Pathogen-Induced Defense Signaling and Signal Crosstalk in Arabidopsis

Erwinia carotovora subsp. carotovora is a bacterial phytopathogen that causes soft rot in various agronomically important crop plants. A genetically specified resistance to E. carotovora has not been defined, and plant resistance to this pathogen is established through nonspecific activation of basal defense responses. This, together with the broad host range, makes this pathogen a good model for studying the activation of plant defenses. Production and secretion of plant cell wall-degrading enzymes (PCWDE) are central to the virulence of E. carotovora. It also possesses the type III secretion system (TTSS) utilized by many Gram-negative bacteria to secrete virulence- promoting effector proteins to plant cells. This study elucidated the role of E. carotovora HrpN (HrpNEcc), an effector protein secreted through TTSS, and the contribution of this protein in the virulence of E. carotovora. Treatment of plants with HrpNEcc was demonstrated to induce a hypersensitive response (HR) as well as resistance to E. carotovora. Resistance induced by HrpNEcc required both salicylic acid (SA)- and jasmonate/ethylene (JA/ET)-dependent defense signaling in Arabidopsis. Simultaneous treatment of Arabidopsis with HrpNEcc and PCWDE polygalacturonase PehA elicited accelerated and enhanced induction of defense genes but also increased production of superoxide and lesion formation. This demonstrates mutual amplification of defense signaling by these two virulence factors of E. carotovora. Identification of genes that are rapidly induced in response to a pathogen can provide novel information about the early events occurring in the plant defense response. CHLOROPHYLLASE 1 (AtCLH1) and EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) are both rapidly triggered by E. carotovora in Arabidopsis. Characterization of AtCLH1 encoding chlorophyll-degrading enzyme chlorophyllase indicated that it might have a role in chlorophyll degradation during plant tissue damage. Silencing of this gene resulted in increased accumulation of reactive oxygen species (ROS) in response to pathogen infection in a light-dependent manner. This led to enhanced SA-dependent defenses and resistance to E. carotovora. Moreover, crosstalk between different defense signaling pathways was observed; JA-dependent defenses and resistance to fungal pathogen Alternaria brassicicola were impaired, indicating antagonism between SA- and JA-dependent signaling. Characterization of ERD15 suggested that it is a novel, negative regulator of abscisic acid (ABA) signaling in Arabidopsis. Overexpression of ERD15 resulted in insensitivity to ABA and reduced tolerance of the plants to dehydration stress. However, simultaneously, the resistance of the plants to E. carotovora was enhanced. Silencing of ERD15 improved freezing and drought tolerance of transgenic plants. This, together with the reducing effect of ABA on seed germination, indicated hypersensitivity to this phytohormone. ERD15 was hypothesized to act as a capacitor that controls the appropriate activation of ABA responses in Arabidopsis.Tässä väitöskirjatyössä pyrittiin selvittämään taudinaiheuttajan aikaansaamaa puolustussignalointia ja signaalireittien välillä tapahtuvaa vuorovaikutusta kasveissa. Tällaista tietoa on mahdollista tulevaisuudessa hyödyntää vastustuskykyisempien viljelykasvien kehittämistyössä. Bakteeri Erwinia carotovora aiheuttaa märkämätää useissa hedelmissä, vihanneksissa ja juureksissa sekä kasvukaudella että varastoimisvaiheessa. Näistä suuri osa on taloudellisesti merkittäviä viljelykasveja, kuten peruna (Solanum tuberosum) ja porkkana (Daucus carota). E. carotovora aikaansaa taudin myös lituruohossa (Arabidopsis thaliana), jota käytettiin tutkimuksen mallikasvina. E. carotovoran tehokas taudinaiheuttamiskyky perustuu suurelta osin sen tuottamiin ja erittämiin kasvin soluseinää hajottaviin entsyymeihin. Näiden lisäksi se erittää kasvisoluun niin kutsuttuja vaikuttajaproteiineja tyyppi III eritysjärjestelmän kautta. Vastineena E. carotovora infektioon kasvissa aktivoituvat epäspesifiset puolustusjärjestelmät; geneettisesti määriteltyä, spesifistä, resistenssiä tätä taudinaiheuttajaa vastaan ei ole löydetty. Koska E. carotovoran erittämät, kasvin soluseinää hajottavat entsyymit peittävät aiheuttamansa kudoksen hajoamisen myötä alleen kaikki muut kasvissa bakteerin vaikutuksesta mahdollisesti tapahtuvat vasteet, työssä tutkittiin erikseen yhden E. carotovoran erittämän vaikuttajaproteiinin, HrpN:n, aikaansaamaa puolustusreaktiota Arabidopsiksessa. HrpN aikaansai hypersensitiivisen reaktion (HR), minkä lisäksi se aktivoi sekä salisyyli (SA)- että jasmonihappoon (JA) perustuvat kasvipuolustusreitit, tehden käsitellystä kasvista vastustuskykyisen myöhempää E. carotovora infektiota vastaan. Työ lisäsi tietoa yksittäisten vaikuttajaproteiinien roolista bakteerin taudinaiheuttamiskyvyssä sekä niiden vaikutuksesta aktivoituvista kasvin puolustusreiteistä. Kasvissa tapahtuvista aikaisen vaiheen puolustusreaktioista voidaan saada tärkeää tietoa identifioimalla geenejä, joiden toiminta aktivoituu nopeasti taudinaiheuttajan hyökkäyksen jälkeen. Chlorophyllase 1 (AtCLH1) geenin koodaaman proteiinin todettiin mahdollisesti osallistuvan kasvin kudosvaurion jälkeiseen klorofyllin hajotukseen. Tämän geenin hiljentäminen kasvissa RNAi tekniikalla johti valoriippuvaiseen oksidatiivisen stressin lisääntymiseen sekä parantuneeseen SA-riippuvaiseen puolustukseen ja vastustuskykyyn E. carotovoraa vastaan. Tämä työ osoitti myös puolustusreittien välillä tapahtuvan vuorovaikutusta: SA-puolustuksen vahvistumisen hintana AtCLH1RNAi-hiljennetyissä kasveissa oli JA riippuvaisen puolustuksen heikkeneminen ja kasvin puolustuskyvyn menetys Altenaria brassicicolalle, joka on sieniin kuuluva taudinaiheuttaja. Early responsive to dehydration (ERD15) geenin karakterisointi osoitti sen olevan kasvin abskissihappo (ABA) signaloinnin negatiivinen säätelijä. Kyseisen geenin hiljentäminen johti parantuneeseen kylmän- ja kuivankestävyyteen kun taas sen ylituotto teki kasveista vastustuskykyisempiä E. carotovoraa vastaan parantamalla SA-riippuvaista puolustussignalointia

Short oligogalacturonides induce pathogen resistance-associated gene expression in Arabidopsis thaliana

Background Oligogalacturonides (OGs) are important components of damage-associated molecular pattern (DAMP) signaling and influence growth regulation in plants. Recent studies have focused on the impact of long OGs (degree of polymerization (DP) from 10–15), demonstrating the induction of plant defense signaling resulting in enhanced defenses to necrotrophic pathogens. To clarify the role of trimers (trimeric OGs, DP3) in DAMP signaling and their impact on plant growth regulation, we performed a transcriptomic analysis through the RNA sequencing of Arabidopsis thaliana exposed to trimers. Results The transcriptomic data from trimer-treated Arabidopsis seedlings indicate a clear activation of genes involved in defense signaling, phytohormone signaling and a down-regulation of genes involved in processes related to growth regulation and development. This is further accompanied with improved defenses against necrotrophic pathogens triggered by the trimer treatment, indicating that short OGs have a clear impact on plant responses, similar to those described for long OGs. Conclusions Our results demonstrate that trimers are indeed active elicitors of plant defenses. This is clearly indicated by the up-regulation of genes associated with plant defense signaling, accompanied with improved defenses against necrotrophic pathogens. Moreover, trimers simultaneously trigger a clear down-regulation of genes and gene sets associated with growth and development, leading to stunted seedling growth in Arabidopsis. Keywords Plant signaling Arabidopsis thaliana Oligogalacturonides OG Trimers Transcriptomics Defense induction Growth inhibition Disease resistance Pectobacterium carotovorum Botrytis cinereaPeer reviewe

Short oligogalacturonides induce pathogen resistance-associated gene expression in Arabidopsis thaliana

Background Oligogalacturonides (OGs) are important components of damage-associated molecular pattern (DAMP) signaling and influence growth regulation in plants. Recent studies have focused on the impact of long OGs (degree of polymerization (DP) from 10–15), demonstrating the induction of plant defense signaling resulting in enhanced defenses to necrotrophic pathogens. To clarify the role of trimers (trimeric OGs, DP3) in DAMP signaling and their impact on plant growth regulation, we performed a transcriptomic analysis through the RNA sequencing of Arabidopsis thaliana exposed to trimers. Results The transcriptomic data from trimer-treated Arabidopsis seedlings indicate a clear activation of genes involved in defense signaling, phytohormone signaling and a down-regulation of genes involved in processes related to growth regulation and development. This is further accompanied with improved defenses against necrotrophic pathogens triggered by the trimer treatment, indicating that short OGs have a clear impact on plant responses, similar to those described for long OGs. Conclusions Our results demonstrate that trimers are indeed active elicitors of plant defenses. This is clearly indicated by the up-regulation of genes associated with plant defense signaling, accompanied with improved defenses against necrotrophic pathogens. Moreover, trimers simultaneously trigger a clear down-regulation of genes and gene sets associated with growth and development, leading to stunted seedling growth in Arabidopsis. Keywords Plant signaling Arabidopsis thaliana Oligogalacturonides OG Trimers Transcriptomics Defense induction Growth inhibition Disease resistance Pectobacterium carotovorum Botrytis cinereaPeer reviewe

Peroxidase-Generated Apoplastic ROS Impair Cuticle Integrity and Contribute to DAMP-Elicited Defenses

Cuticular defects trigger a battery of reactions including enhanced reactive oxygen species (ROS) production and resistance to necrotrophic pathogens. However, the source of ROS generated by such impaired cuticles has remained elusive. Here, we report the characterization of Arabidopsis thaliana ohyl mutant, a Peroxidase 57 (PER57) - overexpressing line that demonstrates enhanced defense responses that result both from increased accumulation of ROS and permeability of the leaf cuticle. The ohyl mutant was identified in a screen of A. thaliana seedlings for oligogalacturonides (OGs) insensitive/hypersensitive mutants that exhibit altered growth retardation in response to exogenous OGs. Mutants impaired in OG sensitivity were analyzed for disease resistance/susceptibility to the necrotrophic phytopathogens Botrytis cinerea and Pectobacterium carotovorum. In the ohyl line, the hypersensitivity to OGs was associated with resistance to the tested pathogens. This PER57 overexpressing line exhibited a significantly more permeable leaf cuticle than wild-type plants and this phenotype could be recapitulated by overexpressing other class III peroxidases. Such peroxidase overexpression was accompanied by the suppressed expression of cutin biosynthesis genes and the enhanced expression of genes associated with OG-signaling. Application of ABA completely removed ROS, restored the expression of genes associated with cuticle biosynthesis and led to decreased permeability of the leaf cuticle, and finally, abolished immunity to B. cinerea. Our work demonstrates that increased peroxidase activity increases permeability of the leaf cuticle. The loss of cuticle integrity primes plant defenses to necrotrophic pathogens via the activation of DAMP-responses.Peer reviewe

Differential role of MAX2 and strigolactones in pathogen, ozone, and stomatal responses

Strigolactones are a group of phytohormones that control developmental processes including shoot branching and various plant-environment interactions in plants. We previously showed that the strigolactone perception mutant more axillary branches 2 (max2) has increased susceptibility to plant pathogenic bacteria. Here we show that both strigolactone biosynthesis (max3 and max4) and perception mutants (max2 and dwarf14) are significantly more sensitive to Pseudomonas syringae DC3000. Moreover, in response to P. syringae infection, high levels of SA accumulated in max2 and this mutant was ozone sensitive. Further analysis of gene expression revealed no major role for strigolactone in regulation of defense gene expression. In contrast, guard cell function was clearly impaired in max2 and depending on the assay used, also in max3, max4, and d14 mutants. We analyzed stomatal responses to stimuli that cause stomatal closure. While the response to abscisic acid (ABA) was not impaired in any of the mutants, the response to darkness and high CO2 was impaired in max2 and d14-1 mutants, and to CO2 also in strigolactone synthesis (max3, max4) mutants. To position the role of MAX2 in the guard cell signaling network, max2 was crossed with mutants defective in ABA biosynthesis or signaling. This revealed that MAX2 acts in a signaling pathway that functions in parallel to the guard cell ABA signaling pathway. We propose that the impaired defense responses of max2 are related to higher stomatal conductance that allows increased entry of bacteria or air pollutants like ozone. Furthermore, as MAX2 appears to act in a specific branch of guard cell signaling (related to CO2 signaling), this protein could be one of the components that allow guard cells to distinguish between different environmental conditions.Peer reviewe

The F-box protein MAX2 contributes to resistance to bacterial phytopathogens in Arabidopsis thaliana

Peer reviewe

Pathogenicity of and plant immunity to soft rot pectobacteria

Soft rot pectobacteria are broad host range enterobacterial pathogens that cause disease on a variety of plant species including the major crop potato. Pectobacteria are aggressive necrotrophs that harbor a large arsenal of plant cell wall-degrading enzymes as their primary virulence determinants. These enzymes together with additional virulence factors are employed to macerate the host tissue and promote host cell death to provide nutrients for the pathogens. In contrast to (hemi)biotrophs such as Pseudomonas, type III secretion systems (T3SS) and T3 effectors do not appear central to pathogenesis of pectobacteria. Indeed, recent genomic analysis of several Pectobacterium species including the emerging pathogen Pectobacterium wasabiae has shown that many strains lack the entire T3SS as well as the T3 effectors. Instead, this analysis has indicated the presence of novel virulence determinants. Resistance to broad host range pectobacteria is complex and does not appear to involve single resistance genes. Instead, activation of plant innate immunity systems including both SA (salicylic acid) and JA (jasmonic acid)/ET (ethylene)-mediated defenses appears to play a central role in attenuation of Pectobacterium virulence. These defenses are triggered by detection of pathogen-associated molecular patterns (PAMPs) or recognition of modified-self such as damage-associated molecular patterns (DAMPs) and result in enhancement of basal immunity (PAMP/DAMP-triggered immunity or pattern-triggered immunity, PTI). In particular plant cell wall fragments released by the action of the degradative enzymes secreted by pectobacteria are major players in enhanced immunity toward these pathogens. Most notably bacterial pectin-degrading enzymes release oligogalacturonide (OG) fragments recognized as DAMPs activating innate immune responses. Recent progress in understanding OG recognition and signaling allows novel genetic screens for OG-insensitive mutants and will provide new insights into plant defense strategies against necrotrophs such as pectobacteria