Respiratory Burst Oxidase Homolog D Research Articles

DGK5β-derived phosphatidic acid regulates ROS production in plant immunity by stabilizing NADPH oxidase

In plant immunity, phosphatidic acid (PA) regulates reactive oxygen species (ROS) by binding to respiratory burst oxidase homolog D (RBOHD), an NADPH oxidase responsible for ROS production. Here, we analyze the influence of PA binding on RBOHD activity and the mechanism of RBOHD-bound PA generation. PA binding enhances RBOHD protein stability by inhibiting vacuolar degradation, thereby increasing chitin-induced ROS production. Mutations in diacylglycerol kinase 5 (DGK5), which phosphorylates diacylglycerol to produce PA, impair chitin-induced PA and ROS production. The DGK5 transcript DGK5β (but not DGK5α) complements reduced PA and ROS production in dgk5-1 mutants, as well as resistance to Botrytis cinerea. Phosphorylation of S506 residue in the C-terminal calmodulin-binding domain of DGK5β contributes to the activation of DGK5β to produce PA. These findings suggest that DGK5β-derived PA regulates ROS production by inhibiting RBOHD protein degradation, elucidating the role of PA-ROS interplay in immune response regulation.

Dual phosphorylation of DGK5-mediated PA burst regulates ROS in plant immunity

Phosphatidic acid (PA) and reactive oxygen species (ROS) are crucial cellular messengers mediating diverse signaling processes in metazoans and plants. How PA homeostasis is tightly regulated and intertwined with ROS signaling upon immune elicitation remains elusive. We report here that Arabidopsis diacylglycerol kinase 5 (DGK5) regulates plant pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). The pattern recognition receptor (PRR)-associated kinase BIK1 phosphorylates DGK5 at Ser-506, leading to a rapid PA burst and activation of plant immunity, whereas PRR-activated intracellular MPK4 phosphorylates DGK5 at Thr-446, which subsequently suppresses DGK5 activity and PA production, resulting in attenuated plant immunity. PA binds and stabilizes the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), regulating ROS production in plant PTI and ETI, and their potentiation. Our data indicate that distinct phosphorylation of DGK5 by PRR-activated BIK1 and MPK4 balances the homeostasis of cellular PA burst that regulates ROS generation in coordinating two branches of plant immunity.

The phagocytosis oxidase/Bem1p domain-containing protein PB1CP negatively regulates the NADPH oxidase RBOHD in plant immunity.

Perception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors activates RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) through direct phosphorylation by BOTRYTIS-INDUCED KINASE 1 (BIK1) and induces the production of reactive oxygen species (ROS). RBOHD activity must be tightly controlled to avoid the detrimental effects of ROS, but little is known about RBOHD downregulation. To understand the regulation of RBOHD, we used co-immunoprecipitation of RBOHD with mass spectrometry analysis and identified PHAGOCYTOSIS OXIDASE/BEM1P (PB1) DOMAIN-CONTAINING PROTEIN (PB1CP). PB1CP negatively regulates RBOHD and the resistance against the fungal pathogen Colletotrichum higginsianum. PB1CP competes with BIK1 for binding to RBOHD invitro. Furthermore, PAMP treatment enhances the PB1CP-RBOHD interaction, thereby leading to the dissociation of phosphorylated BIK1 from RBOHD invivo. PB1CP localizes at the cell periphery and PAMP treatment induces relocalization of PB1CP and RBOHD to the same small endomembrane compartments. Additionally, overexpression of PB1CP in Arabidopsis leads to a reduction in the abundance of RBOHD protein, suggesting the possible involvement of PB1CP in RBOHD endocytosis. We found PB1CP, a novel negative regulator of RBOHD, and revealed its possible regulatory mechanisms involving the removal of phosphorylated BIK1 from RBOHD and the promotion of RBOHD endocytosis.

XYLEM CYSTEINE PEPTIDASE 1 and its inhibitor CYSTATIN 6 regulate pattern-triggered immunity by modulating the stability of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D.

Plants produce a burst of reactive oxygen species (ROS) after pathogen infection to successfully activate immune responses. During pattern-triggered immunity (PTI), ROS are primarily generated by the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD). RBOHD is degraded in the resting state to avoid inappropriate ROS production; however, the enzyme mediating RBOHD degradation and how to prevent RBOHD degradation after pathogen infection is unclear. In this study, we identified an Arabidopsis (Arabidopsis thaliana) vacuole-localized papain-like cysteine protease, XYLEM CYSTEINE PEPTIDASE 1 (XCP1), and its inhibitor CYSTATIN 6 (CYS6). Pathogen-associated molecular pattern-induced ROS burst and resistance were enhanced in the xcp1 mutant but were compromised in the cys6 mutant, indicating that XCP1 and CYS6 oppositely regulate PTI responses. Genetic and biochemical analyses revealed that CYS6 interacts with XCP1 and depends on XCP1 to enhance PTI. Further experiments showed that XCP1 interacts with RBOHD and accelerates RBOHD degradation in a vacuole-mediated manner. CYS6 inhibited the protease activity of XCP1 toward RBOHD, which is critical for RBOHD accumulation upon pathogen infection. As CYS6, XCP1, and RBOHD are conserved in all plant species tested, our findings suggest the existence of a conserved strategy to precisely regulate ROS production under different conditions by modulating the stability of RBOHD.

A PDLP-NHL3 complex integrates plasmodesmal immune signaling cascades

The plant immune system relies on the perception of molecules that signal the presence of a microbe threat. This triggers signal transduction that mediates a range of cellular responses via a collection of molecular machinery including receptors, small molecules, and enzymes. One response to pathogen perception is the restriction of cell-to-cell communication by plasmodesmal closure. We previously found that while chitin and flg22 trigger specialized immune signaling cascades in the plasmodesmal plasma membrane, both execute plasmodesmal closure via callose synthesis at the plasmodesmata. Therefore, the signaling pathways ultimately converge at or upstream of callose synthesis. To establish the hierarchy of signaling at plasmodesmata and characterize points of convergence in microbe elicitor-triggered signaling, we profiled the dependence of plasmodesmal responses triggered by different elicitors on a range of plasmodesmal signaling machinery. We identified that, like chitin, flg22 signals via RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) to induce plasmodesmal closure. Further, we found that PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1), PDLP5, and CALLOSE SYNTHASE 1 (CALS1) are common to microbe- and salicylic acid (SA)-triggered responses, identifying PDLPs as a candidate signaling nexus. To understand how PDLPs relay a signal to CALS1, we screened for PDLP5 interactors and found NON-RACE SPECIFIC DISEASE RESISTANCE/HIN1 HAIRPIN-INDUCED-LIKE protein 3 (NHL3), which is also required for chitin-, flg22- and SA-triggered plasmodesmal responses and PDLP-mediated activation of callose synthesis. We conclude that a PDLP-NHL3 complex acts as an integrating node of plasmodesmal signaling cascades, transmitting multiple immune signals to activate CALS1 and plasmodesmata closure.

Conservation of the PBL-RBOH immune module in land plants

The rapid production of reactive oxygen species (ROS) is a key signaling output in plant immunity. In the angiosperm model species Arabidopsis thaliana (hereafter Arabidopsis), recognition of non- or altered-self elicitor patterns by cell-surface immune receptors activates the receptor-like cytoplasmic kinases (RLCKs) of the AVRPPHB SUSCEPTIBLE 1 (PBS1)-like (PBL) family, particularly BOTRYTIS-INDUCED KINASE1 (BIK1).1,2,3 BIK1/PBLs in turn phosphorylate the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) to induce apoplastic ROS production.4,5 PBL and RBOH functions in plant immunity have been extensively characterized in flowering plants. Much less is known about the conservation of pattern-triggered ROS signaling pathways in non-flowering plants. In this study, we show that in the liverwort Marchantia polymorpha (hereafter Marchantia), single members of the RBOH and PBL families, namely MpRBOH1 and MpPBLa, are required for chitin-induced ROS production. MpPBLa directly interacts with and phosphorylates MpRBOH1 at specific, conserved sites within its cytosolic N terminus, and this phosphorylation is essential for chitin-induced MpRBOH1-mediated ROS production. Collectively, our work reveals the functional conservation of the PBL-RBOH module that controls pattern-triggered ROS production in land plants.

FLS2-RBOHD module regulates changes in the metabolome of Arabidopsis in response to abiotic stress.

Through crosstalk, FLAGELLIN SENSITIVE 2 (FLS2) and RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) are involved in regulating the homeostasis of cellular reactive oxygen species (ROS) and are linked to the metabolic response of plants toward both biotic and abiotic stress. In the present study, we examined the metabolome of Arabidopsis seedlings under drought and salt conditions to better understand the potential role of FLS2 and RBOHD-dependent signaling in the regulation of abiotic stress response. We identified common metabolites and genes that are regulated by FLS2 and RBOHD, and are involved in the response to drought and salt stress. Under drought conditions, D-aspartic acid and the expression of associated genes, such as ASPARAGINE SYNTHASE 2 (ASN2), increased in both fls2 and robed/f double mutants. The accumulation of amino acids, carbohydrates, and hormones, such as L-proline, D-ribose, and indoleacetaldehyde increased in both fls2 and rbohd/f double mutants under salt conditions, as did the expression of related genes, such as PROLINE IMINOPEPTIDASE, PHOSPHORIBOSYL PYROPHOSPHATE SYNTHASE 5, and NITRILASE 3. Collectively, these results indicate that the FLS2-RBOHD module regulates plant response to drought and salt stress through ROS signaling by adjusting the accumulation of metabolites and expression of genes related to metabolite synthesis.

Overexpression of an Antioxidant Enzyme APX1 in cpr5 Mutant Restores its Pleiotropic Growth Phenotype.

Breeding crops with enhanced immunity is an effective strategy to reduce yield loss caused by pathogens. The constitutive expresser of pathogenesis-related genes (cpr5) mutant shows enhanced pathogen resistance but retarded growth; thus, it restricts the application of cpr5 in breeding crops with disease resistance. Reactive oxygen species (ROS) play important roles in plant growth and defense. In this study, we determined that the cpr5 mutant exhibited excessive ROS accumulation. However, the mutation of respiratory burst oxidase homolog D (RBOHD), a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase responsible for the production of ROS signaling in plant immunity, did not suppress excessive ROS levels in cpr5. Furthermore, the cpr5 mutant showed low levels of ascorbate peroxidase 1 (APX1), an important cytosolic ROS-scavenging enzyme. APX1 overexpression in the cpr5 background removed excessive ROS and restored the pleiotropic growth phenotype. Notably, APX1 overexpression did not reduce the resistance of cpr5 mutant to virulent strain Pseudomonas syringae pv. tomato (Pst) DC3000 and avirulent strain Pst DC3000 (avrRpt2). These results suggest that the removal of excessive ROS by APX1 overexpression restored the cpr5 growth phenotype while conserving pathogen resistance. Hence, our study provides a theoretical and empirical basis for utilizing CPR5 in the breeding of crops with disease resistance by effective oxidative stress management via APX1 expression.

Organelle-targeted biosensors reveal distinct oxidative events during pattern-triggered immune responses.

Reactive oxygen species are produced in response to pathogens and pathogen-associated molecular patterns, as exemplified by the rapid extracellular oxidative burst dependent on the NADPH oxidase isoform RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). We used the H2O2 biosensor roGFP2-Orp1 and the glutathione redox state biosensor GRX1-roGFP2 targeted to various organelles to reveal unsuspected oxidative events during the pattern-triggered immune response to flagellin (flg22) and after inoculation with Pseudomonas syringae. roGFP2-Orp1 was oxidized in a biphasic manner 1 and 6 h after treatment, with a more intense and faster response in the cytosol compared to chloroplasts, mitochondria, and peroxisomes. Peroxisomal and cytosolic GRX1-roGFP2 were also oxidized in a biphasic manner. Interestingly, our results suggested that bacterial effectors partially suppress the second phase of roGFP2-Orp1 oxidation in the cytosol. Pharmacological and genetic analyses indicated that the pathogen-associated molecular pattern-induced cytosolic oxidation required the BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) and BOTRYTIS-INDUCED KINASE 1 (BIK1) signaling components involved in the immune response but was largely independent of NADPH oxidases RBOHD and RESPIRATORY BURST OXIDASE HOMOLOG F (RBOHF) and apoplastic peroxidases peroxidase 33 (PRX33) and peroxidase 34 (PRX34). The initial apoplastic oxidative burst measured with luminol was followed by a second oxidation burst, both of which preceded the two waves of cytosolic oxidation. In contrast to the cytosolic oxidation, these bursts were RBOHD-dependent. Our results reveal complex oxidative sources and dynamics during the pattern-triggered immune response, including that cytosolic oxidation is largely independent of the preceding extracellular oxidation events.

Melatonin delays ABA-induced leaf senescence via H2 O2 -dependent calcium signalling.

Precocious leaf senescence can reduce crop yield and quality by limiting the growth stage. Melatonin has been shown to delay leaf senescence; however, the underlying mechanism remains obscure. Here, we show that melatonin offsets abscisic acid (ABA) to protect photosystem II and delay the senescence of attached old leaves under the light. Melatonin induced H2 O2 accumulationaccompanied by an upregulation of melon respiratory burst oxidase homologD (CmRBOHD) under ABA-induced stress. Both melatonin and H2 O2 induced the accumulation of cytoplasmic-free Ca2+ ([Ca2+ ]cyt ) in response to ABA, while blocking of Ca2+ influx channels attenuated melatonin- and H2 O2 -induced ABA tolerance. CmRBOHD overexpression induced [Ca2+ ]cyt accumulation and delayed leaf senescence, whereasdeletion of Arabidopsis AtRBOHD, a homologous gene of CmRBOHD, compromised the melatonin-induced [Ca2+ ]cyt accumulation and delay of leaf senescence in Arabidopsis under ABA stress. Furthermore, melatonin, H2 O2 and Ca2+ attenuated ABA-induced K+ efflux and subsequent cell death. CmRBOHD overexpression and AtRBOHD deletion alleviated and aggravated the ABA-induced K+ efflux, respectively. Taken together, our study unveils a new mechanism by which melatonin offsets ABA action to delay leaf senescence via RBOHD-dependent H2 O2 production that triggers [Ca2+ ]cyt accumulation and subsequently inhibits K+ efflux and delays cell death/leaf senescence in response to ABA.

Spermine inhibits PAMP-induced ROS and Ca2+ burst and reshapes the transcriptional landscape of PAMP-triggered immunity in Arabidopsis.

Polyamines are small polycationic amines whose levels increase during defense. Previous studies support the contribution of the polyamine spermine to defense responses. However, the potential contribution of spermine to pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) has not been completely established. Here, we compared the contribution of spermine and putrescine to early and late PTI responses in Arabidopsis. We found that putrescine and spermine have opposite effects on PAMP-elicited reactive oxygen species (ROS) production, with putrescine increasing and spermine lowering the flg22-stimulated ROS burst. Through genetic and pharmacological approaches, we found that the inhibitory effect of spermine on flg22-elicited ROS production is independent of polyamine oxidation, nitric oxide, and salicylic acid signaling but resembles chemical inhibition of RBOHD (RESPIRATORY BURST OXIDASE HOMOLOG D). Spermine can also suppress ROS elicited by FLS2-independent but RBOHD-dependent pathways, thus pointing to compromised RBOHD activity. Consistent with this, we found that spermine but not putrescine dampens flg22-stimulated cytosolic Ca2+ influx. Finally, we found that both polyamines differentially reshape transcriptional responses during PTI and disease resistance to Pseudomonas syringae. Overall, we provide evidence for the differential contributions of putrescine and spermine to PTI, with an impact on plant defense.

EXTRA LARGE G-PROTEIN2 mediates cell death and hyperimmunity in the chitin elicitor receptor kinase 1-4 mutant.

Heterotrimeric G-proteins are signal transduction complexes that comprised three subunits, Gα, Gβ, and Gγ, and are involved in many aspects of plant life. The noncanonical Gα subunit EXTRA LARGE G-PROTEIN2 (XLG2) mediates pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species (ROS) generation and immunity downstream of pattern recognition receptors. A mutant of the chitin receptor component CHITIN ELICITOR RECEPTOR KINASE1 (CERK1), cerk1-4, maintains normal chitin signaling capacity but shows excessive cell death upon infection with powdery mildew fungi. We identified XLG2 mutants as suppressors of the cerk1-4 phenotype. Mutations in XLG2 complex partners ARABIDOPSIS Gβ1 (AGB1) and Gγ1 (AGG1) have a partial cerk1-4 suppressor effect. Contrary to its role in PAMP-induced immunity, XLG2-mediated control of ROS production by RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) is not critical for cerk1-4-associated cell death and hyperimmunity. The cerk1-4 phenotype is also independent of the co-receptor/adapter kinases BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and SUPPRESSOR OF BIR1 1 (SOBIR1), but requires the E3 ubiquitin ligase PLANT U-BOX 2 (PUB2). XLG2 localizes to both the cell periphery and nucleus, and the cerk1-4 cell death phenotype is mediated by the cell periphery pool of XLG2. Integrity of the XLG2 N-terminal domain, but not its phosphorylation, is essential for correct XLG2 localization and formation of the cerk1-4 phenotype. Our results support a model in which XLG2 acts downstream of an unknown cell surface receptor that activates an NADPH oxidase-independent cell death pathway in Arabidopsis (Arabidopsis thaliana).

Iron oxide nanoparticles and selenium supplementation improve growth and photosynthesis by modulating antioxidant system and gene expression of chlorophyll synthase (CHLG) and protochlorophyllide oxidoreductase (POR) in arsenic-stressed Cucumis melo

Current research reveals the positive role of iron oxide nanoparticles (IONPs) and selenium (Se) in extenuation of arsenic (As) induced toxicity in Cucumis melo. C. melo plants grown in As spiked soil (20 mg kg−1 As) showed reduced growth, chlorophyll (Chl) content, photosynthetic rate, stomatal conductivity and transpiration. On the other hand, the alone applications of IONPs or Se improved growth and physiochemical parameters of C. melo plants. Additionally, exogenous application IONPs and Se synergistically improved the activity of antioxidative enzymes and glyoxalase system in C. melo plants. In addition, the collective treatment of IONPs and Se reduced As uptake, enhanced rate of photosynthesis and increased gas exchange attributes of C. melo plants under As stress. Interactive effect of IONPs and Se regulated reduced glutathione (GSH), oxidized glutathione (GSSG) and ascorbate (AsA) content in C. melo plants exposed to As-contaminated Soil. IONPs and Se treatment also regulated expression of respiratory burst oxidase homologue D (RBOHD) gene, chlorophyll synthase (CHLG) and protochlorophyllide oxidoreductase (POR). Therefore, the combined treatment of IONPs and Se may enhance the growth of crop plants by alleviating As stress.

Upregulated expression of RESPIRATORY BURST OXIDASE HOMOLOG D underlies lesion-mimic phenotype in dark-treated Arabidopsis pheide a oxygenase mutant leaves.

Upregulated expression of RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) encoding a plasma membrane NADPH oxidase is responsible for the lesion-mimic phenotype in detached Arabidopsis leaves with mutation of PHEIDE a OXYGENASE during extended darkness. Chlorophyll degradation is an indispensable process in leaf senescence, either age-dependent or dark-induced. Besides higher chlorophyll retention, a lesion-mimic phenotype (abbreviated as LMP afterwards) was exhibited in Arabidopsis leaves with mutation of PHEIDE a OXYGENASE (PaO) involved in chlorophyll degradation during dark incubation, but the associated mechanism remains elusive. We found that dark-treated pao leaves showed higher membrane damage and H2O2 accumulation, while scavenging H2O2 by its chemical scavenger diminished LMP. RBOHD which encodes NADPH oxidase was strikingly up-regulated in pao leaves during dark treatment. Chemical inhibition of NADPH oxidase or mutation of RBOHD in pao leaves suppressed LMP. Thus, our study suggests that up-regulated RBOHD transcription is responsible for the formation of LMP in dark-treated pao leaves and there may be a retrograde signaling pathway mediating upregulation of RBOHD which remains to be elucidated.

Sphingolipids with 2-hydroxy fatty acids aid in plasma membrane nanodomain organization and oxidative burst.

Plant sphingolipids mostly possess 2-hydroxy fatty acids (HFA), the synthesis of which is catalyzed by FA 2-hydroxylases (FAHs). In Arabidopsis (Arabidopsis thaliana), two FAHs (FAH1 and FAH2) have been identified. However, the functions of FAHs and sphingolipids with HFAs (2-hydroxy sphingolipids) are still unknown because of the lack of Arabidopsis lines with the complete deletion of FAH1. In this study, we generated a FAH1 mutant (fah1c) using CRISPR/Cas9-based genome editing. Sphingolipid analysis of fah1c, fah2, and fah1cfah2 mutants revealed that FAH1 hydroxylates very long-chain FAs (VLCFAs), whereas the substrates of FAH2 are VLCFAs and palmitic acid. However, 2-hydroxy sphingolipids are not completely lost in the fah1cfah2 double mutant, suggesting the existence of other enzymes catalyzing the hydroxylation of sphingolipid FAs. Plasma membrane (PM) analysis and molecular dynamics simulations revealed that hydroxyl groups of sphingolipid acyl chains play a crucial role in the organization of nanodomains, which are nanoscale liquid-ordered domains mainly formed by sphingolipids and sterols in the PM, through hydrogen bonds. In the PM of the fah1cfah2 mutant, the expression levels of 26.7% of the proteins, including defense-related proteins such as the pattern recognition receptors (PRRs) brassinosteroid insensitive 1-associated receptor kinase 1 and chitin elicitor receptor kinase 1, NADPH oxidase respiratory burst oxidase homolog D (RBOHD), and heterotrimeric G proteins, were lower than that in the wild-type. In addition, reactive oxygen species (ROS) burst was suppressed in the fah1cfah2 mutant after treatment with the pathogen-associated molecular patterns flg22 and chitin. These results indicated that 2-hydroxy sphingolipids are necessary for the organization of PM nanodomains and ROS burst through RBOHD and PRRs during pattern-triggered immunity.

The receptor-like cytosolic kinase RIPK activates NADP-malic enzyme 2 to generate NADPH for fueling ROS production

Reactive oxygen species (ROS) production is a conserved immune response in Arabidopsis primarily mediated by respiratory burst oxidase homolog D (RBOHD), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase associated with the plasma membrane. A rapid increase in NADPH is necessary to fuel RBOHD proteins and thus maintain ROS production. However, the molecular mechanism by which NADPH is generated to fuel RBOHD remains unclear. In this study, we isolated a new mutant allele of FLAGELLIN-INSENSITIVE 4 (FIN4), which encodes the first enzyme in de novo NAD biosynthesis. fin4 mutants show reduced NADPH levels and impaired ROS production. However, FIN4 and other genes involved in NAD- and NADPH-generating pathways are not highly upregulated upon elicitor treatment, raising a possibility that a cytosolic NADP-linked dehydrogenase might be post-transcriptionally activated to maintain the NADPH supply close to RBOHD. To verify this possibility, we isolated the proteins associated with RPM1-INDUCED PROTEIN KINASE (RIPK), a receptor-like cytoplasmic kinase that regulates broad-spectrum ROS signaling in plant immunity, and identified NADP-malic enzyme 2 (NADP-ME2), an NADPH-generating enzyme. Compared with wild-type plants, nadp-me2 mutants display decreased NADP-ME activity, lower NADPH levels, and reduced ROS production in response to immune elicitors. Furthermore, we found that RIPK can directly phosphorylate NADP-ME2 and enhance its activity in vitro. The phosphorylation of the NADP-ME2 S371 residue contributes to ROS production upon immune elicitor treatment and susceptibility to the necrotrophic bacterium Pectobacterium carotovorum. Collectively, our study suggests that RIPK phosphorylates and activates NADP-ME2 to rapidly increase cytosolic NADPH, thus fueling RBOHD to sustain ROS production in plant immunity.

RIPK: a crucial ROS signaling component in plants.

Reactive oxygen species (ROS) are involved in plant development and stress acclimation. Recently, Li et al. reported that ROS production is controlled by receptor-like cytoplasmic kinase (RLCK)-mediated respiratory burst oxidase homolog D (RBOHD) phosphorylation, which subsequently regulates pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), damage-associated molecular pattern (DAMP)-triggered immunity (DTI), effector-triggered immunity (ETI), and systemic acquired resistance (SAR) in plants, thus enhancing our understanding of biotic stress tolerance.

Vascular Bundles Mediate Systemic Reactive Oxygen Signaling during Light Stress.

Systemic signaling and systemic acquired acclimation (SAA) are essential for plant survival during episodes of environmental stress. Recent studies highlighted a key role for reactive oxygen species (ROS) signaling in mediating systemic responses and SAA during light stress in Arabidopsis (Arabidopsis thaliana). These studies further identified the RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) protein as a key player in mediating rapid systemic ROS responses. Here, we report that tissue-specific expression of RBOHD in phloem or xylem parenchyma cells of the rbohD mutant restores systemic ROS signaling, systemic stress-response transcript expression, and SAA to a local treatment of light stress. We further demonstrate that RBOHD and RBOHF are both required for local and systemic ROS signaling at the vascular bundles of Arabidopsis. Taken together, our findings highlight a key role for RBOHD-driven ROS production at the vascular bundles of Arabidopsis in mediating light stress-induced systemic signaling and SAA. In addition, they suggest that the integration of ROS, calcium, electric, and hydraulic signals, during systemic signaling, occurs at the vascular bundles.

Oxathiapiprolin, a Novel Chemical Inducer Activates the Plant Disease Resistance

Oxathiapiprolin was developed as a specific plant pathogenic oomycete inhibitor, previously shown to have highly curative and protective activities against the pepper Phytophthora blight disease under field and greenhouse tests. Therefore, it was hypothesized that oxathiapiprolin might potentially activate the plant disease resistance against pathogen infections. This study investigated the potential and related mechanism of oxathiapiprolin to activate the plant disease resistance using the bacterium Pseudomonas syringae pv tomato (Pst) and plant Arabidopsis interaction as the targeted system. Our results showed that oxathiapiprolin could activate the plant disease resistance against Pst DC3000, a non-target pathogen of oxathiapiprolin, in Arabidopsis, tobacco, and tomato plants. Our results also showed the enhanced callose deposition and H2O2 accumulation in the oxathiapiprolin-treated Arabidopsis under the induction of flg22 as the pathogen-associated molecular pattern (PAMP) treatment. Furthermore, increased levels of free salicylic acid (SA) and jasmonic acid (JA) were detected in the oxathiapiprolin-treated Arabidopsis plants compared to the mock-treated ones under the challenge of Pst DC3000. Besides, the gene expression results confirmed that at 24 h after the infiltration with Pst DC3000, the oxathiapiprolin-treated Arabidopsis plants had upregulated expression levels of the respiratory burst oxidase homolog D (RBOHD), JA-responsive gene (PDF1.2), and SA-responsive genes (PR1, PR2, and PR5) compared to the control. Taken together, oxathiapiprolin is identified as a novel chemical inducer which activates the plant disease resistance against Pst DC3000 by enhancing the callose deposition, H2O2 accumulation, and hormone SA and JA production.

CRK2 and C-terminal Phosphorylation of NADPH Oxidase RBOHD Regulate Reactive Oxygen Species Production in Arabidopsis.

Reactive oxygen species (ROS) are important messengers in eukaryotic organisms, and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase-dependent signaling networks. Here, we show that CYSTEINE-RICH RLK2 (CRK2) kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). Functional CRK2 is required for the full elicitor-induced ROS burst, and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment, and substitution of S703 with Ala reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in the C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating microbe-associated molecular pattern-triggered ROS production.