The plant perception of pathogen-associated molecular patterns triggers a plethora of cellular immune responses. One of these responses is a rapid and transient burst of reactive oxygen species (ROS) ...mediated by plasma membrane-localized NADPH oxidases. The ROS burst requires a functional receptor complex and the contribution of several additional regulatory components. In laboratory conditions, the ROS burst can be detected a few minutes after the treatment with an immunogenic microbial elicitor. For these reasons, the elicitor-triggered ROS burst has been often exploited as readout to probe the contribution of plant components to early immune responses. Here, we describe a detailed protocol for the measurement of elicitor-triggered ROS burst in a simple, fast, and easy manner.
Nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins function as sensors that perceive pathogen molecules and activate immunity. In plants, the accumulation and activation of ...NLRs is regulated by SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1). In this work, we found that an effector protein named RipAC, secreted by the plant pathogen Ralstonia solanacearum, associates with SGT1 to suppress NLR-mediated SGT1-dependent immune responses, including those triggered by another R. solanacearum effector, RipE1. RipAC does not affect the accumulation of SGT1 or NLRs, or their interaction. However, RipAC inhibits the interaction between SGT1 and MAP kinases, and the phosphorylation of a MAPK target motif in the C-terminal domain of SGT1. Such phosphorylation is enhanced upon activation of immune signaling and contributes to the activation of immune responses mediated by the NLR RPS2. Additionally, SGT1 phosphorylation contributes to resistance against R. solanacearum. Our results shed light onto the mechanism of activation of NLR-mediated immunity, and suggest a positive feedback loop between MAPK activation and SGT1-dependent NLR activation.
The internal C:N balance must be tightly controlled for the normal growth and development of plants. However, the underlying mechanisms, by which plants sense and balance the intracellular C:N status ...correspondingly to exogenous C:N availabilities remain elusive. In this study, we use comparative gene expression analysis to identify genes that are responsive to imbalanced C:N treatments in the aerial parts of rice seedlings. Transcripts of rice seedlings treated with four C:N availabilities (1:1, 1:60, 60:1 and 60:60) were compared and two groups of genes were classified: high C:low N responsive genes and low C:high N responsive genes. Our analysis identified several functional correlated genes including chalcone synthase (CHS), chlorophyll a-b binding protein (CAB) and other genes that are implicated in C:N balancing mechanism, such as alternative oxidase 1B (OsAOX1B), malate dehydrogenase (OsMDH) and lysine and histidine specific transporter 1 (OsLHT1). Additionally, six jasmonate synthetic genes and key regulatory genes involved in abiotic and biotic stresses, such as OsMYB4, autoinhibited calcium ATPase 3 (OsACA3) and pleiotropic drug resistance 9 (OsPDR9), were differentially expressed under high C:low N treatment. Gene ontology analysis showed that high C:low N up-regulated genes were primarily enriched in fatty acid biosynthesis and defense responses. Coexpression network analysis of these genes identified eight jasmonate ZIM domain protein (OsJAZ) genes and several defense response related regulators, suggesting that high C:low N status may act as a stress condition, which induces defense responses mediated by jasmonate signaling pathway. Our transcriptome analysis shed new light on the C:N balancing mechanisms and revealed several important regulators of C:N status in rice seedlings.
Summary
The subversion of plant cellular functions is essential for bacterial pathogens to proliferate in host plants and cause disease. Most bacterial plant pathogens employ a type III secretion ...system to inject type III effector (T3E) proteins inside plant cells, where they contribute to the pathogen‐induced alteration of plant physiology. In this work, we found that the Ralstonia solanacearum T3E RipAY suppresses plant immune responses triggered by bacterial elicitors and by the phytohormone salicylic acid. Further biochemical analysis indicated that RipAY associates in planta with thioredoxins from Nicotiana benthamiana and Arabidopsis. Interestingly, RipAY displays γ‐glutamyl cyclotransferase (GGCT) activity to degrade glutathione in plant cells, which is required for the reported suppression of immune responses. Given the importance of thioredoxins and glutathione as major redox regulators in eukaryotic cells, RipAY activity may constitute a novel and powerful virulence strategy employed by R. solanacearum to suppress immune responses and potentially alter general redox signalling in host cells.
Effector proteins delivered inside plant cells are powerful weapons for bacterial pathogens, but this exposes the pathogen to potential recognition by the plant immune system. Therefore, the effector ...repertoire of a given pathogen must be balanced for a successful infection. Ralstonia solanacearum is an aggressive pathogen with a large repertoire of secreted effectors. One of these effectors, RipE1, is conserved in most R. solanacearum strains sequenced to date. In this work, we found that RipE1 triggers immunity in N. benthamiana, which requires the immune regulator SGT1, but not EDS1 or NRCs. Interestingly, RipE1-triggered immunity induces the accumulation of salicylic acid (SA) and the overexpression of several genes encoding phenylalanine-ammonia lyases (PALs), suggesting that the unconventional PAL-mediated pathway is responsible for the observed SA biosynthesis. Surprisingly, RipE1 recognition also induces the expression of jasmonic acid (JA)-responsive genes and JA biosynthesis, suggesting that both SA and JA may act cooperatively in response to RipE1. We further found that RipE1 expression leads to the accumulation of glutathione in plant cells, which precedes the activation of immune responses. R. solanacearum secretes another effector, RipAY, which is known to inhibit immune responses by degrading cellular glutathione. Accordingly, RipAY inhibits RipE1-triggered immune responses. This work shows a strategy employed by R. solanacearum to counteract the perception of its effector proteins by plant immune system.
The type-III secreted effector RipE1, from Ralstonia solanacearum, triggers immune responses in Arabidopsis and Nicotiana benthamiana. Such immune responses correlate with an activation of signaling mediated by salicylic acid and jasmonic acid. RipE1-triggered immunity is suppressed by another effector in R. solanacearum, RipAY, showing a bacterial strategy to counteract effector-triggered immunity.
Most bacterial pathogens subvert plant cellular functions using effector proteins delivered inside plant cells. In the plant pathogen
, several of these effectors contain domains with predicted ...enzymatic activities, including acetyltransferases, phosphatases, and proteases, among others. How these enzymatic activities get activated inside plant cells, but not in the bacterial cell, remains unknown in most cases. In this work, we found that the
effector RipAY is phosphorylated in plant cells. One phosphorylated serine residue, S131, is required for the reported gamma-glutamyl cyclotransferase activity of RipAY, responsible for the degradation of gamma-glutamyl compounds (such as glutathione) inside host cells. Accordingly, non-phosphorylable mutants in S131 abolish RipAY-mediated degradation of glutathione in plant cells and the subsequent suppression of plant immune responses. In this article, we examine our results in relation to the recent reports on the biochemical activities of RipAY, and discuss the potential implications of phosphorylation in plant cells as a mechanism to modulate the enzymatic activity of RipAY.
Pyridoxal phosphate (PLP), a vitamin B
6 vitamer, is an essential cofactor for numerous enzymes. Pyridoxine/pyridoxamine phosphate oxidase (PPOX) catalyzes the synthesis of pyridoxal phosphate from ...pyridoxine phosphate (PNP) and/or pyridoxamine phosphate (PMP). The
At5g49970 locus in
Arabidopsis thaliana encodes an
AtPPOX, a PNP/PMP oxidase. The expression of the
AtPPOX gene varied in different tissues of Arabidopsis examined, being up-regulated by light, heat shock, ABA, and ethylene treatments, and down-regulated by exposure to drought and NaCl. Monoclonal antibodies raised against two different domains of
AtPPOX recognized different sizes of
AtPPOX, suggesting that
AtPPOX proteins are produced as splice variants of the
AtPPOX gene in Arabidopsis. Phylogenetic analysis of
AtPPOX across all domains of life demonstrated that plant
AtPPOX homologs have an additional Yjef_N domain preceding the Pyridox_Oxidase domain at the C-terminal end of the protein, while
AtPPOX homologs from bacteria, fungi and animals have only Pyridox_Oxidase domain. The presence of the Yjef_N domain in plant
AtPPOX homologs suggests that acquisition of this domain, and its fusion with the pyridox_oxidase domain began with the endosymbiotic acquisition of the chloroplast. Bioinformatic analysis suggested that
AtPPOX is localized in chloroplast, but the monoclonal antibody could not be used for subcellular localization of this protein. A
GFP–AtPPOX fusion construct introduced into the Arabidospsis protoplast confirmed localization of
AtPPOX into the chloroplast. An RNAi mutant of
AtPPOX showed sensitivity to high light suggesting a role for PPOX in resistance to photooxidative damage, and alteration in root growth in the presence of sucrose suggests a role for PPOX in root development.
►
AtPPOX gene is regulated by abiotic stress and plant hormones. ►
AtPPOX protein is localized in chloroplast. ► Two sizes of
AtPPOX in root and shoot provide evidence for splice variants. ► RNAi mutant of
AtPPOX are sensitive to high light intensity and high sucrose.
Carbon (C) and nitrogen (N) are two essential nutrients affecting plant growth and development. Plants are non-motile organisms and have evolved highly sophisticated and complex sensing and signaling ...mechanisms to respond to the dynamic changes of C and N nutrients in their surroundings. C and N metabolism are tightly coordinated to maintain intracellular C/N homeostasis. However, the regulatory mechanism underlying C/N coordination and balancing in plants remains to be elucidated. It has been suggested that C and N metabolism are modulated by the interaction of C signaling with N signaling or by C/N ratio signaling. This review focuses on cell signaling studies that provide insight into the regulation mechanism of C/N balancing in plants.
Pyridoxine (pyridoxamine) 5′-phosphate oxidase (PPOX) catalyzes the oxidative conversion of pyridoxamine 5′-phosphate (PMP) or pyridoxine 5′-phosphate (PNP) to pyridoxal 5′-phosphate (PLP). The
...At5g49970 gene of
Arabidopsis thaliana shows homology to PPOX’s from a number of organisms including the
Saccharomyces cerevisiae PDX3 gene. A cDNA corresponding to putative
A. thaliana PPOX (
AtPPOX) was obtained using reverse transcriptase-polymerase chain reaction and primers landing at the start and stop codons of
At5g49970. The putative
AtPPOX is 530 amino acid long and predicted to contain three distinct parts: a 64 amino acid long N-terminal putative chloroplast transit peptide, followed by a long Yjef_N domain of unknown function and a C-terminal Pyridox_oxidase domain. Recombinant proteins representing the C-terminal domain of
AtPPOX and
AtPPOX without transit peptide were expressed in
E. coli and showed PPOX enzyme activity. The
PDX3 knockout yeast deficient in PPOX activity exhibited sensitivity to oxidative stress. Constructs of
AtPPOX cDNA of different lengths complemented the
PDX3 knockout yeast for oxidative stress. The role of the Yjef_N domain of
AtPPOX was not determined, but it shows homology with a number of conserved hypothetical proteins of unknown function.
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