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1.  Arabidopsis Putative Deacetylase AtSRT2 Regulates Basal Defense by Suppressing PAD4, EDS5 and SID2 Expression 
Plant and Cell Physiology  2010;51(8):1291-1299.
The silent information regulator protein (Sir2) and its homologs are NAD+-dependent deacetylase enzymes that play important roles in a variety of physiological processes. However, the functions of the Sir2 family in plants are poorly understood. Here, we report that Arabidopsis AtSRT2, a homolog of yeast Sir2, negatively regulates plant basal defense against the pathogen Pseudomonas syringae pv. tomato DC3000 (PstDC3000). In response to PstDC3000 infection, the expression of AtSRT2 was down-regulated in a salicylic acid (SA)-independent manner. In addition, knock-out of AtSRT2 (srt2) enhanced resistance against PstDC3000 and increased expression of pathogenesis-related gene 1 (PR1). Conversely, overexpression of AtSRT2 resulted in hypersusceptibility to PstDC3000 and impaired PR1 induction. Consistent with this phenotype, expression of PAD4, EDS5 and SID2, three essential genes in the SA biosynthesis pathway, were increased in the srt2 mutant and decreased in AtSRT2-overexpressing plants. Taken together, these results demonstrate that AtSRT2 is a negative regulator of basal defense, possibly by suppressing SA biosynthesis.
PMCID: PMC2920754  PMID: 20573705
AtSRT2; Basal defense; EDS5; PAD4; PstDC3000; SID2
2.  PtrWRKY73, a salicylic acid-inducible poplar WRKY transcription factor, is involved in disease resistance in Arabidopsis thaliana 
Plant Cell Reports  2015;34(5):831-841.
Key message
A salicylic acid-inducible WRKY gene,PtrWRKY73,fromPopulus trichocarpa, was isolated and characterized. Overexpression ofPtrWRKY73inArabidopsis thalianaincreased resistance to biotrophic pathogens but reduced resistance against necrotrophic pathogens.
WRKY transcription factors are commonly involved in plant defense responses. However, limited information is available about the roles of the WRKY genes in poplar defense. In this study, we isolated a salicylic acid (SA)-inducible WRKY gene, PtrWRKY73, from Populus trichocarpa, belonging to group I family and containing two WRKY domains, a D domain and an SP cluster. PtrWRKY73 was expressed predominantly in roots, old leaves, sprouts and stems, especially in phloem and its expression was induced in response to treatment with exogenous SA. PtrWRKY73 was localized to the nucleus of plant cells and exhibited transcriptional activation. Overexpression of PtrWRKY73 in Arabidopsis thaliana resulted in increased resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae (PstDC3000), but more sensitivity to the necrotrophic fungal pathogen Botrytis cinerea. The SA-mediated defense-associated genes, such as PR1, PR2 and PAD4, were markedly up-regulated in transgenic plants overexpressing PtrWRKY73. Arabidopsis non-expressor of PR1 (NPR1) was not affected, whereas a defense-related gene PAL4 had reduced in PtrWRKY73 overexpressor plants. Together, these results indicated that PtrWRKY73 plays a positive role in plant resistance to biotrophic pathogens but a negative effect on resistance against necrotrophic pathogens.
Electronic supplementary material
The online version of this article (doi:10.1007/s00299-015-1745-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4405351  PMID: 25627252
Populus; WRKY; Transcription factor; Pathogen; SA
3.  Factors other than root secreted malic acid that contributes toward Bacillus subtilis FB17 colonization on Arabidopsis roots 
Plant Signaling & Behavior  2013;8(11):e27277.
The plant growth promoting rhizobacterium (PGPR) Bacillus subtilis FB17 (hereafter FB17) induces resistance against broad pathogen including Pseudomonas syringae pv tomato (PstDC3000). The extent of plant protection by FB17 depends on establishment of root colonization followed by biofilm formation. The general convention dictates that beneficial rhizobacterium may suppress the root innate immune system to establish a robust colonization. However, it is still not well understood which genetic targets FB17 affects in plants to facilitate a symbiotic association. Our recent study, involving whole transcriptome analysis of Arabidopsis thaliana roots treated with FB17 post 24 h of treatment showed totally 279 genes that were significantly up- or/ downregulated. Further, we found that the mutants for upregulated and downregulated genes post-FB17 colonization showed a differential phenotype for FB17 root colonization. Interestingly, plants mutated in the FB17-responsive genes showed increased Aluminum activated malate transporter (ALMT1) expression under foliar pathogen PstDC3000, infections, indicating the independent functionality of ALMT1 for bacterial recruitment. Taken together this, present study suggests that the establishment of interaction between the plant host and PGPR is a complex phenomenon which is regulated by multiple genetic components.
PMCID: PMC4092310  PMID: 24310121
Arabidopsis, Bacillus subtilis FB17, Biofilm; Malic acid; Rhizobacteria; Root transcriptome
4.  Influence of Human p53 on Plant Development 
PLoS ONE  2016;11(9):e0162840.
Mammalian p53 is a super tumor suppressor and plays a key role in guarding genome from DNA damage. However, p53 has not been found in plants which do not bear cancer although they constantly expose to ionizing radiation of ultraviolet light. Here we introduced p53 into the model plant Arabidopsis and examined p53-conferred phenotype in plant. Most strikingly, p53 caused early senescence and fasciation. In plants, fasciation has been shown as a result of the elevated homologous DNA recombination. Consistently, a reporter with overlapping segments of the GUS gene (1445) showed that the frequency of homologous recombination was highly induced in p53-transgenic plants. In contrast to p53, SUPPRESSOR OF NPR1-1 INDUCIBLE 1 (SNI1), as a negative regulator of homologous recombination in plants, is not present in mammals. Comet assay and clonogenic survival assay demonstrated that SNI1 inhibited DNA damage repair caused by either ionizing radiation or hydroxyurea in human osteosarcoma U2OS cancer cells. RAD51D is a recombinase in homologous recombination and functions downstream of SNI1 in plants. Interestingly, p53 rendered the sni1 mutants madly branching of inflorescence, a phenotype of fasciation, whereas rad51d mutant fully suppressed the p53-induced phenotype, indicating that human p53 action in plant is mediated by the SNI1-RAD51D signaling pathway. The reciprocal species-swap tests of p53 and SNI1 in human and Arabidopsis manifest that these species-specific proteins play a common role in homologous recombination across kingdoms of animals and plants.
PMCID: PMC5029891  PMID: 27648563
5.  Purple Acid Phosphatase5 is required for maintaining basal resistance against Pseudomonas syringae in Arabidopsis 
BMC Plant Biology  2013;13:107.
Plants have evolved an array of constitutive and inducible defense strategies to restrict pathogen ingress. However, some pathogens still manage to invade plants and impair growth and productivity. Previous studies have revealed several key regulators of defense responses, and efforts have been made to use this information to develop disease resistant crop plants. These efforts are often hampered by the complexity of defense signaling pathways. To further elucidate the complexity of defense responses, we screened a population of T-DNA mutants in Colombia-0 background that displayed altered defense responses to virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000).
In this study, we demonstrated that the Arabidopsis Purple Acid Phosphatse5 (PAP5) gene, induced under prolonged phosphate (Pi) starvation, is required for maintaining basal resistance to certain pathogens. The expression of PAP5 was distinctly induced only under prolonged Pi starvation and during the early stage of Pst DC3000 infection (6 h.p.i). T-DNA tagged mutant pap5 displayed enhanced susceptibility to the virulent bacterial pathogen Pst DC3000. The pap5 mutation greatly reduced the expression of pathogen inducible gene PR1 compared to wild-type plants. Similarly, other defense related genes including ICS1 and PDF1.2 were impaired in pap5 plants. Moreover, application of BTH (an analog of SA) restored PR1 expression in pap5 plants.
Taken together, our results demonstrate the requirement of PAP5 for maintaining basal resistance against Pst DC3000. Furthermore, our results provide evidence that PAP5 acts upstream of SA accumulation to regulate the expression of other defense responsive genes. We also provide the first experimental evidence indicating the role PAP5 in plant defense responses.
PMCID: PMC3751912  PMID: 23890153
Arabidopsis; Plant defense responses; PAP5; Pseudomonas syringae; Phosphate starvation
6.  Exchanging the as-1-like element of the PR-1 promoter by the as-1 element of the CaMV 35S promoter abolishes salicylic acid responsiveness and regulation by NPR1 and SNI1 
Plant Signaling & Behavior  2010;5(12):1669-1671.
The plant defense hormone salicylic acid (SA) activates gene expression through a number of different mechanisms. In Arabidopsis thaliana, the SA-induced PATHOGENESIS RELATED (PR)-1 promoter is regulated through TGA transcription factors binding to the two TGACG motifs of the so called as-1 (activation sequence-1)-like element which is located between base pair positions -665 and -641. Activation is mediated by the transcriptional co-activator NPR1 (NON EXPRESSOR OF PR GENES1), which physically interacts with TGA factors. Moreover, the promoter is under the control of the negative regulator SNI1 (SUPPRESSOR OF NPR1, INDUCIBLE1). We have recently reported that SNI1-mediated repression of basal promoter activities and NPR1-dependent induction are maintained in a truncated PR-1 promoter that contains sequences between -816 and -573 upstream of the -68 promoter region. In this addendum, we report that the expression characteristics of this truncated PR-1 promoter is changed profoundly when its as-1-like element is replaced by the as-1 element of Cauliflower Mosaic Virus 35S promoter which also contains two TGACG motifs. The resulting chimeric promoter showed high constitutive activity that was independent from SA, NPR1 and SNI1. Thus, the configuration of two TGA binding sites within the PR-1 promoter determines whether NPR1 can induce and whether SNI1 can repress the promoter.
PMCID: PMC3115131  PMID: 21139438
NPR1; PR-1; salicylic acid; SNI1; TGA factors
7.  Arabidopsis Actin-Depolymerizing Factor-4 Links Pathogen Perception, Defense Activation and Transcription to Cytoskeletal Dynamics 
PLoS Pathogens  2012;8(11):e1003006.
The primary role of Actin-Depolymerizing Factors (ADFs) is to sever filamentous actin, generating pointed ends, which in turn are incorporated into newly formed filaments, thus supporting stochastic actin dynamics. Arabidopsis ADF4 was recently shown to be required for the activation of resistance in Arabidopsis following infection with the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst) expressing the effector protein AvrPphB. Herein, we demonstrate that the expression of RPS5, the cognate resistance protein of AvrPphB, was dramatically reduced in the adf4 mutant, suggesting a link between actin cytoskeletal dynamics and the transcriptional regulation of R-protein activation. By examining the PTI (PAMP Triggered Immunity) response in the adf4 mutant when challenged with Pst expressing AvrPphB, we observed a significant reduction in the expression of the PTI-specific target gene FRK1 (Flg22-Induced Receptor Kinase 1). These data are in agreement with recent observations demonstrating a requirement for RPS5 in PTI-signaling in the presence of AvrPphB. Furthermore, MAPK (Mitogen-Activated Protein Kinase)-signaling was significantly reduced in the adf4 mutant, while no such reduction was observed in the rps5-1 point mutation under similar conditions. Isoelectric focusing confirmed phosphorylation of ADF4 at serine-6, and additional in planta analyses of ADF4's role in immune signaling demonstrates that nuclear localization is phosphorylation independent, while localization to the actin cytoskeleton is linked to ADF4 phosphorylation. Taken together, these data suggest a novel role for ADF4 in controlling gene-for-gene resistance activation, as well as MAPK-signaling, via the coordinated regulation of actin cytoskeletal dynamics and R-gene transcription.
Author Summary
The activation and regulation of the plant immune system requires the coordinated function of numerous pre-formed and inducible cellular responses. Following pathogen perception, plants not only activate specific defense-associated signaling, such as resistance (R) genes, but also redirect basic cellular machinery to support innate immune signaling. Within each of these processes, the actin cytoskeleton has been demonstrated to play a significant role in structural-based defense signaling in plants in response to pathogen infection. Most notably, the actin cytoskeleton of plants has been shown to play a role in structural-based defense signaling following fungal pathogen infection. Recent work from our laboratory has demonstrated that the actin cytoskeleton of Arabidopsis mediates defense signaling following perception of the phytopathogenic bacterium Pseudomonas syringae. Using a combination of genetic and cell biology-based approaches, we found that ADF4, a regulator of actin cytoskeletal dynamics, is required for the specific activation of R-gene-mediated signaling. By analyzing the activation of signaling following pathogen perception, we have identified substantial crosstalk between recognition of pathogen virulence factors (e.g., effector proteins) and the regulation of R-gene transcription. In total, our work highlights the intimate relationship between basic cellular processes and the perception and activation of defense signaling following pathogen infection.
PMCID: PMC3493479  PMID: 23144618
8.  Multiple Candidate Effectors from the Oomycete Pathogen Hyaloperonospora arabidopsidis Suppress Host Plant Immunity 
PLoS Pathogens  2011;7(11):e1002348.
Oomycete pathogens cause diverse plant diseases. To successfully colonize their hosts, they deliver a suite of effector proteins that can attenuate plant defenses. In the oomycete downy mildews, effectors carry a signal peptide and an RxLR motif. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on the model plant Arabidopsis thaliana (Arabidopsis). We investigated if candidate effectors predicted in the genome sequence of Hpa isolate Emoy2 (HaRxLs) were able to manipulate host defenses in different Arabidopsis accessions. We developed a rapid and sensitive screening method to test HaRxLs by delivering them via the bacterial type-three secretion system (TTSS) of Pseudomonas syringae pv tomato DC3000-LUX (Pst-LUX) and assessing changes in Pst-LUX growth in planta on 12 Arabidopsis accessions. The majority (∼70%) of the 64 candidates tested positively contributed to Pst-LUX growth on more than one accession indicating that Hpa virulence likely involves multiple effectors with weak accession-specific effects. Further screening with a Pst mutant (ΔCEL) showed that HaRxLs that allow enhanced Pst-LUX growth usually suppress callose deposition, a hallmark of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We found that HaRxLs are rarely strong avirulence determinants. Although some decreased Pst-LUX growth in particular accessions, none activated macroscopic cell death. Fewer HaRxLs conferred enhanced Pst growth on turnip, a non-host for Hpa, while several reduced it, consistent with the idea that turnip's non-host resistance against Hpa could involve a combination of recognized HaRxLs and ineffective HaRxLs. We verified our results by constitutively expressing in Arabidopsis a sub-set of HaRxLs. Several transgenic lines showed increased susceptibility to Hpa and attenuation of Arabidopsis PTI responses, confirming the HaRxLs' role in Hpa virulence. This study shows TTSS screening system provides a useful tool to test whether candidate effectors from eukaryotic pathogens can suppress/trigger plant defense mechanisms and to rank their effectiveness prior to subsequent mechanistic investigation.
Author Summary
Hyaloperonospora arabidopsidis (Hpa) is an obligate biotroph whose population coevolves with its host, Arabidopsis thaliana. The Hpa isolate Emoy2 genome has been sequenced, allowing the discovery of dozens of secreted candidate effectors. We set out to assign functions to these candidate effectors, investigating if they suppress host defenses. We analyzed a sub-set of Hpa candidate effectors (HaRxLs) that carry the RxLR motif, using a bacterial system for in planta delivery. To our surprise, we found that most of the HaRxLs enhanced plant susceptibility on at least some accessions, while few decreased it. These phenotypes were mostly confirmed on Arabidopsis transgenic lines stably expressing HaRxLs that became more susceptible to compatible Hpa isolates. Furthermore, effectors that conferred enhanced virulence generally suppressed callose deposition, a hallmark of plant defense. This indicates that the “effectorome” of Hpa comprises multiple distinct effectors that can attenuate Arabidopsis immunity. We found that many HaRxLs did not confer enhanced virulence on all host accessions, and also that only ∼50% of the effectors that conferred enhanced Pst growth on Arabidopsis, were able to do so on turnip, a non-host for Hpa. Our data reveal interesting HaRxLs for detailed mechanistic investigation in future experiments.
PMCID: PMC3207932  PMID: 22072967
9.  The Arabidopsis Lectin Receptor Kinase LecRK-V.5 Represses Stomatal Immunity Induced by Pseudomonas syringae pv. tomato DC3000 
PLoS Pathogens  2012;8(2):e1002513.
Stomata play an important role in plant innate immunity by limiting pathogen entry into leaves but molecular mechanisms regulating stomatal closure upon pathogen perception are not well understood. Here we show that the Arabidopsis thaliana L-type lectin receptor kinase-V.5 (LecRK-V.5) negatively regulates stomatal immunity. Loss of LecRK-V.5 function increased resistance to surface inoculation with virulent bacteria Pseudomonas syringae pv tomato DC3000. Levels of resistance were not affected after infiltration-inoculation, suggesting that LecRK-V.5 functions at an early defense stage. By contrast, lines overexpressing LecRK-V.5 were more susceptible to Pst DC3000. Enhanced resistance in lecrk-V.5 mutants was correlated with constitutive stomatal closure, while increased susceptibility phenotypes in overexpression lines were associated with early stomatal reopening. Lines overexpressing LecRK-V.5 also demonstrated a defective stomatal closure after pathogen-associated molecular pattern (PAMP) treatments. LecRK-V.5 is rapidly expressed in stomatal guard cells after bacterial inoculation or treatment with the bacterial PAMP flagellin. In addition, lecrk-V.5 mutants guard cells exhibited constitutive accumulation of reactive oxygen species (ROS) and inhibition of ROS production opened stomata of lecrk-V.5. LecRK-V.5 is also shown to interfere with abscisic acid-mediated stomatal closure signaling upstream of ROS production. These results provide genetic evidences that LecRK-V.5 negatively regulates stomatal immunity upstream of ROS biosynthesis. Our data reveal that plants have evolved mechanisms to reverse bacteria-mediated stomatal closure to prevent long-term effect on CO2 uptake and photosynthesis.
Author Summary
During their lifetime, plants face numerous pathogenic microbes. Plants recognize microbial pathogens via plant receptors and recognition leads to the activation of a general defense response. Some foliar pathogens such as bacteria enter plant leaves through natural surface openings such as stomata. To restrict bacterial entry, plants close stomata upon contact with bacteria. A better understanding of stomatal immunity may lead to development of crops with improved disease resistance. Here, we used the model plant Arabidopsis thaliana to study activation of defense responses after infection by Pseudomonas syringae pv. tomato (Pst) DC3000 bacteria. We found that a gene not previously known to function in the defense response, LecRK-V.5, is modulating Arabidopsis resistance. By studying plants with mutations in or overexpressing this gene, we show that LecRK-V.5 negatively regulates plant stomatal immunity to Pst DC3000. In addition, LecRK-V.5 is rapidly expressed at stomata upon activation of the general defense response. Plants with mutations in LecRK-V.5 also demonstrated constitutive accumulation of reactive oxygen species in stomatal guard cells. We conclude that LecRK-V.5 is a protein that negatively regulates closure of stomata upon bacterial infection.
PMCID: PMC3276567  PMID: 22346749
10.  Modulation of ROS production and hormone levels by AHK5 during abiotic and biotic stress signaling 
Plant Signaling & Behavior  2012;7(8):893-897.
Histidine kinases have been shown to mediate responses to endogenous and exogenous stimuli in organisms such as yeast, bacteria and plants. In the model plant Arabidopsis, histidine kinases have been shown to function in hormone signaling, and abiotic and biotic stress responses. More recently, the least characterized of the Arabidopsis histidine kinases, AHK5, was demonstrated to function in resistance toward the virulent bacterium Pseudomonas syringae pv tomato DC3000 (PstDC3000) and the necrotrophic fungus Botrytis cinerea, and as a negative regulator of tolerance toward salinity. Here, we present data which indicate that AHK5 also impacts on drought stress resistance and on the outcome of an incompatible interaction with avrRpm1-expressing PstDC3000 (PstDC3000 (avrRpm1)). We present a model which proposes a role for reactive oxygen species (ROS) and hormones in integrating abiotic and biotic stress responses via AHK5.
PMCID: PMC3474678  PMID: 22827948
histidine kinase; drought stress; hormone signalling; reactive oxygen species
11.  AvrBsT Acetylates Arabidopsis ACIP1, a Protein that Associates with Microtubules and Is Required for Immunity 
PLoS Pathogens  2014;10(2):e1003952.
Bacterial pathogens of plant and animals share a homologous group of virulence factors, referred to as the YopJ effector family, which are translocated by the type III secretion (T3S) system into host cells during infection. Recent work indicates that some of these effectors encode acetyltransferases that suppress host immunity. The YopJ-like protein AvrBsT is known to activate effector-triggered immunity (ETI) in Arabidopsis thaliana Pi-0 plants; however, the nature of its enzymatic activity and host target(s) has remained elusive. Here we report that AvrBsT possesses acetyltransferase activity and acetylates ACIP1 (for ACETYLATED INTERACTING PROTEIN1), an unknown protein from Arabidopsis. Genetic studies revealed that Arabidopsis ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) infection. Microscopy studies revealed that ACIP1 is associated with punctae on the cell cortex and some of these punctae co-localize with microtubules. These structures were dramatically altered during infection. Pst DC3000 or Pst DC3000 AvrRpt2 infection triggered the formation of numerous, small ACIP1 punctae and rods. By contrast, Pst DC3000 AvrBsT infection primarily triggered the formation of large GFP-ACIP1 aggregates, in an acetyltransferase-dependent manner. Our data reveal that members of the ACIP family are new components of the defense machinery required for anti-bacterial immunity. They also suggest that AvrBsT-dependent acetylation in planta alters ACIP1's defense function, which is linked to the activation of ETI.
Author Summary
How host disease resistance pathways are activated in response to pathogens remains a fundamental question in host-pathogen interactions. In this work, we used the Pseudomonas-Arabidopsis pathosystem to study how the AvrBsT effector activates plant immune signaling. AvrBsT belongs to the YopJ effector family, a group of virulence proteins shared by bacterial pathogens of plants and animals. Bacteria inject these effectors into plant or animal host cells to promote pathogenesis. Recent biochemical studies show that several members of the YopJ family encode acetyltransferases that acetylate host proteins to suppress immune signaling. How the immune system specifically recognizes this family of effectors and/or monitors host acetylation is poorly understood. In this work, we provide biochemical evidence that AvrBsT is an acetyltransferase. We also report the identification and characterization of ACIP1, an Arabidopsis protein of unknown function that is an AvrBsT substrate. We provide evidence that ACIP1 is required for plant immunity and its association with microtubules changes during infection. Moreover, our work suggests that AvrBsT acetyltransferase in planta leads to dramatic changes in ACIP1 localization, which coincides with the activation of strong defense responses. This study highlights an important link between ACIP1 and the microtubule network during anti-bacterial immunity.
PMCID: PMC3930583  PMID: 24586161
12.  Predicting genome-scale Arabidopsis-Pseudomonas syringae interactome using domain and interolog-based approaches 
BMC Bioinformatics  2014;15(Suppl 11):S13.
Every year pathogenic organisms cause billions of dollars' worth damage to crops and livestock. In agriculture, study of plant-microbe interactions is demanding a special attention to develop management strategies for the destructive pathogen induced diseases that cause huge crop losses every year worldwide. Pseudomonas syringae is a major bacterial leaf pathogen that causes diseases in a wide range of plant species. Among its various strains, pathovar tomato strain DC3000 (PstDC3000) is asserted to infect the plant host Arabidopsis thaliana and thus, has been accepted as a model system for experimental characterization of the molecular dynamics of plant-pathogen interactions. Protein-protein interactions (PPIs) play a critical role in initiating pathogenesis and maintaining infection. Understanding the PPI network between a host and pathogen is a critical step for studying the molecular basis of pathogenesis. The experimental study of PPIs at a large scale is very scarce and also the high throughput experimental results show high false positive rate. Hence, there is a need for developing efficient computational models to predict the interaction between host and pathogen in a genome scale, and find novel candidate effectors and/or their targets.
In this study, we used two computational approaches, the interolog and the domain-based to predict the interactions between Arabidopsis and PstDC3000 in genome scale. The interolog method relies on protein sequence similarity to conduct the PPI prediction. A Pseudomonas protein and an Arabidopsis protein are predicted to interact with each other if an experimentally verified interaction exists between their respective homologous proteins in another organism. The domain-based method uses domain interaction information, which is derived from known protein 3D structures, to infer the potential PPIs. If a Pseudomonas and an Arabidopsis protein contain an interacting domain pair, one can expect the two proteins to interact with each other. The interolog-based method predicts ~0.79M PPIs involving around 7700 Arabidopsis and 1068 Pseudomonas proteins in the full genome. The domain-based method predicts 85650 PPIs comprising 11432 Arabidopsis and 887 Pseudomonas proteins. Further, around 11000 PPIs have been identified as interacting from both the methods as a consensus.
The present work predicts the protein-protein interaction network between Arabidopsis thaliana and Pseudomonas syringae pv. tomato DC3000 in a genome wide scale with a high confidence. Although the predicted PPIs may contain some false positives, the computational methods provide reasonable amount of interactions which can be further validated by high throughput experiments. This can be a useful resource to the plant community to characterize the host-pathogen interaction in Arabidopsis and Pseudomonas system. Further, these prediction models can be applied to the agriculturally relevant crops.
PMCID: PMC4251041  PMID: 25350354
Plant-pathogen interactions; Bioinformatics; Unsupervised learning; Arabidopsis; Pseudomonas syringae; Interactome; Computational prediction
13.  Arabidopsis AtERF15 positively regulates immunity against Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea 
Upon pathogen infection, activation of immune response requires effective transcriptional reprogramming that regulates inducible expression of a large set of defense genes. A number of ethylene-responsive factor transcription factors have been shown to play critical roles in regulating immune responses in plants. In the present study, we explored the functions of Arabidopsis AtERF15 in immune responses against Pseudomonas syringae pv. tomato (Pst) DC3000, a (hemi)biotrophic bacterial pathogen, and Botrytis cinerea, a necrotrophic fungal pathogen. Expression of AtERF15 was induced by infection of Pst DC3000 and B. cinerea and by treatments with salicylic acid (SA) and methyl jasmonate. Biochemical assays demonstrated that AtERF15 is a nucleus-localized transcription activator. The AtERF15-overexpressing (AtERF15-OE) plants displayed enhanced resistance while the AtERF15-RNAi plants exhibited decreased resistance against Pst DC3000 and B. cinerea. Meanwhile, Pst DC3000- or B. cinerea-induced expression of defense genes was upregulated in AtERF15-OE plants but downregulated in AtERF15-RNAi plants, as compared to the expression in wild type plants. In response to infection with B. cinerea, the AtERF15-OE plants accumulated less reactive oxygen species (ROS) while the AtERF15-RNAi plants accumulated more ROS. The flg22- and chitin-induced oxidative burst was abolished and expression levels of the pattern-triggered immunity-responsive genes AtFRK1 and AtWRKY53 were suppressed in AtER15-RNAi plants upon treatment with flg22 or chitin. Furthermore, SA-induced defense response was also partially impaired in the AtERF15-RNAi plants. These data demonstrate that AtERF15 is a positive regulator of multiple layers of the immune responses in Arabidopsis.
PMCID: PMC4559647  PMID: 26388886
Arabidopsis thaliana; ethylene-responsive factor (ERF) transcription factors; immune response; pattern-triggered immunity; Pseudomonas syringae pv. tomato DC3000; Botrytis cinerea
14.  The Bacterial Effector HopX1 Targets JAZ Transcriptional Repressors to Activate Jasmonate Signaling and Promote Infection in Arabidopsis 
PLoS Biology  2014;12(2):e1001792.
A bacterial effector protein, HopX1, targets host plant JAZ transcriptional repressors for degradation to activate the jasmonate pathway, thereby promoting bacterial pathogenesis by suppressing host defense responses.
Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome.
Author Summary
Bacterial plant pathogens secrete toxins and inject effector proteins into the host cells to promote infection, and the identification of the individual functions of these molecules is essential to understand the infective process. Remarkably, some Pseudomonas strains have evolved a sophisticated strategy for manipulating hormonal balance by producing the toxin coronatine (COR), which mimics the plant hormone jasmonate-isoleucine (JA-Ile). The JA-Ile pathway plays a key role in plant immunity by activating defenses against fungal pathogens, while promoting bacterial growth by inhibiting the salicylic acid (SA)-dependent defenses required for Pseudomonas resistance. Here, we report that the effector HopX1 from a Pseudomonas syringae strain that does not produce COR exploits an alternative evolutionary strategy to activate the JA-Ile pathway. We show that HopX1 encodes a cysteine protease that interacts with and promotes the degradation of key JA pathway repressors, the JAZ proteins. Correspondingly, ectopically expressing HopX1 in the model plant Arabidopsis induces the expression of JA-dependent genes, and natural infection with Pseudomonas producing HopX1 promotes bacterial growth in a similar fashion to COR. Our results highlight a novel example by which a bacterial effector directly manipulates core regulators of hormone signaling to facilitate infection.
PMCID: PMC3928049  PMID: 24558350
15.  Host-plant-mediated effects of Nadefensin on herbivore and pathogen resistance in Nicotiana attenuata 
BMC Plant Biology  2008;8:109.
The adage from Shakespeare, "troubles, not as single spies, but in battalions come," holds true for Nicotiana attenuata, which is commonly attacked by both pathogens (Pseudomonas spp.) and herbivores (Manduca sexta) in its native habitats. Defense responses targeted against the pathogens can directly or indirectly influence the responses against the herbivores. Nadefensin is an effective induced defense gene against the bacterial pathogen Pseudomonas syringae pv tomato (PST DC3000), which is also elicited by attack from M. sexta larvae, but whether this defense protein influences M. sexta's growth and whether M. sexta-induced Nadefensin directly or indirectly influences PST DC3000 resistance are unknown.
M. sexta larvae consumed less on WT and on Nadefensin-silenced N. attenuata plants that had previously been infected with PST DC3000 than on uninfected plants. WT plants infected with PST DC3000 showed enhanced resistance to PST DC3000 and decreased leaf consumption by M. sexta larvae, but larval mass gain was unaffected. PST DC3000-infected Nadefensin-silenced plants were less resistant to subsequent PST DC3000 challenge, and on these plants, M. sexta larvae consumed less and gained less mass. WT and Nadefensin-silenced plants previously damaged by M. sexta larvae were better able to resist subsequent PST DC3000 challenges than were undamaged plants.
These results demonstrate that Na-defensin directly mediates defense against PST DC3000 and indirectly against M. sexta in N. attenuata. In plants that were previously infected with PST DC3000, the altered leaf chemistry in PST DC3000-resistant WT plants and PST DC3000-susceptible Nadefensin-silenced plants differentially reduced M. sexta's leaf consumption and mass gain. In plants that were previously damaged by M. sexta, the combined effect of the altered host plant chemistry and a broad spectrum of anti-herbivore induced metabolomic responses was more effective than Nadefensin alone in resisting PST DC3000.
PMCID: PMC2613890  PMID: 18950524
16.  The Arabidopsis miR472-RDR6 Silencing Pathway Modulates PAMP- and Effector-Triggered Immunity through the Post-transcriptional Control of Disease Resistance Genes 
PLoS Pathogens  2014;10(1):e1003883.
RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) is a key RNA silencing factor initially characterized in transgene silencing and virus resistance. This enzyme also contributes to the biosynthesis of endogenous short interfering RNAs (siRNAs) from non-coding RNAs, transposable elements and protein-coding transcripts. One class of protein-coding transcripts that have recently emerged as major sources of RDR6-dependent siRNAs are nucleotide-binding leucine-rich repeat (NB-LRR) proteins, a family of immune-receptors that perceive specific pathogen effector proteins and mount Effector-Triggered Immunity (ETI). Nevertheless, the dynamic post-transcriptional control of NB-LRR transcripts during the plant immune response and the functional relevance of NB-LRRs in signaling events triggered by Pathogen-Associated Molecular Patterns (PAMPs) remain elusive. Here, we show that PTI is constitutive and sensitized in the Arabidopsis rdr6 loss-of-function mutant, implicating RDR6 as a novel negative regulator of PTI. Accordingly, rdr6 mutant exhibits enhanced basal resistance towards a virulent Pseudomonas syringae strain. We further provide evidence that dozens of CC-NB-LRRs (CNLs), including the functionally characterized RPS5 gene, are post-transcriptionally controlled by RDR6 both constitutively and during PTI. These CNL transcripts are also regulated by the Arabidopsis microRNA miR472 and knock-down of this miRNA recapitulates the PTI and basal resistance phenotypes observed in the rdr6 mutant background. Furthermore, both miR472 and rdr6 mutants were more resistant to Pto DC3000 expressing AvrPphB, a bacterial effector recognized by the disease resistance protein RPS5, whereas transgenic plants overexpressing miR472 were more susceptible to this bacterial strain. Finally, we show that the enhanced basal and RPS5-mediated resistance phenotypes observed in the rdr6 mutant are dependent on the proper chaperoning of NB-LRR proteins, and might therefore be due to the enhanced accumulation of CNL proteins whose cognate mRNAs are no longer controlled by RDR6-dependent siRNAs. Altogether, this study supports a model whereby the miR472- and RDR6-mediated silencing pathway represents a key regulatory checkpoint modulating both PTI and ETI responses through the post-transcriptional control of disease resistance genes.
Author Summary
Virus resistance relies in some plant-viral interactions on the RNA-DEPENDANT RNA POLYMERASE 6 (RDR6), a major actor of RNA silencing that acts at the post-transcriptional level. Here, we demonstrate that RDR6 also plays a role in basal defense and race-specific resistance. RDR6 and the microRNA miR472, which targets the mRNAs of disease resistance genes of coiled-coil nucleotide-binding leucine-rich-repeats family (e.g. RPS5), act in cooperation to control post-transcriptionally these immune receptors. Induction of these resistance genes is primed in rdr6- and miR472-elicited mutants and this effect is associated with an enhanced basal and race-specific immunity in these backgrounds.
PMCID: PMC3894208  PMID: 24453975
17.  Characterization of a JAZ7 activation-tagged Arabidopsis mutant with increased susceptibility to the fungal pathogen Fusarium oxysporum  
Journal of Experimental Botany  2016;67(8):2367-2386.
A JAZ7 T-DNA activation mutant confers increased JA-sensitivity, up-regulated defense and JA-mediated gene expression, and increased susceptibility to two pathogens that disrupt host JA-responses, Fusarium oxysporum and Pst DC3000.
In Arabidopsis, jasmonate (JA)-signaling plays a key role in mediating Fusarium oxysporum disease outcome. However, the roles of JASMONATE ZIM-domain (JAZ) proteins that repress JA-signaling have not been characterized in host resistance or susceptibility to this pathogen. Here, we found most JAZ genes are induced following F. oxysporum challenge, and screening T-DNA insertion lines in Arabidopsis JAZ family members identified a highly disease-susceptible JAZ7 mutant (jaz7-1D). This mutant exhibited constitutive JAZ7 expression and conferred increased JA-sensitivity, suggesting activation of JA-signaling. Unlike jaz7 loss-of-function alleles, jaz7-1D also had enhanced JA-responsive gene expression, altered development and increased susceptibility to the bacterial pathogen Pst DC3000 that also disrupts host JA-responses. We also demonstrate that JAZ7 interacts with transcription factors functioning as activators (MYC3, MYC4) or repressors (JAM1) of JA-signaling and contains a functional EAR repressor motif mediating transcriptional repression via the co-repressor TOPLESS (TPL). We propose through direct TPL recruitment, in wild-type plants JAZ7 functions as a repressor within the JA-response network and that in jaz7-1D plants, misregulated ectopic JAZ7 expression hyper-activates JA-signaling in part by disturbing finely-tuned COI1-JAZ-TPL-TF complexes.
PMCID: PMC4809290  PMID: 26896849
Defense; ERF-associated amphiphilic repressor; fungal pathogen; methyl jasmonate; Pseudomonas syringae; TOPLESS; transcriptional repression.
18.  A Genetic Screen Reveals Arabidopsis Stomatal and/or Apoplastic Defenses against Pseudomonas syringae pv. tomato DC3000 
PLoS Pathogens  2011;7(10):e1002291.
Bacterial infection of plants often begins with colonization of the plant surface, followed by entry into the plant through wounds and natural openings (such as stomata), multiplication in the intercellular space (apoplast) of the infected tissues, and dissemination of bacteria to other plants. Historically, most studies assess bacterial infection based on final outcomes of disease and/or pathogen growth using whole infected tissues; few studies have genetically distinguished the contribution of different host cell types in response to an infection. The phytotoxin coronatine (COR) is produced by several pathovars of Pseudomonas syringae. COR-deficient mutants of P. s. tomato (Pst) DC3000 are severely compromised in virulence, especially when inoculated onto the plant surface. We report here a genetic screen to identify Arabidopsis mutants that could rescue the virulence of COR-deficient mutant bacteria. Among the susceptible to coronatine-deficient Pst DC3000 (scord) mutants were two that were defective in stomatal closure response, two that were defective in apoplast defense, and four that were defective in both stomatal and apoplast defense. Isolation of these three classes of mutants suggests that stomatal and apoplastic defenses are integrated in plants, but are genetically separable, and that COR is important for Pst DC3000 to overcome both stomatal guard cell- and apoplastic mesophyll cell-based defenses. Of the six mutants defective in bacterium-triggered stomatal closure, three are defective in salicylic acid (SA)-induced stomatal closure, but exhibit normal stomatal closure in response to abscisic acid (ABA), and scord7 is compromised in both SA- and ABA-induced stomatal closure. We have cloned SCORD3, which is required for salicylic acid (SA) biosynthesis, and SCORD5, which encodes an ATP-binding cassette (ABC) protein, AtGCN20/AtABCF3, predicted to be involved in stress-associated protein translation control. Identification of SCORD5 begins to implicate an important role of stress-associated protein translation in stomatal guard cell signaling in response to microbe-associated molecular patterns and bacterial infection.
Author Summary
Pathogen entry into host tissue is a critical first step in causing infection. For foliar bacterial plant pathogens, natural surface openings, such as stomata, are important entry sites into the leaf apoplast (internal intercellular spaces). Recent studies have shown that plants respond to surface-inoculated bacterial pathogens by reducing stomatal aperture as part of the innate immune response to restrict bacterial invasion. Once inside plant tissue, bacteria encounter defenses in the apoplast. To counter host defenses during invasion and in the apoplast, bacterial pathogens produce a variety of virulence factors, such as the polyketide toxin coronatine produced by Pseudomonas syringae pv. tomato (Pst) DC3000. Coronatine-deficient Pst DC3000 mutants are compromised in virulence, especially when inoculated onto the plant surface. In this study, we conducted a random genetic screen to identify Arabidopsis mutants that could rescue the virulence of coronatine-deficient mutant bacteria and obtained three classes of Arabidopsis mutants: those that are defective in stomatal closure only, those defective in apoplastic defense only, and those compromised in both stomatal closure and apoplastic defenses. The isolation of these host mutants highlight the important role of COR, a molecular mimic of the plant hormone jasmonate, in overcoming both stomatal and apoplastic defenses during Pst DC3000 infection.
PMCID: PMC3188540  PMID: 21998587
19.  Jasmonate ZIM-Domain (JAZ) Protein Regulates Host and Nonhost Pathogen-Induced Cell Death in Tomato and Nicotiana benthamiana 
PLoS ONE  2013;8(9):e75728.
The nonhost-specific phytotoxin coronatine (COR) produced by several pathovars of Pseudomonas syringae functions as a jasmonic acid-isoleucine (JA-Ile) mimic and contributes to disease development by suppressing plant defense responses and inducing reactive oxygen species in chloroplast. It has been shown that the F-box protein CORONATINE INSENSITIVE 1 (COI1) is the receptor for COR and JA-Ile. JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators for JA signaling in Arabidopsis. However, the physiological significance of JAZ proteins in P. syringae disease development and nonhost pathogen-induced hypersensitive response (HR) cell death is not completely understood. In this study, we identified JAZ genes from tomato, a host plant for P. syringae pv. tomato DC3000 (Pst DC3000), and examined their expression profiles in response to COR and pathogens. Most JAZ genes were induced by COR treatment or inoculation with COR-producing Pst DC3000, but not by the COR-defective mutant DB29. Tomato SlJAZ2, SlJAZ6 and SlJAZ7 interacted with SlCOI1 in a COR-dependent manner. Using virus-induced gene silencing (VIGS), we demonstrated that SlJAZ2, SlJAZ6 and SlJAZ7 have no effect on COR-induced chlorosis in tomato and Nicotiana benthamiana. However, SlJAZ2-, SlJAZ6- and SlJAZ7-silenced tomato plants showed enhanced disease-associated cell death to Pst DC3000. Furthermore, we found delayed HR cell death in response to the nonhost pathogen Pst T1 or a pathogen-associated molecular pattern (PAMP), INF1, in SlJAZ2- and SlJAZ6-silenced N. benthamiana. These results suggest that tomato JAZ proteins regulate the progression of cell death during host and nonhost interactions.
PMCID: PMC3785428  PMID: 24086622
20.  TaNAC1 acts as a negative regulator of stripe rust resistance in wheat, enhances susceptibility to Pseudomonas syringae, and promotes lateral root development in transgenic Arabidopsis thaliana 
Plant-specific NAC transcription factors (TFs) constitute a large family and play important roles in regulating plant developmental processes and responses to environmental stresses, but only some of them have been investigated for effects on disease reaction in cereal crops. Virus-induced gene silencing (VIGS) is an effective strategy for rapid functional analysis of genes in plant tissues. In this study, TaNAC1, encoding a new member of the NAC1 subgroup, was cloned from bread wheat and characterized. It is a TF localized in the cell nucleus, and contains an activation domain in its C-terminal. TaNAC1 was strongly expressed in wheat roots and was involved in responses to infection by the obligate pathogen Puccinia striiformis f. sp. tritici and defense-related hormone treatments such as salicylic acid (SA), methyl jasmonate, and ethylene. Knockdown of TaNAC1 with barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) enhanced stripe rust resistance. TaNAC1-overexpression in Arabidopsis thaliana plants gave enhanced susceptibility, attenuated systemic-acquired resistance to Pseudomonas syringae DC3000, and promoted lateral root development. Jasmonic acid-signaling pathway genes PDF1.2 and ORA59 were constitutively expressed in transgenic plants. TaNAC1 overexpression suppressed the expression levels of resistance-related genes PR1 and PR2 involved in SA signaling and AtWRKY70, which functions as a connection node between the JA- and SA-signaling pathways. Collectively, TaNAC1 is a novel NAC member of the NAC1 subgroup, negatively regulates plant disease resistance, and may modulate plant JA- and SA-signaling defense cascades.
PMCID: PMC4342887  PMID: 25774162
wheat; stripe rust; disease resistance; BSMV-VIGS; jasmonic acid
21.  Bacillus cereus AR156 primes induced systemic resistance by suppressing miR825/825* and activating defense-related genes in Arabidopsis 
Small RNAs play an important role in plant immune responses. However, their regulatory function in induced systemic resistance (ISR) is nascent. Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces ISR in Arabidopsis against bacterial infection. Here, by comparing small RNA profiles of Pseudomonas syringae pv. tomato (Pst) DC3000-infected Arabidopsis with and without AR156 pretreatment, we identified a group of Arabidopsis microRNAs (miRNAs) that are differentially regulated by AR156 pretreatment. miR825 and miR825* are two miRNA generated from a single miRNA gene. Northern blot analysis indicated that they were significantly downregulated in Pst DC3000-infected plants pretreated with AR156, in contrast to the plants without AR156 pretreatment.miR825 targets two ubiquitin-protein ligases, while miR825* targets toll-interleukin-like receptor (TIR)-nucleotide binding site (NBS) and leucine-rich repeat (LRR) type resistance (R) genes. The expression of these target genes negatively correlated with the expression of miR825 and miR825*. Moreover, transgenic plants showing reduced expression of miR825 and miR825* displayed enhanced resistance to Pst DC3000 infection, whereas transgenic plants overexpressing miR825 and miR825* were more susceptible. Taken together, our data indicates that Bacillus cereus AR156 pretreatment primes ISR to Pst infection by suppressing miR825 and miR825* and activating the defense related genes they targeted.
PMCID: PMC5028193  PMID: 26526683
Induced systemic resistance; ISR; microRNA; plant innate immunity; small RNA
22.  Arabidopsis TNL-WRKY domain receptor RRS1 contributes to temperature-conditioned RPS4 auto-immunity 
In plant effector-triggered immunity (ETI), intracellular nucleotide binding-leucine rich repeat (NLR) receptors are activated by specific pathogen effectors. The Arabidopsis TIR (Toll-Interleukin-1 receptor domain)-NLR (denoted TNL) gene pair, RPS4 and RRS1, confers resistance to Pseudomonas syringae pv tomato (Pst) strain DC3000 expressing the Type III-secreted effector, AvrRps4. Nuclear accumulation of AvrRps4, RPS4, and the TNL resistance regulator EDS1 is necessary for ETI. RRS1 possesses a C-terminal “WRKY” transcription factor DNA binding domain suggesting that important RPS4/RRS1 recognition and/or resistance signaling events occur at the nuclear chromatin. In Arabidopsis accession Ws-0, the RPS4Ws/RRS1Ws allelic pair governs resistance to Pst/AvrRps4 accompanied by host programed cell death (pcd). In accession Col-0, RPS4Col/RRS1Col effectively limits Pst/AvrRps4 growth without pcd. Constitutive expression of HA-StrepII tagged RPS4Col (in a 35S:RPS4-HS line) confers temperature-conditioned EDS1-dependent auto-immunity. Here we show that a high (28°C, non-permissive) to moderate (19°C, permissive) temperature shift of 35S:RPS4-HS plants can be used to follow defense-related transcriptional dynamics without a pathogen effector trigger. By comparing responses of 35S:RPS4-HS with 35S:RPS4-HS rrs1-11 and 35S:RPS4-HS eds1-2 mutants, we establish that RPS4Col auto-immunity depends entirely on EDS1 and partially on RRS1Col. Examination of gene expression microarray data over 24 h after temperature shift reveals a mainly quantitative RRS1Col contribution to up- or down-regulation of a small subset of RPS4Col-reprogramed, EDS1-dependent genes. We find significant over-representation of WRKY transcription factor binding W-box cis-elements within the promoters of these genes. Our data show that RRS1Col contributes to temperature-conditioned RPS4Col auto-immunity and are consistent with activated RPS4Col engaging RRS1Col for resistance signaling.
PMCID: PMC3797954  PMID: 24146667
resistance gene pair; temperature shift; EDS1 signaling; biotic stress; programed cell death; transcriptional reprograming
23.  Voltage-gated sodium channel expression in mouse DRG after SNI leads to re-evaluation of projections of injured fibers 
Molecular Pain  2014;10:19.
Dysregulation of voltage-gated sodium channels (Navs) is believed to play a major role in nerve fiber hyperexcitability associated with neuropathic pain. A complete transcriptional characterization of the different isoforms of Navs under normal and pathological conditions had never been performed on mice, despite their widespread use in pain research. Navs mRNA levels in mouse dorsal root ganglia (DRG) were studied in the spared nerve injury (SNI) and spinal nerve ligation (SNL) models of neuropathic pain. In the SNI model, injured and non-injured neurons were intermingled in lumbar DRG, which were pooled to increase the tissue available for experiments.
A strong downregulation was observed for every Navs isoform expressed except for Nav1.2; even Nav1.3, known to be upregulated in rat neuropathic pain models, was lower in the SNI mouse model. This suggests differences between these two species. In the SNL model, where the cell bodies of injured and non-injured fibers are anatomically separated between different DRG, most Navs were observed to be downregulated in the L5 DRG receiving axotomized fibers. Transcription was then investigated independently in the L3, L4 and L5 DRG in the SNI model, and an important downregulation of many Navs isoforms was observed in the L3 DRG, suggesting the presence of numerous injured neurons there after SNI. Consequently, the proportion of axotomized neurons in the L3, L4 and L5 DRG after SNI was characterized by studying the expression of activating transcription factor 3 (ATF3). Using this marker of nerve injury confirmed that most injured fibers find their cell bodies in the L3 and L4 DRG after SNI in C57BL/6 J mice; this contrasts with their L4 and L5 DRG localization in rats. The spared sural nerve, through which pain hypersensitivity is measured in behavioral studies, mostly projects into the L4 and L5 DRG.
The complex regulation of Navs, together with the anatomical rostral shift of the DRG harboring injured fibers in C57BL/6 J mice, emphasize that caution is necessary and preliminary anatomical experiments should be carried out for gene and protein expression studies after SNI in mouse strains.
PMCID: PMC4007621  PMID: 24618114
Activating transcription factor 3 (ATF3); Dorsal root ganglia (DRG); Nerve injury; Neuropathic pain; Quantitative real time polymerase chain reaction (qRT-PCR); Sciatic nerve; Spared nerve injury (SNI); Spinal nerve ligation (SNL); Voltage-gated sodium channels (Navs)
24.  The Nuclear Immune Receptor RPS4 Is Required for RRS1SLH1-Dependent Constitutive Defense Activation in Arabidopsis thaliana  
PLoS Genetics  2014;10(10):e1004655.
Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific “avirulent” pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector.
Author Summary
How plant NB-LRR resistance proteins and the related mammalian Nod-like receptors (NLRs) activate defense is poorly understood. Plant and animal immune receptors can function in pairs. Two Arabidopsis nuclear immune receptors, RPS4 and RRS1, confer recognition of the unrelated bacterial effectors, AvrRps4 and PopP2, and activate defense. Using delivery of PopP2 into Arabidopsis leaf cells via Pseudomonas type III secretion, we define early transcriptional changes upon RPS4/RRS1-dependent PopP2 recognition. We show an auto-active allele of RRS1, RRS1SLH1, triggers transcriptional reprogramming of defense genes that are also reprogrammed by AvrRps4 or PopP2 in an RPS4/RRS1-dependent manner. To discover genetic requirements for RRS1SLH1 auto-activation, we conducted a suppressor screen. Many suppressor of slh1 immunity (sushi) mutants that are impaired in RRS1SLH1-mediated auto-activation carry loss-of-function mutations in RPS4. This suggests that RPS4 functions as a signaling component together with or downstream of RRS1-activated immunity, in contrast to earlier hypotheses, significantly advancing our understanding of how immune receptors activate defense in plants.
PMCID: PMC4207616  PMID: 25340333
25.  Functional analysis of the theobroma cacao NPR1 gene in arabidopsis 
BMC Plant Biology  2010;10:248.
The Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1) that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA) accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response.
A putative Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica, Arabidopsis and Carica papaya. The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors (BTB/POZ and ankyrin repeat protein-protein interaction domains and a nuclear localization sequence (NLS)). To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants.
Our data indicate that the TcNPR1 is a functional ortholog of Arabidopsis NPR1, and is likely to play a major role in defense response in cacao. This fundamental knowledge can contribute to breeding of disease resistant cacao varieties through the application of molecular markers or the use of transgenic strategies.
PMCID: PMC3095330  PMID: 21078185

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