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.
AtSRT2; Basal defense; EDS5; PAD4; PstDC3000; SID2
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.
NPR1; PR-1; salicylic acid; SNI1; TGA factors
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.
Arabidopsis, Bacillus subtilis FB17, Biofilm; Malic acid; Rhizobacteria; Root transcriptome
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.
Arabidopsis; Plant defense responses; PAP5; Pseudomonas syringae; Phosphate starvation
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.
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.
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.
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.
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.
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.
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.
histidine kinase; drought stress; hormone signalling; reactive oxygen species
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.
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.
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.
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.
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.
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)
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.
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.
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.
Plant-pathogen interactions; Bioinformatics; Unsupervised learning; Arabidopsis; Pseudomonas syringae; Interactome; Computational prediction
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.
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.
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.
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.
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.
resistance gene pair; temperature shift; EDS1 signaling; biotic stress; programed cell death; transcriptional reprograming
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.
Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B), which has been previously implicated in constitutive clathrin-mediated endocytosis (CME), functions in responses to flg22 (the active peptide derivative of bacterial flagellin) and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto) DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI), drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC−, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD), the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca2+-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC−. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2), was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects observed in drp2b. In conclusion, this study adds DRP2B to the relatively short list of known vesicular trafficking proteins with roles in flg22-signaling and PTI in plants.
Plants have developed effective mechanisms for protection against pathogens including bacteria, but if a plant is unable to induce defenses, pathogenic bacteria invade and colonize the host, which can lead to reduced yield and nutritional quality of crops. An important aspect of engineering durable crop resistance against bacteria is elucidating and manipulating resistance pathways in the model plant Arabidopsis thaliana. The plant receptor FLAGELLIN SENSING 2 (FLS2) recognizes the bacterial protein flagellin to initiate host defense responses contributing to immunity. Here, we identify Dynamin-Related Protein 2B (DRP2B), previously implicated in membrane trafficking in plants, as a novel component of defense responses against flagellin and bacterial Pseudomonas syringae strains in Arabidopsis thaliana. More specifically, DRP2B functioned in the first line of defense against bacteria, namely in pattern-triggered immunity. We also demonstrated that DRP2B has different roles in three distinct branches of the flg22-signaling network that could be separated by their genetic requirement for the NADPH oxidase RbohD. In drp2b mutant plants, impaired ligand-induced endocytosis of FLS2 may contribute in part to the non-canonical combination of immune defects. Our findings highlight the importance of a functional vesicular trafficking network for plant immune responses and effective immunity against bacteria.
Biotic and abiotic stresses are major unfavorable factors that affect crop productivity worldwide. NAC proteins comprise a large family of transcription factors that play important roles in plant growth and development as well as in responses to biotic and abiotic stresses. In a virus-induced gene silencing-based screening to identify genes that are involved in defense response against Botrytis cinerea, we identified a tomato NAC gene SlSRN1 (Solanum lycopersicum
Stress-related NAC1). SlSRN1 is a plasma membrane-localized protein with transactivation activity in yeast. Expression of SlSRN1 was significantly induced by infection with B. cinerea or Pseudomonas syringae pv. tomato (Pst) DC3000, leading to 6–8 folds higher than that in the mock-inoculated plants. Expression of SlSRN1 was also induced by salicylic acid, jasmonic acid and 1-amino cyclopropane-1-carboxylic acid and by drought stress. Silencing of SlSRN1 resulted in increased severity of diseases caused by B. cinerea and Pst DC3000. However, silencing of SlSRN1 resulted in increased tolerance against oxidative and drought stresses. Furthermore, silencing of SlSRN1 accelerated accumulation of reactive oxygen species but attenuated expression of defense genes after infection by B. cinerea. Our results demonstrate that SlSRN1 is a positive regulator of defense response against B. cinerea and Pst DC3000 but is a negative regulator for oxidative and drought stress response in tomato.
Pathogen infection of higher plants often induces a rapid production of phosphatic acid (PA) and changes in lipid profiles, but the enzymatic basis and the function of the lipid change in pathogen-plant interactions are not well understood.Infection of PLDβ1-deficient plants by Pseudomonas syringae pv. DC3000 resulted in less bacterial growth than in wild-type plants, and the effect was more profound in virulent Pst DC3000 than avirulent Pst DC3000 (avrRpt2) infection. The expression levels of salicylic acid (SA)-inducible genes were higher, but those inducible by jasmonic acid (JA) were lower in PLDβ1 mutants than in wild-type plants.However, PLDβ1-deficient plants were more susceptible than wild-type plants to the fungus Botrytis cinerea. The PLDβ1-deficient plants had lower levels of PA, JA and JA-related defense gene expression after B. cinerea inoculation.PLDβ1 plays a positive role in pathogen-induced JA production and plant resistance to necrotrophic fungal pathogen B. cinerea, but a negative role in the SA-dependent signaling pathway and plant tolerance to the infection of biotrophic Pst DC3000. PLDβ1 is responsible for the major part of PA increased in response to necrotrophic B. cinerea and virulent Pst DC3000 infection, but contributes less to the avirulent Pst DC3000 (avrRpt2)-induced PA production.
Botrytis cinerea; Pseudomonas syringae; Arabidopsis thaliana; phospholipase Dβ1; pathogeneses; phosphatidic acid; lysophospholipids; lipid signaling
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.
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.
A common feature of plant defense responses is the transcriptional regulation of a large number of genes upon pathogen infection or treatment with pathogen elicitors. A large body of evidence suggests that plant WRKY transcription factors are involved in plant defense including transcriptional regulation of plant host genes in response to pathogen infection. However, there is only limited information about the roles of specific WRKY DNA-binding transcription factors in plant defense.
We analyzed the role of the WRKY25 transcription factor from Arabidopsis in plant defense against the bacterial pathogen Pseudomonas syringae. WRKY25 protein recognizes the TTGACC W-box sequences and its translational fusion with green fluorescent protein is localized to the nucleus. WRKY25 expression is responsive to general environmental stress. Analysis of stress-induced WRKY25 in the defense signaling mutants npr1, sid2, ein2 and coi1 further indicated that this gene is positively regulated by the salicylic acid (SA) signaling pathway and negatively regulated by the jasmonic acid signaling pathway. Two independent T-DNA insertion mutants for WRKY25 supported normal growth of a virulent strain of P. syringae but developed reduced disease symptoms after infection. By contrast, Arabidopsis constitutively overexpressing WRKY25 supported enhanced growth of P. syringae and displayed increased disease symptom severity as compared to wild-type plants. These WRKY25-overexpressing plants also displayed reduced expression of the SA-regulated PR1 gene after the pathogen infection, despite normal levels of free SA.
The nuclear localization and sequence-specific DNA-binding activity support that WRKY25 functions as a transcription factor. Based on analysis of both T-DNA insertion mutants and transgenic overexpression lines, stress-induced WRKY25 functions as a negative regulator of SA-mediated defense responses to P. syringae. This proposed role is consistent with the recent finding that WRKY25 is a substrate of Arabidopsis MAP kinase 4, a repressor of SA-dependent defense responses.
The SR/CAMTA proteins represent a small family of transcription activators that play important roles in plant responses to biotic and abiotic stresses. Seven SlSR/CAMTA genes were identified in tomato as tomato counterparts of SR/CAMTA; however, the involvement of SlSRs/CAMTAs in biotic and abiotic stress responses is not clear. In this study, we performed functional analysis of the SlSR/CAMTA family for their possible functions in defense response against pathogens and tolerance to drought stress.
Expression of SlSRs was induced with distinct patterns by Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000. Virus-induced gene silencing (VIGS)-based knockdown of either SlSR1 or SlSR3L in tomato resulted in enhanced resistance to B. cinerea and Pst DC3000 and led to constitutive accumulation of H2O2, elevated expression of defense genes, marker genes for pathogen-associated molecular pattern-triggered immunity, and regulatory genes involved in the salicylic acid- and ethylene-mediated signaling pathways. Furthermore, the expression of SlSR1L and SlSR2L in detached leaves and whole plants was significantly induced by drought stress. Silencing of SlSR1L led to decreased drought stress tolerance, accelerated water loss in leaves, reduced root biomass and attenuated expression of drought stress responsive genes in tomato. The SlSR1 and SlSR3L proteins were localized in the nucleus of plant cells when transiently expressed in Nicotiana benthamiana and had transcriptional activation activity in yeast.
VIGS-based functional analyses demonstrate that both SlSR1 and SlSR3L in the tomato SlSR/CAMTA family are negative regulators of defense response against B. cinerea and Pst DC3000 while SlSR1L is a positive regulator of drought stress tolerance in tomato.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0286-3) contains supplementary material, which is available to authorized users.
Tomato (Solanum lycopersicum); SR/CAMTA; Disease resistance response; Drought stress
The SUPPRESSOR OF rps4-RLD1 (SRFR1) gene was identified based on enhanced AvrRps4-triggered resistance in the naturally susceptible Arabidopsis accession RLD. No other phenotypic effects were recorded, and the extent of SRFR1 involvement in regulating effector-triggered immunity was unknown. Here we show that mutations in SRFR1 in the accession Columbia-0 (Col-0) lead to severe stunting and constitutive expression of the defense gene PR1. These phenotypes were temperature-dependent. A cross between srfr1-1 (RLD background) and srfr1-4 (Col-0) showed that stunting was caused by a recessive locus in Col-0. Mapping and targeted crosses identified the Col-0-specific resistance gene SNC1 as the locus that causes stunting. SRFR1 was proposed to function as a transcriptional repressor, and SNC1 is indeed overexpressed in srfr1-4. Interestingly, co-regulated genes in the SNC1 cluster are also upregulated in the srfr1-4 snc1-11 double mutant, indicating that the overexpression of SNC1 is not a secondary effect of constitutive defense activation. In addition, a Col-0 RPS4 mutant showed full susceptibility to bacteria expressing avrRps4 at 24°C but not at 22°C, while RLD susceptibility was not temperature-dependent. The rps4-2 snc1-11 double mutant showed increased, but not full, susceptibility at 22°C, indicating that additional cross-talk between resistance pathways may exist. Intriguingly, when transiently expressed in Nicotiana benthamiana, SRFR1, RPS4 and SNC1 are in a common protein complex in a cytoplasmic microsomal compartment. Our results highlight SRFR1 as a convergence point in at least a subset of TIR-NBS-LRR protein-mediated immunity in Arabidopsis. Based on the cross-talk evident from our results, they also suggest that reports of constitutive resistance phenotypes in Col-0 need to consider the possible involvement of SNC1.
Plants, like humans, have an immune system to defend against disease. This immune system seeks out the presence of disease-causing microbes and other invaders by detecting non-plant molecules and proteins. Plants rely on this surveillance to activate an antimicrobial response of appropriate strength at the right time; as with humans, an overactive immune system can be harmful to plants. We study how plants achieve an appropriate balance, using genetics and the interaction between the reference plant Arabidopsis thaliana and the bacterial plant pathogen Pseudomonas syringae. So-called plant resistance proteins are important activators of immunity that directly or indirectly intercept foreign proteins deployed by pathogens. Resistance proteins are generally thought to be highly specific detectors that only respond to a single pathogen protein. However, while working with a negative regulator of plant immunity called SRFR1, we discovered a surprising level of cross-talk between different resistance proteins that becomes evident only under certain environmental conditions such as low temperature. We also show that SRFR1 and these resistance proteins bind to each other, possibly explaining the observed cross-talk. Our work thus highlights linkages between resistance pathways and provides insight into the molecular architecture of the plant innate immune response.