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Nodulation outer protein M (NopM) is an IpaH family type three (T3) effector secreted by the nitrogen-fixing nodule bacterium Sinorhizobium sp. strain NGR234. Previous work indicated that NopM is an E3 ubiquitin ligase required for an optimal symbiosis between NGR234 and the host legume Lablab purpureus. Here, we continued to analyze the function of NopM. Recombinant NopM was biochemically characterized using an in vitro ubiquitination system with Arabidopsis thaliana proteins. In this assay, NopM forms unanchored polyubiquitin chains and possesses auto-ubiquitination activity. In a NopM variant lacking any lysine residues, auto-ubiquitination was not completely abolished, indicating noncanonical auto-ubiquitination of the protein. In addition, we could show intermolecular ubiquitin transfer from NopM to C338A (enzymatically inactive NopM form) in vitro. Bimolecular fluorescence complementation analysis provided clues about NopM–NopM interactions at plasma membranes in planta. NopM, but not C338A, expressed in tobacco cells induced cell death, suggesting that E3 ubiquitin ligase activity of NopM induced effector-triggered immunity responses. Likewise, expression of NopM in Lotus japonicus caused reduced nodule formation, whereas expression of C338A showed no obvious effects on symbiosis. Further experiments indicated that serine residue 26 of NopM is phosphorylated in planta and that NopM can be phosphorylated in vitro by salicylic acid–induced protein kinase (NtSIPK), a mitogen-activated protein kinase (MAPK) of tobacco. Hence, NopM is a phosphorylated T3 effector that can interact with itself, with ubiquitin, and with MAPKs.

Nodulation outer protein M (NopM) is an IpaH family type three (T3) effector secreted by the nitrogen-fixing nodule bacterium Sinorhizobium sp. strain NGR234. Previous work indicated that NopM is an E3 ubiquitin ligase required for an optimal symbiosis between NGR234 and the host legume Lablab purpureus. Here, we continued to analyze the function of NopM. Recombinant NopM was biochemically characterized using an in vitro ubiquitination system with Arabidopsis thaliana proteins. In this assay, NopM forms unanchored polyubiquitin chains and possesses auto-ubiquitination activity. In a NopM variant lacking any lysine residues, auto-ubiquitination was not completely abolished, indicating noncanonical auto-ubiquitination of the protein. In addition, we could show intermolecular ubiquitin transfer from NopM to C338A (enzymatically inactive NopM form) in vitro. Bimolecular fluorescence complementation analysis provided clues about NopM–NopM interactions at plasma membranes in planta. NopM, but not C338A, expressed in tobacco cells induced cell death, suggesting that E3 ubiquitin ligase activity of NopM induced effector-triggered immunity responses. Likewise, expression of NopM in Lotus japonicus caused reduced nodule formation, whereas expression of C338A showed no obvious effects on symbiosis. Further experiments indicated that serine residue 26 of NopM is phosphorylated in planta and that NopM can be phosphorylated in vitro by salicylic acid–induced protein kinase (NtSIPK), a mitogen-activated protein kinase (MAPK) of tobacco. Hence, NopM is a phosphorylated T3 effector that can interact with itself, with ubiquitin, and with MAPKs.

Introduction The involvement of students in the embodiment of university teaching through peer-assisted learning formats is commonly applied. Publications on this topic exclusively focus on strictly defined situations within the curriculum and selected target groups. This study, in contrast, presents and evaluates a large-scale structured and quality-assured peer teaching programme, which offers diverse and targeted courses throughout the preclinical part of the medical curriculum. Methods The large-scale peer teaching programme consists of subject specific and interdisciplinary tutorials that address all scientific, physiological and anatomic subjects of the preclinical curriculum as well as tutorials with contents exceeding the formal curriculum. In the study year 2013/14 a total of 1420 lessons were offered within the programme. Paper-based evaluations were conducted over the full range of courses. Acceptance and benefit of this peer teaching programme were evaluated in a retrospective study covering the period 2012 to 2014. Usage of tutorials by students who commenced their studies in 2012/13 (n=959) was analysed from 2012 till 2014. Based on the results of 13 first assessments in the preclinical subjects anatomy, biochemistry and physiology, the students were assigned to one of five groups. These groups were compared according to participation in the tutorials. To investigate the benefit of tutorials of the peer teaching programme, the results of biochemistry re-assessments of participants and non-participants of tutorials in the years 2012 till 2014 (n=188, 172 and 204, respectively) were compared using Kolmogorov-Smirnov- and Chi-square tests as well as the effect size Cohen's d . Results Almost 70 % of the students attended the voluntary additional programme during their preclinical studies. The students participating in the tutorials had achieved different levels of proficiency in first assessments. The acceptance of different kinds of tutorials appears to correlate with their performance in first assessments. 94% of the students participating in tutorials offered in the study year 2013/14 rated the tutorials as "excellent" or "good". An objective benefit has been shown by a significant increase in re-assessment scores with an effect size between the medium and large magnitudes for participants of tutorials compared to non-participants in the years 2012, 2013 and 2014. In addition, significantly higher pass rates of re-assessments could be observed. Conclusion Acceptance, utilisation and benefit of the assessed peer teaching programme are high. Beyond the support of students, a contribution to the individualisation of studies and teaching is made. Further studies are necessary to investigate possible influences of large-scale peer teaching programmes, for example on the reduction of study length and drop-off rates, as well as additional effects on academic achievements.

Our current understanding of pathogen-associated molecular pattern (PAMP)-triggered immunity signaling pathways in plants is limited due to the redundancy of several components or the lethality of mutants in Arabidopsis (Arabidopsis thaliana). To overcome this, we used a virus-induced gene silencing-based approach in combination with pharmacological studies to decipher links between early PAMP-triggered immunity events and their roles in immunity following PAMP perception in Nicotiana benthamiana. Two different calcium influx inhibitors suppressed the reactive oxygen species (ROS) burst: activation of the mitogen-activated protein kinases (MAPKs) and PAMP-induced gene expression. The calcium burst was unaffected in plants specifically silenced for components involved in ROS generation or for MAPKs activated by PAMP treatment. Importantly, the ROS burst still occurred in plants silenced for the two major defense-associated MAPK genes NbSIPK (for salicylic acid-induced protein kinase) and NbWIPK (for wound-induced protein kinase) or for both genes simultaneously, demonstrating that these MAPKs are dispensable for ROS production. We further show that NbSIPK silencing is sufficient to prevent PAMP-induced gene expression but that both MAPKs are required for bacterial immunity against two virulent strains of Pseudomonas syringae and their respective nonpathogenic mutants. These results suggest that the PAMP-triggered calcium burst is upstream of separate signaling branches, one leading to MAPK activation and then gene expression and the other to ROS production. In addition, this study highlights the essential roles of NbSIPK and NbWIPK in antibacterial immunity. Unexpectedly, negative regulatory mechanisms controlling the intensity of the PAMP-triggered calcium and ROS bursts were also revealed by this work.

The major virulence strategy for plant pathogenic bacteria is deployment of effector molecules within the host cytoplasm. Each bacterial strain possesses a set of 20-30 effectors which have overlapping activities, are functionally interchangeable, and diverge in composition between strains. Effectors target host molecules to suppress immunity. Two main strategies are apparent. Effectors that target host proteins seem to attack conserved structural domains but otherwise lack specificity. On the other hand, those that influence host gene transcription directly do so with extreme specificity. In both cases, examples are known where the host has exploited effector-target affinities to establish immune recognition of effectors. The molecular activity of each effector links virulence and immune outcomes.

Plant pathogenic bacteria inject about 30 virulence effector proteins into the host cell using a specialized secretion apparatus. Bacteria which are unable to do this elicit host immunity and cannot grow inside living plant tissue. Thus, the primary function of the effectors is to suppress host immunity. The identity of individual effectors within each complement varies even between closely related bacterial strains, and effectors themselves act redundantly and are apparently interchangeable. Many effectors are known to target components of plant defense pathways, but it is difficult to study their role in molecular terms. For some of them, there is controversy about their mode of action. We propose that effectors act promiscuously by targeting host molecules with low specificity and affinity.

SummaryPlant innate immunity relies on a set of pattern recognition receptors (PRRs) that respond to ligands known as pathogen-associated molecular patterns (PAMPs) [1–3]. To overcome such immunity, phytopathogenic bacteria deliver virulence molecules called effector proteins into the plant cell that collectively promote pathogenesis [4–7]. The vast majority of PRRs controlling PAMP-triggered immunity (PTI) and the mechanisms used by specific effectors to suppress these pathways are mostly unknown. Here, we show that the Arabidopsis LysM receptor kinase CERK1 [8, 9], which is critical for chitin elicitor signaling and resistance to fungal pathogens, plays an essential role in restricting bacterial growth on plants. This is supported by the fact that CERK1 is a target of the bacterial type III effector protein AvrPtoB, which blocks all defense responses through this receptor. AvrPtoB ubiquitinates the CERK1 kinase domain in vitro and targets CERK1 for degradation in vivo. We show that CERK1 is a determinant of bacterial immunity, but its contribution is overcome by bacteria expressing AvrPtoB. Our results reveal a new pathway for plant immunity against bacteria and a role for AvrPtoB E3-ligase activity in suppressing PTI.

In pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), plant cell surface receptors sense potential microbial pathogens by recognizing elicitors called PAMPs. Although diverse PAMPs trigger PTI through distinct receptors, the resulting intracellular responses overlap extensively. Despite this, a common component(s) linking signal perception with transduction remains unknown. In this study, we identify SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)3/brassinosteroid-associated kinase (BAK)1, a receptor-like kinase previously implicated in hormone signaling, as a component of plant PTI. In Arabidopsis thaliana , AtSERK3/BAK1 rapidly enters an elicitor-dependent complex with FLAGELLIN SENSING 2 (FLS2), the receptor for the bacterial PAMP flagellin and its peptide derivative flg22. In the absence of AtSERK3/BAK1 , early flg22-dependent responses are greatly reduced in both A. thaliana and Nicotiana benthamiana . Furthermore, N. benthamiana Serk3/Bak1 is required for full responses to unrelated PAMPs and, importantly, for restriction of bacterial and oomycete infections. Thus, SERK3/BAK1 appears to integrate diverse perception events into downstream PAMP responses, leading to immunity against a range of invading microbes.

The plant's innate immune system detects potential biotic threats through recognition of microbe-associated molecular patterns (MAMPs) or danger-associated molecular patterns (DAMPs) by pattern recognition receptors (PRR). A central regulator of pattern-triggered immunity (PTI) is the BRI1-associated kinase 1 (BAK1), which undergoes complex formation with PRR upon ligand binding. Although viral patterns inducing PTI are well known from animal systems, nothing similar has been reported for plants. Rather, antiviral defense in plants is thought to be mediated by post-transcriptional gene silencing of viral RNA or through effector-triggered immunity, i.e., recognition of virus-specific effectors by resistance proteins. Nevertheless, infection by compatible viruses can also lead to the induction of defense gene expression, indicating that plants may also recognize viruses through PTI. Here, we show that PTI, or at least the presence of the regulator BAK1, is important for antiviral defense of Arabidopsis plants. Arabidopsis bak1 mutants show increased susceptibility to three different RNA viruses during compatible interactions. Furthermore, crude viral extracts but not purified virions induce several PTI marker responses in a BAK1-dependent manner. Overall, we conclude that BAK1-dependent PTI contributes to antiviral resistance in plants.

Conserved microbial molecules known as PAMPs (pathogen-associated molecular patterns) elicit defence responses in plants through extracellular receptor proteins. One important PAMP is the flagellin protein derived from motile bacteria. We show here that the solanaceous species Nicotiana benthamiana perceives the flagellin proteins of both pathogenic and non-host species of Pseudomonas syringae. The response to flagellin required a gene closely related to that encoding the Arabidopsis thaliana flagellin receptor that we designated NbFls2. In addition, silencing of NbFls2 led to increased growth of compatible, non-host and non-pathogenic strains of P. syringae. Thus, flagellin perception restricts growth of P. syringae strains on N. benthamiana. Pathogenic bacteria secrete effector proteins into the plant cell to enhance virulence. We tested the ability of several unrelated effectors to suppress PAMP-mediated defences. The effector proteins AvrPto and AvrPtoB, but not AvrRps4, suppressed all responses tested including the hypersensitive response induced by non-host flagellins and the oomycete elicitor INF1. Strikingly, transient expression of avrPto or avrPtoB stimulated the growth of non-pathogenic Agrobacterium tumefaciensin planta, suggesting that multiplication of this species is also restricted by PAMP perception. Unexpectedly, AvrPtoB but not AvrPto required the defence-associated genes Rar1, Sgt1 and Eds1 for suppression. This observation separates the respective mechanisms of the two effectors, and suggests that AvrPtoB may target the defence machinery directly for its suppressive effect.

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., Author Summary Many Gram-negative bacterial pathogens possess type 3 secretion systems, which deliver effector proteins into eukaryotic host cells through needle-like structures. Effectors manipulate the host cell and many of them suppress host defense responses. Interestingly, certain symbiotic strains of rhizobia also possess such secretion systems. Rhizobia infect legume roots and induce root nodules, where the bacteria convert atmospheric nitrogen into ammonia. Here, we characterize the effector NopM of Rhizobium sp. strain NGR234. We demonstrate that NopM possesses E3 ubiquitin ligase activity, indicating that NopM can “tag" proteins with ubiquitin, and thus target them for proteasome-dependent degradation. Using a mutant approach, we demonstrate that enzymatically active NopM promotes establishment of symbiosis with Lablab purpureus, the host plant from which NGR234 was originally isolated. We further examine effects of NopM when directly expressed in eukaryotic cells and show that NopM interferes with specific signaling pathways. NopM expressed in the model plant Nicotiana benthamiana dampened generation of reactive oxygen species (ROS), which are formed in response to the bacterial flagellin peptide flg22. We suggest that NopM promotes nodule initiation by reducing the levels of harmful ROS during the infection process.

Plant pathogenic bacteria adhere to cell walls and remain external to the cell throughout the pathogenic lifecycle, where they elicit host immunity through host plasma membrane localised receptors. To be successful pathogens, bacteria must suppress these defence responses, which they do by secreting a suite of virulence effector molecules into the host cytoplasm. However, effectors themselves can act as elicitors after perception by intracellular host immune receptors, thus, re-activating plant immunity. Bacterial effectors generally target host molecules through specific molecular activities to defeat plant defence responses. Although effectors can be used as tools to elucidate components of plant immunity, only a handful of these molecular targets are known and much remains to be learnt about effector strategies for bacterial pathogenicity. This review highlights recent advances in our understanding of the mode of action of bacterial effectors, which in the future will lead to improvements in agriculture.