MAPK signaling regulates nitric oxide and NADPH oxidase-dependent oxidative bursts in Nicotiana benthamiana

Nitric oxide (NO) and reactive oxygen species (ROS) act as signals in innate immunity in plants. The radical burst is induced by INF1 elicitin, produced by the oomycete pathogen Phytophthora infestans. NO ASSOCIATED1 (NOA1) and NADPH oxidase participate in the radical burst. Here, we show that mitogen-activated protein kinase (MAPK) cascades MEK2-SIPK/NTF4 and MEK1-NTF6 participate in the regulation of the radical burst. NO generation was induced by conditional activation of SIPK/NTF4, but not by NTF6, in Nicotiana benthamiana leaves. INF1- and SIPK/NTF4-mediated NO bursts were compromised by the knockdown of NOA1. However, ROS generation was induced by either SIPK/NTF4 or NTF6. INF1- and MAPK-mediated ROS generation was eliminated by silencing Respiratory Burst Oxidase Homolog B (RBOHB), an inducible form of the NADPH oxidase. INF1-induced expression of RBOHB was compromised in SIPK/NTF4/NTF6-silenced leaves. These results indicated that INF1 regulates NOA1-mediated NO and RBOHB-dependent ROS generation through MAPK cascades. NOA1 silencing induced high susceptibility to Colletotrichum orbiculare but not to P. infestans; conversely, RBOHB silencing decreased resistance to P. infestans but not to C. orbiculare. These results indicate that the effects of the radical burst on the defense response appear to be diverse in plant-pathogen interactions.     

Molecular mechanisms of generation for nitric oxide and reactive oxygen species, and role of the radical burst in plant immunity

Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in the regulation of innate immunity in plants. A potato calcium-dependent protein kinase (StCDPK5) activates an NADPH oxidase StRBOHA to D by direct phosphorylation of N-terminal regions, and heterologous expression of StCDPK5 and StRBOHs in Nicotiana benthamiana results in oxidative burst. The transgenic potato plants that carry a constitutively active StCDPK5 driven by a pathogen-inducible promoter of the potato showed high resistance to late blight pathogen Phytophthora infestans accompanied by HR-like cell death and H₂O₂ accumulation in the attacked cells. In contrast, these plants showed high susceptibility to early blight necrotrophic pathogen Alternaria solani, suggesting that oxidative burst confers high resistance to biotrophic pathogen, but high susceptibility to necrotrophic pathogen. NO and ROS synergistically function in defense responses. Two MAPK cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NbRBOHB gene in N. benthamiana. On the other hand, NO burst is regulated by the MEK2-SIPK cascade. Conditional activation of SIPK in potato plants induces oxidative and NO bursts, and confers resistance to both biotrophic and necrotrophic pathogens, indicating the plants may have obtained during evolution the signaling pathway which regulates both NO and ROS production to adapt to wide-spectrum pathogens.     

Reactive oxygen species and their role in plant defence and cell wall metabolism

Harnessing the toxic properties of reactive oxygen species (ROS) to fight off invading pathogens can be considered a major evolutionary success story. All aerobic organisms have evolved the ability to regulate the levels of these toxic intermediates, whereas some have evolved elaborate signalling pathways to dramatically increase the levels of ROS and use them as weapons in mounting a defence response, a process commonly referred to as the oxidative burst. The balance between steady state levels of ROS and the exponential increase in these levels during the oxidative burst has begun to shed light on complex signalling networks mediated by these molecules. Here, we discuss the different sources of ROS that are present in plant cells and review their role in the oxidative burst. We further describe two well-studied ROS generating systems, the NADPH oxidase and apoplastic peroxidase proteins, and their role as the primary producers of ROS during pathogen invasion. We then discuss what is known about the metabolic and proteomic fluxes that occur in plant cells during the oxidative burst and after pathogen recognition, and try to highlight underlying biochemical processes that may provide more insight on the complex regulation of ROS in plants.     

MAPK phosphatase MKP2 mediates disease responses in Arabidopsis and functionally interacts with MPK3 and MPK6

Mitogen‐activated protein kinase (MAPK) cascades have important functions in plant stress responses and development and are key players in reactive oxygen species (ROS) signalling and in innate immunity. In Arabidopsis, the transmission of ROS and pathogen signalling by MAPKs involves the coordinated activation of MPK6 and MPK3; however, the specificity of their negative regulation by phosphatases is not fully known. Here, we present genetic analyses showing that MAPK phosphatase 2 (MKP2) regulates oxidative stress and pathogen defence responses and functionally interacts with MPK3 and MPK6. We show that plants lacking a functional MKP2 gene exhibit delayed wilting symptoms in response to Ralstonia solanacearum and, by contrast, acceleration of disease progression during Botrytis cinerea infection, suggesting that this phosphatase plays differential functions in biotrophic versus necrotrophic pathogen‐induced responses. MKP2 function appears to be linked to MPK3 and MPK6 regulation, as indicated by BiFC experiments showing that MKP2 associates with MPK3 and MPK6 in vivo and that in response to fungal elicitors MKP2 exerts differential affinity versus both kinases. We also found that MKP2 interacts with MPK6 in HR‐like responses triggered by fungal elicitors, suggesting that MPK3 and MPK6 are subject to differential regulation by MKP2 in this process. We propose that MKP2 is a key regulator of MPK3 and MPK6 networks controlling both abiotic and specific pathogen responses in plants.     

Oxidative burst and cell death in ozone-exposed plants

The phytotoxic air pollutant ozone spontaneously generates reactive oxygen species (ROS) in the leaf apoplast, provokes hypersensitive response-like lesions and induces defence reactions that significantly overlap with pathogen and other oxidative stress responses. Consequently, ozone has been used as a tool to unravel in planta ROS-induced plant defence and cell death mechanisms. Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide or superoxide anions in distinct species. Accumulation of these ROS precedes the induction of cell death, and both responses co-occur spatially in the periveinal regions of the leaves. The review summarizes some of the recent results that have been obtained concerning the molecular basis of apoplastic ROS production in monocot and dicot species. Signal molecules, in particular ethylene and salicylic acid, control and potentiate the oxidative burst and subsequent cell death in its initiation and propagation phases while jasmonate leads to lesion containment. Amplification mechanisms that result in the production of excess ROS and hypersensitive cell death are discussed as major factors in ozone sensitivity of plant species and cultivars.     

Recent advances in plant MAP kinase signalling

Mitogen activated protein kinases (MAPK) are important mediators in signal transmission, connecting the perception of external stimuli to cellular responses. MAPK cascades are involved in signalling various biotic and abiotic stresses, like wounding and pathogen infection, temperature stress or drought, but are also involved in mediating the action of some plant hormones, such as ethylene and auxin. Moreover, MAPKs have been implicated in cell cycle and developmental processes. In Arabidopsis mutant screens and in vivo assays several components of plant MAPK cascades have been identified. This review gives an update of recent advances in plant MAPK signalling and discusses the emerging mechanisms of some selected MAPK pathways.     

Intercepting host MAPK signaling cascades by bacterial type III effectors

The evolutionarily conserved MAP kinase (MAPK) cascades play essential roles in plant and animal innate immunity. A recent explosion of research has uncovered a myriad of virulence strategies used by pathogenic bacteria to intercept MAPK signaling through diverse type III effectors injected into host cells. Here, we review the latest literature and discuss the various mechanisms that pathogenic bacteria use to manipulate host MAPK signaling cascades.     

Discovery of oxidative burst in the field of plant immunity: Looking back at the early pioneering works and towards the future development

This article is introductory to the series of works presented in this special issue on the homeostasis and the signaling roles of reactive oxygen species (ROS) in plants. Upper half of this article briefly describes the history of the ROS study in the field of plant immunity research initiated by the observation that the attacks by pathogenic microorganisms possibly stimulate the burst of ROS production in the plant tissues. The topics covered in the series of works presented here include the plants'' responses to abiotic oxidative stress (atmospheric ozone), regulation of seed germination, chemical interaction between parasitic and host plants and the draught tolerance, all controlled through homeostasis of ROS at biochemical and molecular biological levels. Lastly a discussion forum was proposed to further deepen our understanding of ROS behaviors in plants.Key words: hypersensitive response, NADPH oxidase, oxidative burst, plant immunity, reactive oxygen species     

Dynamic changes in the localization of MAPK cascade components controlling pathogenesis-related (PR) gene expression during innate immunity in parsley

The activation of mitogen-activated protein kinase (MAPK) cascades is an important mechanism for stress adaptation through the control of gene expression in mammals, yeast, and plants. MAPK activation has emerged as a common mechanism by which plants trigger pathogen defense responses following innate immune recognition of potential microbial pathogens. We are studying the non-host plant defense response of parsley to attempted infection by Phytophthora species using an experimental system of cultured parsley cells and the Phytophthora-derived Pep-13 peptide elicitor. Following receptor-mediated recognition of this peptide, parsley cells trigger a multifaceted innate immune response, involving the activation of three MAPKs that have been shown to function in the oxidative burst-independent activation of defense gene expression. Using this same experimental model we now report the identification of a MAPK kinase (MAPKK) that functions upstream in this pathway. This kinase, referred to as PcMKK5 based on sequence similarity to Arabidopsis thaliana AtMKK5, is activated in parsley cells following Pep-13 treatment and functions as an in vivo activator of all three MAPKs previously shown to be involved in this response. Gain- and loss-of-function mutant versions of PcMKK5, when used in protoplast co-transfection assays, demonstrated that kinase activity of PcMKK5 is required for PR gene promoter activation following Pep-13 treatment. Furthermore, using specific antibodies and immunofluorescent labeling, we demonstrate that activation of MAPKs in parsley cells correlates with an increase in their nuclear localization, which is not detectable for activated PcMKK5. These results suggest that activation of gene expression through MAPK cascades during innate immune responses in plants involves dynamic changes in the localization of the proteins involved, which may reflect the distribution of key protein substrates for the activated MAPKs.     

Stimulation of p42 and p44 Mitogen-Activated Protein Kinases by Reactive Oxygen Species and Nitric Oxide in Hippocampus

Abstract: Reactive oxygen species (ROS) have been suggested to act as cellular messengers that mediate signal transduction cascades in various cell types. However, little is known about their role in this capacity in the nervous system. We have begun to investigate the role of ROS, and that of nitric oxide (NO), in mediating mitogen-activated protein kinase (MAPK) signaling in rat hippocampal slices. Our studies have revealed that direct exposure of hippocampal slices to hydrogen peroxide, xanthine/xanthine oxidase (a superoxide-generating system), sodium nitroprusside (an NO donor compound), S -nitroso- N -acetylpenicillamine (an NO donor compound), or 3-morpholinosydnonimine (a compound that produces NO and superoxide) results in an enhancement in tyrosine phosphorylation of several proteins, including proteins with apparent molecular masses of 42 and 44 kDa. We investigated the possibility that these proteins correspond to the active forms of p42 MAPK and p44 MAPK. Hippocampal slices exposed to various ROS and NO donors resulted in increases in levels of the active forms of both p42 MAPK and p44 MAPK. The ROS- and NO-enhanced tyrosine phosphorylation and activation of p42 MAPK and p44 MAPK were inhibited by pretreatment with the antioxidant N -acetyl- l -cysteine. Our observations indicate that ROS and NO can mediate protein tyrosine phosphorylation and MAPK signaling in the hippocampus via a redox-sensitive mechanism and suggest a potential cellular mechanism for their effects in the nervous system.     

ASK Family Proteins in Stress Response and Disease

Cells are continuously exposed to reactive oxygen species (ROS) generated by aerobic metabolism. Excessively generated ROS causes severe dysfunctions to cells as oxidative stress. On the other hand, there is increasing evidence that ROS plays important roles as a signaling intermediate that induces a wide variety of cellular responses such as proliferation, differentiation, senescence, and apoptosis. To transmit physiological ROS-mediated signals and to adapt to oxidative stress, cells are equipped with various intracellular signal transduction systems, represented by mitogen-activated protein kinase (MAPK) cascades. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream regulator of the stress-activated MAPK cascades and has been shown to play critical roles in ROS-mediated cellular responses. Here, we highlight the roles of members of the ASK family, which consists of ASK1 and newly characterized ASK2, in ROS signaling with their possible involvement in human diseases.     

Candida albicans cell surface superoxide dismutases degrade host-derived reactive oxygen species to escape innate immune surveillance

Mammalian innate immune cells produce reactive oxygen species (ROS) in the oxidative burst reaction to destroy invading microbial pathogens. Using quantitative real-time ROS assays, we show here that both yeast and filamentous forms of the opportunistic human fungal pathogen Candida albicans trigger ROS production in primary innate immune cells such as macrophages and dendritic cells. Through a reverse genetic approach, we demonstrate that coculture of macrophages or myeloid dendritic cells with C. albicans cells lacking the superoxide dismutase (SOD) Sod5 leads to massive extracellular ROS accumulation in vitro . ROS accumulation was further increased in coculture with fungal cells devoid of both Sod4 and Sod5. Survival experiments show that C. albicans mutants lacking Sod5 and Sod4 exhibit a severe loss of viability in the presence of macrophages in vitro . The reduced viability of sod5 Δ/Δ and sod4 Δ/Δ sod5 Δ/Δ mutants relative to wild type is not evident with macrophages from gp91phox ^{−/ −} mice defective in the oxidative burst activity, demonstrating a ROS-dependent killing activity of macrophages targeting fungal pathogens. These data show a physiological role for cell surface SODs in detoxifying ROS, and suggest a mechanism whereby C. albicans , and perhaps many other microbial pathogens, can evade host immune surveillance in vivo .     

Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase

Reactive oxygen species (ROS) are implicated in plant innate immunity. NADPH oxidase (RBOH; for Respiratory Burst Oxidase Homolog) plays a central role in the oxidative burst, and EF-hand motifs in the N terminus of this protein suggest possible regulation by Ca(2+). However, regulatory mechanisms are largely unknown. We identified Ser-82 and Ser-97 in the N terminus of potato (Solanum tuberosum) St RBOHB as potential phosphorylation sites. An anti-phosphopeptide antibody (pSer82) indicated that Ser-82 was phosphorylated by pathogen signals in planta. We cloned two potato calcium-dependent protein kinases, St CDPK4 and St CDPK5, and mass spectrometry analyses showed that these CDPKs phosphorylated only Ser-82 and Ser-97 in the N terminus of St RBOHB in a calcium-dependent manner. Ectopic expression of the constitutively active mutant of St CDPK5, St CDPK5VK, provoked ROS production in Nicotiana benthamiana leaves. The CDPK-mediated ROS production was disrupted by knockdown of Nb RBOHB in N. benthamiana. The loss of function was complemented by heterologous expression of wild-type potato St RBOHB but not by a mutant (S82A/S97A). Furthermore, the heterologous expression of St CDPK5VK phosphorylated Ser-82 of St RBOHB in N. benthamiana. These results suggest that St CDPK5 induces the phosphorylation of St RBOHB and regulates the oxidative burst.     

Reactive-Oxygen-Species-Mediated P. aeruginosa Killing Is Functional in Human Cystic Fibrosis Macrophages

Pseudomonas aeruginosa is the most common pathogen for chronic lung infection in cystic fibrosis (CF) patients. About 80% of adult CF patients have chronic P. aeruginosa infection, which accounts for much of the morbidity and most of the mortality. Both bacterial genetic adaptations and defective innate immune responses contribute to the bacteria persistence. It is well accepted that CF transmembrane conductance regulator (CFTR) dysfunction impairs the airways-epithelium-mediated lung defence; however, other innate immune cells also appear to be affected, such as neutrophils and macrophages, which thus contribute to this infectious pathology in the CF lung. In macrophages, the absence of CFTR has been linked to defective P. aeruginosa killing, increased pro-inflammatory cytokine secretion, and reduced reactive oxygen species (ROS) production. To learn more about macrophage dysfunction in CF patients, we investigated the generation of the oxidative burst and its impact on bacterial killing in CF macrophages isolated from peripheral blood or lung parenchyma of CF patients, after P. aeruginosa infection. Our data demonstrate that CF macrophages show an oxidative response of similar intensity to that of non-CF macrophages. Intracellular ROS are recognized as one of the earliest microbicidal mechanisms against engulfed pathogens that are activated by macrophages. Accordingly, NADPH inhibition resulted in a significant increase in the intracellular bacteria survival in CF and non-CF macrophages, both as monocyte-derived macrophages and as lung macrophages. These data strongly suggest that the contribution of ROS to P. aeruginosa killing is not affected by CFTR mutations.     

Plant MAP kinase pathways: how many and what for?

Mitogen activated protein kinases (MAPK) are important mediators in signal transmission, connecting the perception of external stimuli to cellular responses. MAPK cascades are involved in signalling various biotic and abiotic stresses, like wounding and pathogen infection, temperature stress or drought, but also some plant hormones, such as ethylene and auxin. Moreover, MAPKs have been implicated in cell cycle and developmental processes. In Arabidopsis mutant screens and in vivo assays several components of plant MAPK cascades have been identified. This review compares results obtained from functional analyses of MAPK cascades in plants with recent data obtained from searching the complete Arabidopsis genome. This analysis reveals that plants have an overall of 24 MAPK pathways of which only a small subset has been studied so far.     

Rapid ascorbate response to bacterial elicitor treatment in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> cells

An early event of the incompatible plant–pathogen interactions is an oxidative burst. On one hand, the ROS generated during oxidative burst is advantageous. ROS can serve as secondary messengers mediating defence gene activation and establishment of additional defences. On the other hand, the concentration of ROS must be carefully regulated to avoid undesired cellular cytotoxicity. The major water soluble, low molecular weight antioxidant, ascorbic acid plays a crucial role in ROS balancing (scavenging). The regulation of ascorbate level, therefore, can be an important point of the fine-tuning of ROS level during the early phase of plant–pathogen interaction. To evaluate how this interaction affects the biosynthesis, the recycling, and the level of ascorbate, we challenged Arabidopsis thaliana cells with two different harpin proteins (HrpZ_pto and HrpW_pto). HrpZ_pto and HrpW_pto treatments caused a well-defined ROS peak. The expression of the alternative oxidase (AOX1a) and vtc5, one of the paralog genes that encode the rate limiting enzyme of ascorbate biosynthesis, followed the elevation of ROS. Similarly, the activity of ascorbate peroxidase and galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) (GLDH), the enzyme catalysing the ultimate, mitochondria coupled step of ascorbate biosynthesis and the level of ascorbate and glutathione also followed the elevation of ROS due to harpin treatment. The enhanced expression of AOX1a, the elevated activity of GLDH, and the increased level of ascorbate and glutathione all can contribute to the mitigation or absence of programmed cell death. Finally, a new function, the fine-tuning of redox balance during plant–pathogen interaction, can be proposed to vtc5.     

Salicylic acid-induced superoxide generation catalyzed by plant peroxidase in hydrogen peroxide-independent manner

It has been reported that salicylic acid (SA) induces both immediate spike and long lasting phases of oxidative burst represented by the generation of reactive oxygen species (ROS) such as superoxide anion radical (O₂^•−). In general, in the earlier phase of oxidative burst, apoplastic peroxidase are likely involved and in the late phase of the oxidative burst, NADPH oxidase is likely involved. Key signaling events connecting the 2 phases of oxidative burst are calcium channel activation and protein phosphorylation events. To date, the known earliest signaling event in response to exogenously added SA is the cell wall peroxidase-catalyzed generation of O₂^•− in a hydrogen peroxide (H₂O₂)-dependent manner. However, this model is incomplete since the source of the initially required H₂O₂ could not be explained. Based on the recently proposed role for H₂O₂-independent mechanism for ROS production catalyzed by plant peroxidases (Kimura et al., 2014, Frontiers in Plant Science), we hereby propose a novel model for plant peroxidase-catalyzed oxidative burst fueled by SA.     

Microbial Pathogens Trigger Host DNA Double-Strand Breaks Whose Abundance Is Reduced by Plant Defense Responses

Immune responses and DNA damage repair are two fundamental processes that have been characterized extensively, but the links between them remain largely unknown. We report that multiple bacterial, fungal and oomycete plant pathogen species induce double-strand breaks (DSBs) in host plant DNA. DNA damage detected by histone γ-H2AX abundance or DNA comet assays arose hours before the disease-associated necrosis caused by virulent Pseudomonas syringae pv. tomato. Necrosis-inducing paraquat did not cause detectable DSBs at similar stages after application. Non-pathogenic E. coli and Pseudomonas fluorescens bacteria also did not induce DSBs. Elevation of reactive oxygen species (ROS) is common during plant immune responses, ROS are known DNA damaging agents, and the infection-induced host ROS burst has been implicated as a cause of host DNA damage in animal studies. However, we found that DSB formation in Arabidopsis in response to P. syringae infection still occurs in the absence of the infection-associated oxidative burst mediated by AtrbohD and AtrbohF. Plant MAMP receptor stimulation or application of defense-activating salicylic acid or jasmonic acid failed to induce a detectable level of DSBs in the absence of introduced pathogens, further suggesting that pathogen activities beyond host defense activation cause infection-induced DNA damage. The abundance of infection-induced DSBs was reduced by salicylic acid and NPR1-mediated defenses, and by certain R gene-mediated defenses. Infection-induced formation of γ-H2AX still occurred in Arabidopsis atr/atm double mutants, suggesting the presence of an alternative mediator of pathogen-induced H2AX phosphorylation. In summary, pathogenic microorganisms can induce plant DNA damage. Plant defense mechanisms help to suppress rather than promote this damage, thereby contributing to the maintenance of genome integrity in somatic tissues.