Recent evidence suggests that the ubiquitin-proteasome system (UPS) is involved in several aspects of plant immunity and a range of plant pathogens subvert the UPS to enhance their virulence. Here, we show that proteasome activity is strongly induced during basal defense in Arabidopsis and mutant lines defective in proteasome subunits RPT2a and RPN12a support increased bacterial growth of virulent Pseudomonas syringae DC3000 (Pst), strains in local leaves. Both proteasome subunits are required for PTI events such as production of reactive oxygen species and mitogen-activated protein kinases signaling as well as for defense gene expression. Furthermore, analysis of bacterial growth after a secondary infection of systemic leaves revealed that the establishment of systemic-acquired resistance (SAR) is impaired in proteasome mutants, suggesting that the proteasome plays an important role in defense priming and SAR. In addition, we show that Pst inhibits proteasome activity in a type-III secretion dependent manner. A systematic screen for type-III effector proteins from Pst for their ability to interfere with proteasome activity revealed HopM1, HopAO1, HopA1 and HopG1 as candidates. Identification of proteins interacting with HopM1 by mass-spectrometry indicate that HopM1 resides in a complex together with several E3 ubiquitin ligases and proteasome subunits, supporting the hypothesis that HopM1 associates with the proteasome leading to its inhibition. We conclude that the proteasome is an essential component of the plant immune system and that some pathogens have developed a general strategy to overcome proteasome-mediated defense.
Phytopathogenic bacteria of the Xanthomonas genus cause severe diseases on hundreds of host plants, including economically important crops, such as bean, cabbage, cassava, citrus, hemp, pepper, rice, sugarcane, tomato or wheat. Diseases occurring in nature comprise bacterial blight, canker, necrosis, rot, scald, spot, streak or wilt. Xanthomonas spp. are distributed worldwide and pathogenic and nonpathogenic strains are essentially found in association to plants. Some phytopathogenic strains are emergent or re-emergent and, consequently, dramatically impact agriculture, economy and food safety. During the last decades, massive efforts were undertaken to decipher Xanthomonas biology. So far, more than one hundred complete or draft genomes from diverse Xanthomonas species have been sequenced (http://www.xanthomonas.org), thus providing powerful tools to study genetic determinants triggering pathogenicity and adaptation to plant habitats. Xanthomonas spp. employ an arsenal of virulence factors to invade its host, including extracellular polysaccharides, plant cell wall-degrading enzymes, adhesins and secreted effectors. In most xanthomonads, type III secretion (T3S) system and secreted effectors (T3Es) are essential to bacterial pathogenicity through the inhibition of plant immunity or the induction of plant susceptibility (S) genes, as reported for Transcription Activation-Like (TAL) effectors. Yet, toxins can also be major virulence determinants in some xanthomonads while nonpathogenic Xanthomonas species do live in sympatry with plant without any T3S systems nor T3Es.
In a context of ever increasing international commercial exchanges and modifications of the climate, monitoring and regulating pathogens spread is of crucial importance for food security. A deep knowledge of the genomic diversity of Xanthomonas spp. is required for scientists to properly identify strains, to help preventing future disease outbreaks and to achieve knowledge-informed sustainable disease resistance in crops.
This Research Topic published in the ‘Plant Biotic Interactions’ section of Frontiers in Plant Science and Frontiers in Microbiology aims at illustrating several of the recent achievements of the Xanthomonas community. We collected twelve manuscripts dealing with comparative genomics or T3E repertoires, including five focusing on TAL effectors which we hope will contribute to advance research on plant pathogenic bacteria.
Xanthomonas euvesicatoria is the causal agent of bacterial spot disease in pepper and tomato. X. euvesicatoria bacteria interfere with plant cellular processes by injecting effector proteins into host cells through the type III secretion (T3S) system. About 35 T3S effectors have been identified in X. euvesicatoria 85-10, and a few of them were implicated in suppression of pattern-triggered immunity (PTI). We used an Arabidopsis thaliana pathogen-free protoplast–based assay to identify X. euvesicatoria 85-10 effectors that interfere with PTI signaling induced by the bacterial peptide flg22. Of 33 tested effectors, 17 inhibited activation of a PTI-inducible promoter. Among them, nine effectors also interfered with activation of an abscisic acid–inducible promoter. However, effectors that inhibited flg22-induced signaling did not affect phosphorylation of mitogen-activated protein (MAP) kinases acting downstream of flg22 perception. Further investigation of selected effectors revealed that XopAJ, XopE2, and XopF2 inhibited activation of a PTI-inducible promoter by the bacterial peptide elf18 in Arabidopsis protoplasts and by flg22 in tomato protoplasts. The effectors XopF2, XopE2, XopAP, XopAE, XopH, and XopAJ inhibited flg22-induced callose deposition in planta and enhanced disease symptoms caused by attenuated Pseudomonas syringae bacteria. Finally, selected effectors were found to localize to various plant subcellular compartments. These results indicate that X. euvesicatoria bacteria utilize multiple T3S effectors to suppress flg22-induced signaling acting downstream or in parallel to MAP kinase cascades and suggest they act through different molecular mechanisms.
The autophagic clearance of 26S proteasomes (proteaphagy) is an important homeostatic mechanism within the ubiquitin system that modulates proteolytic capacity and eliminates damaged particles. Here, we define two proteaphagy routes in yeast that respond to either nitrogen starvation or particle inactivation. Whereas the core autophagic machineries required for Atg8 lipidation and vesiculation are essential for both routes, the upstream Atg1 kinase participates only in starvation-induced proteaphagy. Following inactivation, 26S proteasomes become extensively modified with ubiquitin. Although prior studies with Arabidopsis implicated RPN10 in tethering ubiquitylated proteasomes to ATG8 lining the autophagic membranes, yeast proteaphagy employs the evolutionarily distinct receptor Cue5, which simultaneously binds ubiquitin and Atg8. Proteaphagy of inactivated proteasomes also requires the oligomeric Hsp42 chaperone, suggesting that ubiquitylated proteasomes are directed by Hsp42 to insoluble protein deposit (IPOD)-type structures before encapsulation. Together, Cue5 and Hsp42 provide a quality control checkpoint in yeast directed at recycling dysfunctional 26S proteasomes.
Parasitic plants are a constraint on agriculture worldwide. Cuscuta reflexa is a stem holoparasite that infests most dicotyledonous plants. One exception is tomato, which is resistant to C. reflexa. We discovered that tomato responds to a small peptide factor occurring in Cuscuta spp. with immune responses typically activated after perception of microbe-associated molecular patterns. We identified the cell surface receptor-like protein CUSCUTA RECEPTOR 1 (CuRe1) as essential for the perception of this parasite-associated molecular pattern. CuRe1 is sufficient to confer responsiveness to the Cuscuta factor and increased resistance to parasitic C. reflexa when heterologously expressed in otherwise susceptible host plants. Our findings reveal that plants recognize parasitic plants in a manner similar to perception of microbial pathogens.
Plant disease symptoms exhibit complex spatial and temporal patterns that are challenging to quantify. Image-based phenotyping approaches enable multi-dimensional characterization of host-microbe interactions and are well suited to capture spatial and temporal data that are key to understanding disease progression. We applied image-based methods to investigate cassava bacterial blight, which is caused by the pathogen Xanthomonas axonopodis pv. manihotis (Xam). We generated Xam strains in which individual predicted type III effector (T3E) genes were mutated and applied multiple imaging approaches to investigate the role of these proteins in bacterial virulence. Specifically, we quantified bacterial populations, water-soaking disease symptoms, and pathogen spread from the site of inoculation over time for strains with mutations in avrBs2, xopX, and xopK as compared to wild-type Xam. ∆avrBs2 and ∆xopX both showed reduced growth in planta and delayed spread through the vasculature system of cassava. ∆avrBs2 exhibited reduced water-soaking symptoms at the site of inoculation. In contrast, ∆xopK exhibited enhanced induction of disease symptoms at the site of inoculation but reduced spread through the vasculature. Our results highlight the importance of adopting a multi-pronged approach to plant disease phenotyping to more fully understand the roles of T3Es in virulence. Finally, we demonstrate that the approaches used in this study can be extended to many host-microbe systems and increase the dimensions of phenotype that can be explored.
Programmed cell death (PCD) is a conserved process among eukaryotes that serves a multitude of functional roles during an organism’s natural life cycle. PCD involves the tightly regulated process of cell death cued by specific spatiotemporal stimuli, which confer survival benefits. In eukaryotes, PCD is an essential process involved in senescence, aging, embryo development, cell differentiation, and immunity. In animal systems, morphologically distinct forms of PCD have been described (Figure 1) [1, 2]. Type I, or apoptotic cell death, is the best understood form of PCD and is defined by cell shrinkage, nuclear condensation and fragmentation, and eventual disintegration of the cell into apoptotic bodies that are digested by phagocytes. Type II cell death is an autophagic process that is induced during nutrient deprivation and chronic stress. Autophagic cell death is characterized by the rupture of the lysosome and subsequent release of toxic chemicals that degrade the cell contents. Unlike type I and type II, type III PCD is distinguished by the swelling of organelles and subsequent rupture of the plasma membrane. A programmed necrosis or necroptosis was initially believed to be an uncontrolled process of necrosis, but has been recently reclassified as type III form of cell death. Finally, pyroptosis is another recently categorized form of cell death that is mediated by caspase-1 activity. Morphologically, pyroptotic cells share characteristics of both apoptosis and necrosis . Noteworthy, necroptosis and pyroptosis are pro-inflammatory forms of PCD activated by microbial infections and diverse environmental stimuli.
In plants, PCD is less rigorously classified (Figure 1). One difficulty in distinguishing the forms of PCD in plants and animals comes as a result of the different cellular morphology in plant cells — most notably the presence of the cell wall and chloroplasts. Unlike the plasma membrane, the degradation of the cell wall is not a universal feature of PCD in plants. Additionally, the formation of apoptotic bodies is not observed in plant cells, as there are no circulating phagocytes to engulf them . Instead, plant cells committed to PCD release autolytic compounds stored in the vacuole that degrade cell contents. In these cases, the cell wall may develop perforations for the absorption and recycling of cellular components by neighboring cells. Although not as well characterized as the mitochondria, the chloroplasts have been shown to induce light-dependent PCD through singlet oxygen species (1O2) that may function in parallel to mitochondrial-mediated PCD at an early step in initiating the rupture of the vacuole .
A specialized form of plant cell death called hypersensitive response (HR) is initiated as a defense response to pathogen infection. HR shares morphological features and molecular mechanisms reminiscent of both pyroptosis and necroptosis . Moreover, HR is unique in that it induces a signaling cascade to propagate immunity in neighboring cells as well as priming distal tissues for potential pathogen challenge, a phenomenon known as systemic acquired resistance . Here we will briefly describe diverse plant disease resistance pathways, early molecular events during pathogen perception, and downstream signaling components. We will thoroughly discuss how pathogens have evolved strategies to circumvent and/or suppress diverse immune responses, in particular plant cell death. While many of these mechanisms involve indirect disabling of upstream immune responses to avoid cell death, direct manipulation of PCD regulators by pathogen effectors has not been extensively explored in the literature, and will be the focal point of this article.
Viruses interfere with and usurp host machinery and circumvent defense responses to create a suitable cellular environment for successful infection. This is usually achieved through interactions between viral proteins and host factors. Geminiviruses are a group of plant-infecting DNA viruses, of which some contain a betasatellite, known as DNAβ. Here, we report that Cotton leaf curl Multan virus (CLCuMuV) uses its sole satellite-encoded protein βC1 to regulate the plant ubiquitination pathway for effective infection. We found that CLCuMu betasatellite (CLCuMuB) βC1 interacts with NbSKP1, and interrupts the interaction of NbSKP1s with NbCUL1. Silencing of either NbSKP1s or NbCUL1 enhances the accumulation of CLCuMuV genomic DNA and results in severe disease symptoms in plants. βC1 impairs the integrity of SCFCOI1 and the stabilization of GAI, a substrate of the SCFSYL1 to hinder responses to jasmonates (JA) and gibberellins (GA). Moreover, JA treatment reduces viral accumulation and symptoms. These results suggest that CLCuMuB βC1 inhibits the ubiquitination function of SCF E3 ligases through interacting with NbSKP1s to enhance CLCuMuV infection and symptom induction in plants.
The plant cytoskeleton underpins the function of a multitude of cellular mechanisms, including those associated with developmental- and stress-associated signaling processes. In recent years, the actin cytoskeleton has been demonstrated to play a key role in plant immune signaling, including a recent demonstration that pathogens target actin filaments to block plant defense and immunity. Herein, we quantified spatial changes in host actin filament organization following infection with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), demonstrating that the type III effector HopG1 is required for pathogen-induced changes to actin filament architecture and host disease symptom development during infection. Using a suite of pathogen effector deletion constructs, coupled with high resolution microscopy, we found that deletion of hopG1 from Pst DC3000 resulted in a reduction in actin bundling, a concomitant increase in the density of filament arrays in Arabidopsis, both of which correlate with host disease symptom development. As a mechanism underpinning this activity, we further show that the HopG1 effector interacts with an Arabidopsis mitochondrial-localized kinesin motor protein. Kinesin mutant plants show reduced disease symptoms following pathogen infection, which can be complemented by actin modifying agents. In total, our results support a model in which HopG1 induces changes in the organization of the actin cytoskeleton as part of its virulence function in promoting disease symptom development.
Proteotoxic stress, which is generated by the accumulation of unfolded or aberrant proteins due to environmental or cellular conditions, can be mitigated by several mechanisms, including activation of the unfolded protein response and coordinated increases in protein chaperones and activities that direct proteolysis, such as the 26S proteasome. Using RNA-seq analyses combined with chemical inhibitors or mutants that induce proteotoxic stress by impairing 26S proteasome capacity, we defined the transcriptional network that responds to this stress in Arabidopsis thaliana. This network includes genes encoding core and assembly factors needed to build the complete 26S particle, alternative proteasome capping factors, enzymes involved in protein ubiquitylation/deubiquitylation and cellular detoxification, protein chaperones, autophagy components, and various transcriptional regulators. Many loci in this proteasome-stress regulon contain a consensus cis element upstream of the transcription start site, which was previously identified as a binding site for the NAM/ATAF1/CUC2 (NAC)-78 transcription factor. Double mutants disrupting NAC78 and its closest relative NAC53 are compromised in the activation of this regulon, and notably are strongly hypersensitive to the proteasome inhibitors MG132 and bortezomib. Given that NAC53 and NAC78 homo- and hetero-dimerize, we propose that they work as a pair in activating the expression of numerous factors that help plants survive proteotoxic stress, and thus play a central regulatory role in maintaining protein homeostasis.
The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity.
.Until recently it was not possible to engineer novel recognition specificities of classical plant immune receptors to completely unrelated effectors. In a recent publication, Kim et al. engineered a plant effector target to increase novel recognition specificities by trapping unrelated pathogen-derived proteases in their act .
RESISTANCE TO PSEUDOMONAS SYRINGAE 5 (RPS5) is a plant immune receptor of the Nucleotide-binding, leucine-rich repeat (NLR) type which perceives the Pseudomonas syringae Type-III effector AvrPphB, a papain-like cysteine protease belonging to the Peptidase C58 family . The perception of AvrPphB by RPS5 requires one additional host-derived factor known as AVRPPHB SUSCEPTIBLE 1 (PBS1), which belongs to Subfamily VII of Receptor-like Cytoplasmic Kinases (RLCK VII). Upon bacterial infection, PBS1, which binds to RPS5 in its pre-activation state, is cleaved by AvrPphB. PBS1 cleavage exposes a five amino acid loop in PBS1 that is believed to activate RPS5, triggering an immune response characterized by the hypersensitive response (HR), a form of programmed cell death . Interestingly, RPS5-mediated immune signaling requires both PBS1 fragments, and the conformational change induced by cleavage can be mimicked by insertion of five amino acids in the AvrPphB cleavage site . Therefore, perception of AvrPphB follows a mouse-trap mechanism where cleavage of PBS1 (bait) sets off the trap and activates RPS5, triggering immune responses..
Although nucleotide-binding domain, leucine-rich repeat (NLR) proteins are the major immune receptors in plants, the mechanism that controls their activation and immune signaling remains elusive. Here, we report that the avirulence effector AvrPiz-t from Magnaporthe oryzae targets the rice E3 ligase APIP10 for degradation, but that APIP10, in return, ubiquitinates AvrPiz-t and thereby causes its degradation. Silencing of APIP10 in the non-Piz-t background compromises the basal defense against M. oryzae. Conversely, silencing of APIP10 in the Piz-t background causes cell death, significant accumulation of Piz-t, and enhanced resistance to M. oryzae, suggesting that APIP10 is a negative regulator of Piz-t. We show that APIP10 promotes degradation of Piz-t via the 26S proteasome system. Furthermore, we demonstrate that AvrPiz-t stabilizes Piz-t during M. oryzae infection. Together, our results show that APIP10 is a novel E3 ligase that functionally connects the fungal effector AvrPiz-t to its NLR receptor Piz-t in rice.
•CPR5 is a component of the nuclear pore complex (NPC)
•CPR5 regulates nuclear transport through the selective barrier of the NPC
•The CPR5 homomer is disrupted upon induction of effector-triggered immunity (ETI)
•Conformational change in CPR5 leads to CKI release and NPC permeabilization for ETI
Nuclear transport of immune receptors, signal transducers, and transcription factors is an essential regulatory mechanism for immune activation. Whether and how this process is regulated at the level of the nuclear pore complex (NPC) remains unclear. Here, we report that CPR5, which plays a key inhibitory role in effector-triggered immunity (ETI) and programmed cell death (PCD) in plants, is a novel transmembrane nucleoporin. CPR5 associates with anchors of the NPC selective barrier to constrain nuclear access of signaling cargos and sequesters cyclin-dependent kinase inhibitors (CKIs) involved in ETI signal transduction. Upon activation by immunoreceptors, CPR5 undergoes an oligomer to monomer conformational switch, which coordinates CKI release for ETI signaling and reconfigures the selective barrier to allow significant influx of nuclear signaling cargos through the NPC. Consequently, these coordinated NPC actions result in simultaneous activation of diverse stress-related signaling pathways and constitute an essential regulatory mechanism specific for ETI/PCD induction.
Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed.
Effectors secreted by the type III secretion system are essential for bacterial pathogenesis. Members of the Yersinia outer-protein J (YopJ) family of effectors found in diverse plant and animal pathogens depend on a protease-like catalytic triad to acetylate host proteins and produce virulence. However, the structural basis for this noncanonical acetyltransferase activity remains unknown. Here, we report the crystal structures of the YopJ effector HopZ1a, produced by the phytopathogen Pseudomonas syringae, in complex with the eukaryote-specific cofactor inositol hexakisphosphate (IP6) and/or coenzyme A (CoA). Structural, computational and functional characterizations reveal a catalytic core with a fold resembling that of ubiquitin-like cysteine proteases and an acetyl-CoA-binding pocket formed after IP6-induced structural rearrangements. Modeling-guided mutagenesis further identified key IP6-interacting residues of Salmonella effector AvrA that are required for acetylating its substrate. Our study reveals the structural basis of a novel class of acetyltransferases and the conserved allosteric regulation of YopJ effectors by IP6.
Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component.
Clearance of misfolded and aggregated proteins is central to cell survival. Here, we describe a new pathway for maintaining protein homeostasis mediated by the proteasome shuttle factor UBQLN2. The 26S proteasome degrades polyubiquitylated substrates by recognizing them through stoichiometrically bound ubiquitin receptors, but substrates are also delivered by reversibly bound shuttles. We aimed to determine why these parallel delivery mechanisms exist and found that UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome.
Pathogenic bacteria rely on secreted effector pro-teins to manipulate host signaling pathways, oftenin creative ways. CE clan proteases, specific hydro-lases for ubiquitin-like modifications (SUMO andNEDD8) in eukaryotes, reportedly serve as bacterialeffector proteins with deSUMOylase, deubiquiti-nase, or, even, acetyltransferase activities. Here,we characterize bacterial CE protease activities,revealing K63-linkage-specific deubiquitinases inhuman pathogens, such as Salmonella,Escherichia,and Shigella, as well as ubiquitin/ubiquitin-like cross-reactive enzymes inChlamydia,Rickettsia, and Xanthomonas.Five crystal structures, including ubiquitin/ubiquitin-like complexes, explain substrates pecificities and redefine relationships across the CEclan. Importantly, this work identifies novel family members and provides key discoveries among previously reported effectors, such as the unex-pected deubiquitinase activity in Xanthomonas XopD, contributed by an unstructured ubiquitin binding region. Furthermore, accessory domains regulate properties such as subcellular localization, as exem-plified by a ubiquitin-binding domain in SalmonellaTyphimurium SseL. Our work both highlights and ex-plains the functional adaptations observed among diverse CE clan proteins.
Plasma membrane-localized pattern recognition receptors (PRRs) such as FLAGELLIN SENSING2 (FLS2), EF-TU RECEPTOR (EFR) and CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) recognize microbe-associated molecular patterns (MAMPs) to activate pattern-triggered immunity (PTI). A reverse genetics approach on genes responsive to the priming agent beta-aminobutyric acid (BABA) revealed IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as a critical PTI player. Arabidopsis thaliana ios1 mutants were hyper-susceptible to Pseudomonas syringae bacteria. Accordingly, ios1 mutants showed defective PTI responses, notably delayed up-regulation of the PTI-marker gene FLG22-INDUCED RECEPTOR-LIKE KINASE1 (FRK1), reduced callose deposition and mitogen-activated protein kinase activation upon MAMP treatment. Moreover, Arabidopsis lines over-expressing IOS1 were more resistant to bacteria and showed a primed PTI response. In vitro pull-down, bimolecular fluorescence complementation, co-immunoprecipitation, and mass spectrometry analyses supported the existence of complexes between the membrane-localized IOS1 and BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)-dependent PRRs FLS2 and EFR, as well as with the BAK1-independent PRR CERK1. IOS1 also associated with BAK1 in a ligand-independent manner, and positively regulated FLS2-BAK1 complex formation upon MAMP treatment. In addition, IOS1 was critical for chitin-mediated PTI. Finally, ios1 mutants were defective in BABA-induced resistance and priming. This work reveals IOS1 as a novel regulatory protein of FLS2-, EFR- and CERK1-mediated signaling pathways that primes PTI activation.
Involvement of 26S proteasomal subunits in plant pathogen-interactions, and the roles of each subunit in independently modulating the activity of many intra- and inter-cellular regulators controlling physiological and defense responses of a plant were well reported. In this regard, we aimed to functionally characterize a Solanum lycopersicum 26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity. SlRPT4 could specifically bind to the stem-loop structure of intergenic region (IR), present in both DNA-A and DNA-B molecule of the bipartite viral genome. Lack of secondary structure in replication-associated gene fragment prevented formation of DNA-protein complex suggesting that binding of SlRPT4 with DNA is secondary structure specific. Interestingly, binding of SlRPT4 to IR inhibited the function of RNA Pol-II and subsequently reduced the bi-directional transcription of ToLCNDV genome. Virus-induced gene silencing of SlRPT4 gene incited conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Furthermore, transient overexpression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. Overall, present study highlights non-proteolytic function of SlRPT4 and their participation in defense pathway against virus infection in tomato.
Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp. stewartii, we employed a yeast-two-hybrid screen with non-lethal fragments of WtsE and a synthetic genetic array with full-length WtsE. Together these screens indicate that WtsE targets maize protein phosphatase 2A (PP2A) heterotrimeric enzyme complexes via direct interaction with B’ regulatory subunits. AvrE1, another AvrE-family T3E from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), associates with specific PP2A B’ subunit proteins from its susceptible host Arabidopsis that are homologous to the maize B’ subunits shown to interact with WtsE. Additionally, AvrE1 was observed to associate with the WtsE-interacting maize proteins, indicating that PP2A B’ subunits are likely conserved targets of AvrE-family T3Es. Notably, the ability of AvrE1 to promote bacterial growth and/or suppress callose deposition was compromised in Arabidopsis plants with mutations of PP2A genes. Also, chemical inhibition of PP2A activity blocked the virulence activity of both WtsE and AvrE1 in planta. The function of HopM1, a Pto DC3000 T3E that is functionally redundant to AvrE1, was also impaired in specific PP2A mutant lines, although no direct interaction with B’ subunits was observed. These results indicate that sub-component specific PP2A complexes are targeted by bacterial T3Es, including direct targeting by members of the widely conserved AvrE-family.
Our understanding of plant biotic interactions has grown significantly in recent years with the identification of the mechanisms involved in innate immunity, hormone signaling, and secondary metabolism. The impact of such interactions on primary metabolism and the role of metabolic signals in the response of the plants, however, remain far less explored. The SnRK1 (SNF1-related kinase 1) kinases act as metabolic sensors, integrating very diverse stress conditions, and are key in maintaining energy homeostasis for growth and survival. Consistently, an important role is emerging for these kinases as regulators of biotic stress responses triggered by viral, bacterial, fungal, and oomycete infections as well as by herbivory. While this identifies SnRK1 as a promising target for directed modification or selection for more quantitative and sustainable resistance, its central function also increases the chances of unwanted side effects on growth and fitness, stressing the need for identification and in-depth characterization of the mechanisms and target processes involved.
Virus interactions with plant silencing and innate immunity pathways can potentially alter the susceptibility of virus-infected plants to secondary infections with nonviral pathogens. We found that Arabidopsis plants infected with Cauliflower mosaic virus (CaMV) or transgenic for CaMV silencing suppressor P6 exhibit increased susceptibility to Pseudomonas syringae pv. tomato (Pst) and allow robust growth of the Pst mutant hrcC-, which cannot deploy effectors to suppress innate immunity. The impaired antibacterial defense correlated with the suppressed oxidative burst, reduced accumulation of the defense hormone salicylic acid (SA) and diminished SA-dependent autophagy. The viral protein domain required for suppression of these plant defense responses is dispensable for silencing suppression but essential for binding and activation of the plant target-of-rapamycin (TOR) kinase which, in its active state, blocks cellular autophagy and promotes CaMV translation. Our findings imply that CaMV P6 is a versatile viral effector suppressing both silencing and innate immunity. P6-mediated suppression of oxidative burst and SA-dependent autophagy may predispose CaMV-infected plants to bacterial infection.
The Arabidopsis immune receptor FLS2 perceives bacterial flagellin epitope flg22 to activate defenses through the central cytoplasmic kinase BIK1. The heterotrimeric G proteins composed of the non-canonical Gα protein XLG2, the Gβ protein AGB1, and the Gγ proteins AGG1 and AGG2 are required for FLS2-mediated immune responses through an unknown mechanism. Here we show that in the pre-activation state, XLG2 directly interacts with FLS2 and BIK1, and it functions together with AGB1 and AGG1/2 to attenuate proteasome-mediated degradation of BIK1, allowing optimum immune activation. Following the activation by flg22, XLG2 dissociates from AGB1 and is phosphorylated by BIK1 in the N terminus. The phosphorylated XLG2 enhances the production of reactive oxygen species (ROS) likely by modulating the NADPH oxidase RbohD. The study demonstrates that the G proteins are directly coupled to the FLS2 receptor complex and regulate immune signaling through both pre-activation and post-activation mechanisms.
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