The RIN4-like/NOI proteins NOI10 and NOI11 modulate the response to biotic stresses mediated by RIN4 in Arabidopsis.

KEY MESSAGE: NOI10 and NOI11 are two RIN4-like/NOI proteins that participate in the immune response of the Arabidopsis plant and affect the RIN4-regulated mechanisms involving the R-proteins RPM1 and RPS2. The immune response in plants depends on the regulation of signaling pathways triggered by pathogens and herbivores. RIN4, a protein of the RIN4-like/NOI family, is considered to be a central immune signal in the interactions of plants and pathogens. In Arabidopsis thaliana, four of the 15 members of the RIN4-like/NOI family (NOI3, NOI5, NOI10, and NOI11) were induced in response to the plant herbivore Tetranychus urticae. While overexpressing NOI10 and NOI11 plants did not affect mite performance, opposite callose accumulation patterns were observed when compared to RIN4 overexpressing plants. In vitro and in vivo analyses demonstrated the interaction of NOI10 and NOI11 with the RIN4 interactors RPM1, RPS2, and RIPK, suggesting a role in the context of the RIN4-regulated immune response. Transient expression experiments in Nicotiana benthamiana evidenced that NOI10 and NOI11 differed from RIN4 in their functionality. Furthermore, overexpressing NOI10 and NOI11 plants had significant differences in susceptibility with WT and overexpressing RIN4 plants when challenged with Pseudomonas syringae bacteria expressing the AvrRpt2 or the AvrRpm1 effectors. These results demonstrate the participation of NOI10 and NOI11 in the RIN4-mediated pathway. Whereas RIN4 is considered a guardee protein, NOI10 and NOI11 could act as decoys to modulate the concerted activity of effectors and R-proteins.

The Upregulated Expression of the Citrus RIN4 Gene in HLB Diseased Citrus Aids Candidatus Liberibacter Asiaticus Infection.

The citrus industry has been threatened by Huanglongbing (HLB) for over a century. Here, an HLB-induced Arabidopsis RPM1-interacting protein 4 (RIN4) homologous gene was cloned from Citrus clementina, and its characteristics and function were analyzed to determine its role during citrus-Candidatus Liberibacter asiaticus (CLas) interactions. Quantitative real-time PCR showed that RIN4 was expressed in roots, stems, leaves and flowers, with the greatest expression level in leaves. Its expression was suppressed by gibberellic acid, indole-3-acetic acid, salicylic acid and jasmonic acid treatments, but was induced by abscisic acid and salt treatments, as well as wounding. The transient expression of a RIN4-GFP showed that RIN4 was localized in the cell membrane. RIN4-overexpressing transgenic C. maxima cv. 'Shatianyou' plants were obtained, and some transgenic plants showed greater sensitivity to CLas infection and earlier HLB symptoms appearance than non-transgenic controls. Results obtained in this study indicated that the upregulated expression of RIN4 in HLB diseased citrus may aid CLas infection.

Investigation of natural RIN4 variants reveals a motif crucial for function and provides an opportunity to broaden NLR regulation specificity.

Multiple bacterial effectors target RPM1-INTERACTING PROTEIN4 (RIN4), the biochemical modifications of which are recognized by several plant nucleotide-binding and leucine-rich repeat immune receptor (NLR) proteins. Recently, a comparative study of Arabidopsis and apple (Malus domestica) RIN4s revealed that the RIN4 specificity motif (RSM) is critical for NLR regulation. Here, we investigated the extent to which the RSM contributes to the functions of natural RIN4 variants. Functional analysis of 33 natural RIN4 variants from 28 plant species showed that the RSM is generally required yet sometimes dispensable for the RIN4-mediated suppression of NLR auto-activity or effector-triggered NLR activation. Association analysis of the sequences and fire blight resistance gene originating from Malus × robusta 5 (FB_MR5) activation functions of the natural RIN4 variants revealed H167 to be an indispensable residue for RIN4 function in the regulation of NLRs. None of the tested natural RIN4 variants could suppress RESISTANCE TO PSEUDOMONAS SYRINGAE PV. MACULICOLA1 (RPM1) auto-activity and activate FB_MR5. To engineer RIN4 to carry broader NLR compatibility, we generated chimeric RIN4 proteins, several of which could regulate RPM1, RESISTANT TO PSEUDOMONAS SYRINGAE2 (RPS2), and FB_MR5. We propose that the intrinsically disordered nature of RIN4 provides a flexible platform to broaden pathogen recognition specificity by establishing compatibility with otherwise incompatible NLRs.

RIN4 homologs from important crop species differentially regulate the Arabidopsis NB-LRR immune receptor, RPS2.

KEY MESSAGE: RIN4 homologs from important crop species differ in their ability to prevent ectopic activity of the nucleotide binding-leucine rich repeat resistance protein, RPS2. Pathogens deploy virulence effectors to perturb host processes. Plants utilize intracellular resistance (R) proteins to recognize pathogen effectors either by direct interaction or indirectly via effector-mediated perturbations of host components. RPM1-INTERACTING PROTEIN4 (RIN4) is a plant immune regulator that mediates the indirect activation of multiple, independently evolved R-proteins by multiple, unrelated effector proteins. One of these, RPS2 (RESISTANT TO P. SYRINGAE2), is activated upon cleavage of Arabidopsis (At)RIN4 by the Pseudomonas syringae effector AvrRpt2. To gain insight into the AvrRpt2-RIN4-RPS2 defense-activation module, we compared the function of AtRIN4 with RIN4 homologs present in a diverse range of plant species. We selected seven homologs containing conserved features of AtRIN4, including two NOI (Nitrate induced) domains, each containing a predicted cleavage site for AvrRpt2, and a C-terminal palmitoylation site predicted to mediate membrane tethering of the proteins. Palmitoylation-mediated tethering of AtRIN4 to the plasma membrane and cleavage by AvrRpt2 are required for suppression and activation of RPS2, respectively. While all seven homologs are localized at the plasma membrane, only four suppress RPS2 when transiently expressed in Nicotiana benthamiana. All seven homologs are cleaved by AvrRpt2 and, for those homologs that are able to suppress RPS2, cleavage relieves suppression of RPS2. Further, we demonstrate that the membrane-tethered, C-terminal AvrRpt2-generated cleavage fragment is sufficient for the suppression of RPS2. Lastly, we show that the membrane localization of RPS2 is unaffected by its suppression or activation status.

The resistance associated protein RIN4 promotes the extracellular transport of AtEXO70E2.

RIN4 is an important immunomodulator in Arabidopsis, which is targeted by multiple pathogenic effectors, and consequently guarded by different immune receptors. Although RIN4 plays a significant role in plant immunity, its molecular function is not fully understood. We found that RIN4 interacts with the exocyst subunit EXO70E2. Transiently expressed RIN4 can recruits EXO70E2 vesicles to the plasma membrane, and promote the transport of the vesicles to the extracellular matrix. RIN4 also can decrease the protein level of EXO70E2. Base on the fact that EXO70 proteins positively mediates plant immunity, the function of RIN4 is to promote the extracellular export of defense related vesicles. Pathogens will secret effectors to modify or cleavage it to interfere this exocytosis.

Functions of RPM1-interacting protein 4 in plant immunity.

KEY MESSAGE: We reviewed recent advances related to RIN4, including its involvement in the immune process through posttranslational modifications, PM H+-ATPase activity regulation, interaction with EXO70 and identification of RIN4-associated NLR proteins. RPM1-interacting protein 4 (RIN4) is a conserved plant immunity regulator that has been extensively studied and can be modified by pathogenic effector proteins. RIN4 plays an important role in both PTI and ETI. In this article, we review the functions of the two conserved NOI domains of RIN4, the C-terminal cysteine residues required for membrane localization and the sites targeted and modified by effector proteins during plant immunity. In addition, we discuss the effect of RIN4 on the stomatal virulence of pathogens via the regulation of PM H+-ATPase activity, which is involved in the immune process through interactions with the exocyst subunit EXO70, and progress in the identification of RIN4-related R proteins in multiple species. This review provides new insights enhancing the current understanding of the immune function of RIN4.

Inositol-requiring enzyme 1 (IRE1) plays for AvrRpt2-triggered immunity and RIN4 cleavage in Arabidopsis under endoplasmic reticulum (ER) stress.

Many stresses induce the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum, a phenomenon known as ER stress. In response to ER stress, cells initiate a protective response, known as unfolded protein response (UPR), to maintain cellular homeostasis. The UPR sensor, inositol-requiring enzyme 1 (IRE1), catalyzes the cytoplasmic splicing of bZIP transcription factor-encoding mRNAs to activate the UPR signaling pathway. Recently, we reported that pretreatment of Arabidopsis thaliana plants with tunicamycin, an ER stress inducer, increased their susceptibility to bacterial pathogens; on the other hand, IRE1 deficient mutants were susceptible to Pseudomonas syringae pv. maculicola (Psm) and failed to induce salicylic acid (SA)-mediated systemic acquired resistance. However, the functional relationship of IRE1 with the pathogen and TM treatment remains unknown. In the present study, we showed that bacterial pathogen-associated molecular patterns (PAMPs) induced IRE1 expression; however, PAMP-triggered immunity (PTI) response such as callose deposition, PR1 protein accumulation, or Pst DC3000 hrcC growth was not altered in ire1 mutants. We observed that IRE1 enhanced plant immunity against the bacterial pathogen P. syringae pv. tomato DC3000 (Pst DC3000) under ER stress. Moreover, TM-pretreated ire1 mutants were more susceptible to the avirulent strain Pst DC3000 (AvrRpt2) and showed greater cell death than wild-type plants during effector-triggered immunity (ETI). Additionally, Pst DC3000 (AvrRpt2)-mediated RIN4 degradation was reduced in ire1 mutants under TM-induced ER stress. Collectively, our results reveal that IRE1 plays a pivotal role in the immune signaling pathway to activate plant immunity against virulent and avirulent bacterial strains under ER stress.

Ptr1 evolved convergently with RPS2 and Mr5 to mediate recognition of AvrRpt2 in diverse solanaceous species.

The Ptr1 (Pseudomonas tomato race 1) locus in Solanum lycopersicoides confers resistance to strains of Pseudomonas syringae pv. tomato expressing AvrRpt2 and Ralstonia pseudosolanacearum expressing RipBN. Here we describe the identification and phylogenetic analysis of the Ptr1 gene. A single recombinant among 585 F2 plants segregating for the Ptr1 locus was discovered that narrowed the Ptr1 candidates to eight nucleotide-binding leucine-rich repeat protein (NLR)-encoding genes. From analysis of the gene models in the S. lycopersicoides genome sequence and RNA-Seq data, two of the eight genes emerged as the strongest candidates for Ptr1. One of these two candidates was found to encode Ptr1 based on its ability to mediate recognition of AvrRpt2 and RipBN when it was transiently expressed with these effectors in leaves of Nicotiana glutinosa. The ortholog of Ptr1 in tomato and in Solanum pennellii is a pseudogene. However, a functional Ptr1 ortholog exists in Nicotiana benthamiana and potato, and both mediate recognition of AvrRpt2 and RipBN. In apple and Arabidopsis, recognition of AvrRpt2 is mediated by the Mr5 and RPS2 proteins, respectively. Phylogenetic analysis places Ptr1 in a distinct clade compared with Mr5 and RPS2, and it therefore appears to have arisen by convergent evolution for recognition of AvrRpt2.

Populus euphratica remorin 6.5 activates plasma membrane H+-ATPases to mediate salt tolerance.

Remorins (REMs) play an important role in the ability of plants to adapt to adverse environments. PeREM6.5, a protein of the REM family in Populus euphratica (salt-resistant poplar), was induced by NaCl stress in callus, roots and leaves. We cloned the full-length PeREM6.5 from P. euphratica and transformed it into Escherichia coli and Arabidopsis thaliana. PeREM6.5 recombinant protein significantly increased the H+-ATPase hydrolytic activity and H+ transport activity in P. euphratica plasma membrane (PM) vesicles. Yeast two-hybrid assay showed that P. euphratica REM6.5 interacted with RPM1-interacting protein 4 (PeRIN4). Notably, the PeREM6.5-induced increase in PM H+-ATPase activity was enhanced by PeRIN4 recombinant protein. Overexpression of PeREM6.5 in Arabidopsis significantly improved salt tolerance in transgenic plants in terms of survival rate, root growth, electrolyte leakage and malondialdehyde content. Arabidopsis plants overexpressing PeREM6.5 retained high PM H+-ATPase activity in both in vivo and in vitro assays. PeREM6.5-transgenic plants had reduced accumulation of Na+ due to the Na+ extrusion promoted by the H+-ATPases. Moreover, the H+ pumps caused hyperpolarization of the PM, which reduced the K+ loss mediated by the depolarization-activated channels in the PM of salinized roots. Therefore, we conclude that PeREM6.5 regulated H+-ATPase activity in the PM, thus enhancing the plant capacity to maintain ionic homeostasis under salinity.

Rpa1 mediates an immune response to avrRpm1Psa and confers resistance against Pseudomonas syringae pv. actinidiae.

The type three effector AvrRpm1Pma from Pseudomonas syringae pv. maculicola (Pma) triggers an RPM1-mediated immune response linked to phosphorylation of RIN4 (RPM1-interacting protein 4) in Arabidopsis. However, the effector-resistance (R) gene interaction is not well established with different AvrRpm1 effectors from other pathovars. We investigated the AvrRpm1-triggered immune responses in Nicotiana species and isolated Rpa1 (Resistance to Pseudomonas syringae pv. actinidiae 1) via a reverse genetic screen in Nicotiana tabacum. Transient expression and gene silencing were performed in combination with co-immunoprecipitation and growth assays to investigate the specificity of interactions that lead to inhibition of pathogen growth. Two closely related AvrRpm1 effectors derived from Pseudomonas syringae pv. actinidiae biovar 3 (AvrRpm1Psa ) and Pseudomonas syringae pv. syringae strain B728a (AvrRpm1Psy ) trigger immune responses mediated by RPA1, a nucleotide-binding leucine-rich repeat protein with an N-terminal coiled-coil domain. In a display of contrasting specificities, RPA1 does not respond to AvrRpm1Pma , and correspondingly AvrRpm1Psa and AvrRpm1Psy do not trigger the RPM1-mediated response, demonstrating that separate R genes mediate specific immune responses to different AvrRpm1 effectors. AvrRpm1Psa co-immunoprecipitates with RPA1, and both proteins co-immunoprecipitate with RIN4. In contrast with RPM1, however, RPA1 was not activated by the phosphomimic RIN4T166D and silencing of RIN4 did not affect the RPA1 activity. Delivery of AvrRpm1Psa by Pseudomonas syringae pv. tomato (Pto) in combination with transient expression of Rpa1 resulted in inhibition of the pathogen growth in N. benthamiana. Psa growth was also inhibited by RPA1 in N. tabacum.

A host target of a bacterial cysteine protease virulence effector plays a key role in convergent evolution of plant innate immune system receptors.

Some virulence effectors secreted from pathogens target host proteins and induce biochemical modifications that are monitored by nucleotide-binding and leucine-rich repeat (NLR) immune receptors. Arabidopsis RIN4 protein (AtRIN4: RPM1-interacting protein 4) homologs are present in diverse plant species and targeted by several bacterial type III effector proteins including the cysteine protease AvrRpt2. RIN4 is 'guarded' by several independently evolved NLRs from various plant species, including Arabidopsis RPS2. Recently, it was shown that the MR5 NLR from a wild apple relative can recognize the AvrRpt2 effector from Erwinia amylovora, but the details of this recognition remained unclear. The present contribution reports the mechanism of AvrRpt2 recognition by independently evolved NLRs, MR5 from apple and RPS2, both of which require proteolytically processed RIN4 for activation. It shows that the C-terminal cleaved product of apple RIN4 (MdRIN4) but not AtRIN4 is necessary and sufficient for MR5 activation. Additionally, two polymorphic residues in AtRIN4 and MdRIN4 are identified that are crucial in the regulation of and physical association with NLRs. It is proposed that polymorphisms in RIN4 from distantly related plant species allow it to remain an effector target while maintaining compatibility with multiple NLRs.

Role of RIN4 in Regulating PAMP-Triggered Immunity and Effector-Triggered Immunity: Current Status and Future Perspectives.

As sessile organisms, plants have developed sophisticated system to defend themselves against microbial attack. Since plants do not have specialized immune cells, all plant cells appear to have the innate ability to recognize pathogens and turn on an appropriate defense response. The plant innate immune system has two major branches: PAMPs (pathogen associated molecular patterns)-triggered immunity (PTI) and effector-triggered immunity (ETI). The ability to discriminate between self and non-self is a fundamental feature of living organisms, and it is a prerequisite for the activation of plant defenses specific to microbial infection. Arabidopsis cells express receptors that detect extracellular molecules or structures of the microbes, which are called collectively PAMPs and activate PTI. However, nucleotidebinding site leucine-rich repeats (NB-LRR) proteins mediated ETI is induced by direct or indirect recognition of effector molecules encoded by avr genes. In Arabidopsis, plasmamembrane localized multifunctional protein RIN4 (RPM1interacting protein 4) plays important role in both PTI and ETI. Previous studies have suggested that RIN4 functions as a negative regulator of PTI. In addition, many different bacterial effector proteins modify RIN4 to destabilize plant immunity and several NB-LRR proteins, including RPM1 (resistance to Pseudomonas syringae pv. maculicola 1), RPS2 (resistance to P. syringae 2) guard RIN4. This review summarizes the current studies that have described signaling mechanism of RIN4 function, modification of RIN4 by bacterial effectors and different interacting partner of RIN4 in defense related pathway. In addition, the emerging role of the RIN4 in plant physiology and intercellular signaling as it presents in exosomes will be discussed.

The Ptr1 Locus of Solanum lycopersicoides Confers Resistance to Race 1 Strains of Pseudomonas syringae pv. tomato and to Ralstonia pseudosolanacearum by Recognizing the Type III Effectors AvrRpt2 and RipBN.

Race 1 strains of Pseudomonas syringae pv. tomato, which cause bacterial speck disease of tomato, are becoming increasingly common and no simply inherited genetic resistance to such strains is known. We discovered that a locus in Solanum lycopersicoides, termed Pseudomonas tomato race 1 (Ptr1), confers resistance to race 1 P. syringae pv. tomato strains by detecting the activity of type III effector AvrRpt2. In Arabidopsis, AvrRpt2 degrades the RIN4 protein, thereby activating RPS2-mediated immunity. Using site-directed mutagenesis of AvrRpt2, we found that, like RPS2, activation of Ptr1 requires AvrRpt2 proteolytic activity. Ptr1 also detected the activity of AvrRpt2 homologs from diverse bacteria, including one in Ralstonia pseudosolanacearum. The genome sequence of S. lycopersicoides revealed no RPS2 homolog in the Ptr1 region. Ptr1 could play an important role in controlling bacterial speck disease and its future cloning may shed light on an example of convergent evolution for recognition of a widespread type III effector.

Pathogens and Disease Play Havoc on the Host Epiproteome-The "First Line of Response" Role for Proteomic Changes Influenced by Disorder.

Organisms face stress from multiple sources simultaneously and require mechanisms to respond to these scenarios if they are to survive in the long term. This overview focuses on a series of key points that illustrate how disorder and post-translational changes can combine to play a critical role in orchestrating the response of organisms to the stress of a changing environment. Increasingly, protein complexes are thought of as dynamic multi-component molecular machines able to adapt through compositional, conformational and/or post-translational modifications to control their largely metabolic outputs. These metabolites then feed into cellular physiological homeostasis or the production of secondary metabolites with novel anti-microbial properties. The control of adaptations to stress operates at multiple levels including the proteome and the dynamic nature of proteomic changes suggests a parallel with the equally dynamic epigenetic changes at the level of nucleic acids. Given their properties, I propose that some disordered protein platforms specifically enable organisms to sense and react rapidly as the first line of response to change. Using examples from the highly dynamic host-pathogen and host-stress response, I illustrate by example how disordered proteins are key to fulfilling the need for multiple levels of integration of response at different time scales to create robust control points.

RIN4 recruits the exocyst subunit EXO70B1 to the plasma membrane.

The exocyst is a conserved vesicle-tethering complex with principal roles in cell polarity and morphogenesis. Several studies point to its involvement in polarized secretion during microbial pathogen defense. In this context, we have found an interaction between the Arabidopsis EXO70B1 exocyst subunit, a protein which was previously associated with both the defense response and autophagy, and RPM1 INTERACTING PROTEIN 4 (RIN4), the best studied member of the NOI protein family and a known regulator of plant defense pathways. Interestingly, fragments of RIN4 mimicking the cleavage caused by the Pseudomonas syringae effector protease, AvrRpt2, fail to interact strongly with EXO70B1. We observed that transiently expressed RIN4, but not the plasma membrane (PM) protein aquaporin PIP2, recruits EXO70B1 to the PM. Unlike EXO70B1, RIN4 does not recruit the core exocyst subunit SEC6 to the PM under these conditions. Furthermore, the AvrRpt2 effector protease delivered by P. syringae is able to release both RIN4 and EXO70B1 to the cytoplasm. We present a model for how RIN4 might regulate the localization and putative function of EXO70B1 and speculate on the role the AvrRpt2 protease might have in the regulation of this defense response.

The intrinsically disordered structural platform of the plant defence hub protein RPM1-interacting protein 4 provides insights into its mode of action in the host-pathogen interface and evolution of the nitrate-induced domain protein family.

Arabidopsis thaliana (At) RPM1-interacting protein 4 (RIN4), targeted by many defence-suppressing bacterial type III effectors and monitored by several resistance proteins, regulates plant immune responses to pathogen-associated molecular patterns and type III effectors. Little is known about the overall protein structure of AtRIN4, especially in its unbound form, and the relevance of structure to its diverse biological functions. AtRIN4 contains two nitrate-induced (NOI) domains and is a member of the NOI family. Using experimental and bioinformatic approaches, we demonstrate that the unbound AtRIN4 is intrinsically disordered under physiological conditions. The intrinsically disordered polypeptide chain of AtRIN4 is interspersed with molecular recognition features (MoRFs) and anchor-identified long-binding regions, potentially allowing it to undergo disorder-to-order transitions upon binding to partner(s). A poly-l-proline II structure, often responsible for protein recognition, is also identified in AtRIN4. By performing bioinformatics analyses on RIN4 homologues from different plant species and the NOI proteins from Arabidopsis, we infer the conservation of intrinsic disorder, MoRFs and long-binding regions of AtRIN4 in other plant species and the NOI family. Intrinsic disorder and MoRFs could provide RIN4 proteins with the binding promiscuity and plasticity required to act as hubs in a pivotal position within plant defence signalling cascades.

Investigating the functions of the RIN4 protein complex during plant innate immune responses.

Pathogen recognition by the plant innate immune system invokes a sophisticated signal transduction network that culminates in disease resistance. The Arabidopsis protein RIN4 is a well-known regulator of plant immunity. However, the molecular mechanisms by which RIN4 controls multiple immune responses have remained elusive. in our recently published study, we purified components of the RIN4 protein complex from A. thaliana and identified several novel RIN4-associated proteins.1 we found that one class of RIN4-associated proteins, the plasma membrane H(+)-ATPases AHA1 and AHA2, play a crucial role in resisting pathogen invasion. Plants use RIN4 to regulate H(+)-ATPase activity during immune responses, thereby controlling stomatal apertures during pathogen attack. Stomata were previously identified as active regulators of plant immune responses during pathogen invasion, but how the plant innate immune system coordinates this response was unknown.2,3 Our investigations have revealed a novel function of rin4 during pathogenesis. Here, we discuss the rin4-AHA1/2 interaction and highlight additional RIN4-associated proteins (RAPs) as well as speculate on their potential roles in plant innate immunity.