.

Osteoarthritis (OA) is a chronic degenerative disease that significantly impacts the quality of life of the elderly population. Recently, the pathogenesis of OA has been reported to involve autophagy in chondrocytes. Intriguingly, icariin, one of the main components of epimedium, exerts multiple pharmacological effects, including a protective effect against chondrocyte damage. Thus, we aimed to investigate the therapeutic effect of icariin on OA and its potential underlying mechanism by using a rat model of OA. After treatment with icariin or an autophagy activator (rapamycin) or inhibitor (3-methyladenine), OA chondrocyte viability was measured using the CCK-8 assay, apoptosis in the chondrocytes was evaluated using the acridine orange-propidium iodide assay and flow cytometry, and OA tissue pathological state was assessed using micro-CT scanning and safranin O staining. Furthermore, immunohistochemical staining was used to measure the expression level of Beclin-1 and immunofluorescence labeling was used to visualize LC3 expression, and western blotting was used to determine the expression levels of autophagy proteins and key proteins in the PI3K signaling pathway. The apoptotic rate of OA chondrocytes was markedly elevated by 3-methyladenine and suppressed by rapamycin and icariin; autophagy genes were drastically downregulated in the 3-methyladenine group and upregulated in the rapamycin and icariin groups; and the PI3K/AKT/mTOR signaling pathway was activated by 3-methyladenine and inhibited by rapamycin and icariin. Notably, following treatment with rapamycin and icariin, the severe pathological state in OA cartilage tissues was substantially alleviated, and this was accompanied by activated autophagy and inhibited PI3K signaling in the cartilage tissues. Taken together, these findings indicate that icariin alleviates OA by regulating the autophagy of chondrocytes by mediating PI3K/AKT/mTOR signaling.

Enhanced production of seed oil with improved fatty acid composition by overexpressing NAD+ -dependent glycerol-3-phosphate dehydrogenase in soybean.

There is growing interest in expanding the production of soybean oils (mainly triacylglycerol, or TAG) to meet rising feed demand and address global energy concerns. We report that a plastid-localized glycerol-3-phosphate dehydrogenase (GPDH), encoded by GmGPDHp1 gene, catalyzes the formation of glycerol-3-phosphate (G3P), an obligate substrate required for TAG biosynthesis. Overexpression of GmGPDHp1 increases soybean seed oil content with high levels of unsaturated fatty acids (FAs), especially oleic acid (C18:1), without detectably affecting growth or seed protein content or seed weight. Based on the lipidomic analyses, we found that the increase in G3P content led to an elevated diacylglycerol (DAG) pool, in which the Kennedy pathway-derived DAG was mostly increased, followed by PC-derived DAG, thereby promoting the synthesis of TAG containing relatively high proportion of C18:1. The increased G3P levels induced several transcriptional alterations of genes involved in the glycerolipid pathways. In particular, genes encoding the enzymes responsible for de novo glycerolipid synthesis were largely upregulated in the transgenic lines, in-line with the identified biochemical phenotype. These results reveal a key role for GmGPDHp1-mediated G3P metabolism in enhancing TAG synthesis and demonstrate a strategy to modify the FA compositions of soybean oils for improved nutrition and biofuel.

Lipoxin A4 protects rat skin flaps against ischemia-reperfusion injury through inhibiting cell apoptosis and inflammatory response induced by endoplasmic reticulum stress.

BACKGROUND: The ischemia-reperfusion (I/R) injury of skin flap is a complex pathophysiological process involving many cells and factors. Although endoplasmic reticulum (ER) stress-induced cell apoptosis and inflammatory response are of immense importance in the skin flap ischemia, the treatment for I/R injury induced by ER stress is barely reported. METHODS: Healthy male Wister rats were randomly divided into three groups: sham-operated group, I/R model group and I/R + LXA4 group. I/R-induced injury in skin flaps with or without pre-treatment of Lipoxin A4 (LXA4, 100 µg/kg) was tested by using HE and TUNEL staining. Related factors associated with oxidative stress, apoptosis, inflammatory response, and ER stress were tested by ELISA, biochemical assay, and western blotting, respectively. RESULTS: Our results showed that LXA4 treatment significantly promotes skin flap survival and attenuates I/R injury by inhibiting oxidative stress, apoptosis, and inflammatory factor release, evidenced by the decreased expression of malondialdehyde (MDA), lactate dehydrogenase (LDH), NF-κBp65, tumor necrosis factor α (TNF-α), ET, active Caspase-3 and Bax and up-regulated superoxide dismutase (SOD), glutathione (GSH) level and Bcl-2 expression. Moreover, LXA4 treatment also reverses the increased expression of GRP78, p-PERK, p-eIF2α, ATF4, and CHOP induced by I/R injury. CONCLUSIONS: In conclusion, we showed that ER stress causes cell apoptosis and inflammatory response, resulting in the skin flaps injury. LXA4 exhibits a protective effect on skin flaps against I/R injury through the inhibition of ER stress.

QTL Mapping and Data Mining to Identify Genes Associated With the Sinorhizobium fredii HH103 T3SS Effector NopD in Soybean.

In some legume-rhizobium symbioses, host specificity is influenced by rhizobial type III effectors-nodulation outer proteins (Nops). However, the genes encoding host proteins that interact with Nops remain unknown. In this study, we aimed to identify candidate soybean genes associated with NopD, one of the type III effectors of Sinorhizobium fredii HH103. The results showed that the expression pattern of NopD was analyzed in rhizobia induced by genistein. We also found NopD can be induced by TtsI, and NopD as a toxic effector can induce tobacco leaf death. In 10 soybean germplasms, NopD played a positively effect on nodule number (NN) and nodule dry weight (NDW) in nine germplasms, but not in Kenjian28. Significant phenotype of NN and NDW were identified between Dongnong594 and Charleston, Suinong14 and ZYD00006, respectively. To map the quantitative trait locus (QTL) associated with NopD, a recombinant inbred line (RIL) population derived from the cross between Dongnong594 and Charleston, and chromosome segment substitution lines (CSSLs) derived from Suinong14 and ZYD00006 were used. Two overlapping conditional QTL associated with NopD on chromosome 19 were identified. Two candidate genes were identified in the confident region of QTL, we found that NopD could influence the expression of Glyma.19g068600 (FBD/LRR) and expression of Glyma.19g069200 (PP2C) after HH103 infection. Haplotype analysis showed that different types of Glyma.19g069200 haplotypes could cause significant nodule phenotypic differences, but Glyma.19g068600 (FBD/LRR) was not. These results suggest that NopD promotes S. fredii HH103 infection via directly or indirectly regulating Glyma.19g068600 and Glyma.19g069200 expression during the establishment of symbiosis between rhizobia and soybean plants.

Effect of ß­ecdysterone on glucocorticoid­induced apoptosis and autophagy in osteoblasts.

The aim of the present study was to investigate the effect of glucocorticoids in osteoblasts and to examine the role of ß­ecdysterone in the pathogenesis of glucocorticoid­induced osteoporosis. Osteoblasts were induced from bone marrow mesenchymal stem cells, which were isolated from C57BL/6 mice. Cell viability and apoptosis of osteoblasts were measured by Cell Counting Kit­8 and flow cytometry analysis, respectively. The expression of related genes and proteins was measured by reverse transcription quantitative polymerase chain reaction and western blot analysis respectively. Dose­dependent decreases in the cell proliferation and differentiation were observed in dexamethasone (Dex)­treated osteoblasts, evidenced by downregulation in the activity of alkaline phosphatasedecreased expression levels of Runt­related transcription factor 2 and osteocalcin, and upregulated expression of RANK ligand. Dex also induced apoptosis and inhibited autophagy of osteoblasts, evidenced by upregulated B­cell lymphoma 2 (Bcl­2)­associated X protein/Bcl­2 ratio and the activation of mammalian target of rapamycin (mTOR), and decreased expression levels of Beclin­1, autophagy protein 5 and microtubule­associated protein 1 light chain 3 II. The effects on cell proliferation, apoptosis and autophagy induced by Dex were reversed by ß­ecdysterone in a dose­dependent manner. Therefore, ß­ecdysterone may be a promising candidate drug for the treatment of osteoporosis through inducing osteoblast autophagic activity by inactivating mTOR.

[Surgical treatment of grade III pronation-external rotation fractures of ankle].

Objective: To summarize the short-term effectiveness of the surgical treatment for grade III pronation-external rotation ankle fractures. Methods: Between October 2011 and May 2015, 36 patients with pronation-external rotation ankle fractures (grade III) were treated with internal fixation and repair of the anterior lower tibiofibular ligament, including 21 males and 15 females with an average age of 45.2 years (range, 21-72 years). Injury was caused by sprain in 19 cases, by traffic accident in 6 cases, and by falling from height in 11 cases. All patients had closed fractures, with no blood vessel and nerve injury. The locations were the left ankle in 13 cases and the right ankle in 23 cases. The time from injury to operation was 3 to 10 days (mean, 6.5 days). At last follow-up, ankle function was evaluated by American Orthopaedic Foot and Ankle Society (AOFAS) ankle-foot score, and the improvement of pain was evaluated by visual analogue scale (VAS). Results: Primary healing of incision was obtained in all patients, no incision infection and skin necrosis occurred. Twenty-eight patients were followed up 12-36 months (mean, 17.5 months). X-ray films showed bone union was achieved within 2.6-5 months (mean, 3.2 months). No fracture of internal fixation and disruption of tibiofibular diastasis occurred. At last follow-up, according to AOFAS score for ankle function evaluation, the pain score was 36.3±2.9, the function score was 44.3±3.2, the ligament condition score was 9.2±0.5, and the total score was 89.8±6.6; the results were excellent in 11 cases, good in 15 cases, and fair in 2 cases. VAS score was 1.6±0.5. The range of motion of the ankle was (13±5)° in dorsiflexion and (38±9)° in planteroflexion. Conclusion: Repair of anterior tibial ligament is an effective method to treat tibiofibular diastasis injury in the surgical treatment of grade III pronation-external rotation ankle fractures, with convenient operation, and satisfactory short-term effectiveness.

[Surgical treatment for open tarsometatarsal joint injury].

OBJECTIVE: To discuss the surgical method and clinical efficacy for open tarsometatarsal joint injuries. METHODS: From March 2011 to January 2015, 21 patients with open tarsometatarsal joint injuries were treated with stage-surgery method, including 14 males and 7 females with an average age of 45.6 years old ranging from 20 to 75 years. Injury site occurred in the left foot of 13 cases and right foot of 8 cases. Traffic injury was in 5 cases, crush injury in 6 cases, heavy crushing was in 10 cases. According to Myerson to classify for tarsometatarsal joint injury, 5 cases were type B2, 9 cases were type C1, and 7 cases were type C2. And according to Gustilo to typing for soft tissue injury, 5 cases were type IIB, 10 cases were type IIIA, 6 cases were type IIIB. Fracture healingand complications were observed after operation and clinical effects were evaluated according to the midfoot score of AOFAS. RESULTS: All the patients were followed up from 11 to 40 months with an average of 16.2 months. The fracture healing time was from 10 to 16 weeks with an average of 12.3 weeks. No complications such as deep infection, nonunion and osteomyelitis were found. Midfoot score of AOFAS at last follow-up was 83.0±14.9, 9 cases got excellent results, 8 good, 2 fair, 2 poor. Two patients complicated with severe traumatic arthritis once again underwent tarsometatarsal arthrodesis. CONCLUSIONS: For the treatment of open tarsometatarsal joint injury, reasonable debridement, comprehensive assessment for the soft tissue injury, correctly grasp the surgical indications and time of internal fixation, can reduce the incidence of deep infection and osteomyelitis.

SNP-SNP Interaction Analysis on Soybean Oil Content under Multi-Environments.

Soybean oil content is one of main quality traits. In this study, we used the multifactor dimensionality reduction (MDR) method and a soybean high-density genetic map including 5,308 markers to identify stable single nucleotide polymorphism (SNP)-SNP interactions controlling oil content in soybean across 23 environments. In total, 36,442,756 SNP-SNP interaction pairs were detected, 1865 of all interaction pairs associated with soybean oil content were identified under multiple environments by the Bonferroni correction with p <3.55×10-11. Two and 1863 SNP-SNP interaction pairs detected stable across 12 and 11 environments, respectively, which account around 50% of total environments. Epistasis values and contribution rates of stable interaction (the SNP interaction pairs were detected in more than 2 environments) pairs were detected by the two way ANOVA test, the available interaction pairs were ranged 0.01 to 0.89 and from 0.01 to 0.85, respectively. Some of one side of the interaction pairs were identified with previously research as a major QTL without epistasis effects. The results of this study provide insights into the genetic architecture of soybean oil content and can serve as a basis for marker-assisted selection breeding.

Functional analysis of NopM, a novel E3 ubiquitin ligase (NEL) domain effector of Rhizobium sp. strain NGR234.

Type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS) are not only virulence factors of pathogenic bacteria, but also influence symbiotic interactions between nitrogen-fixing nodule bacteria (rhizobia) and leguminous host plants. In this study, we characterized NopM (nodulation outer protein M) of Rhizobium sp. strain NGR234, which shows sequence similarities with novel E3 ubiquitin ligase (NEL) domain effectors from the human pathogens Shigella flexneri and Salomonella enterica. NopM expressed in Escherichia coli, but not the non-functional mutant protein NopM-C338A, showed E3 ubiquitin ligase activity in vitro. In vivo, NopM, but not inactive NopM-C338A, promoted nodulation of the host plant Lablab purpureus by NGR234. When NopM was expressed in yeast, it inhibited mating pheromone signaling, a mitogen-activated protein (MAP) kinase pathway. When expressed in the plant Nicotiana benthamiana, NopM inhibited one part of the plant's defense response, as shown by a reduced production of reactive oxygen species (ROS) in response to the flagellin peptide flg22, whereas it stimulated another part, namely the induction of defense genes. In summary, our data indicate the potential for NopM as a functional NEL domain E3 ubiquitin ligase. Our findings that NopM dampened the flg22-induced ROS burst in N. benthamiana but promoted defense gene induction are consistent with the concept that pattern-triggered immunity is split in two separate signaling branches, one leading to ROS production and the other to defense gene induction.