Stéphanie Pateyron

Constitutively Active Mitogen-Activated Protein Kinase Versions Reveal Functions of Arabidopsis MPK4 in Pathogen Defense Signaling

Mutations generating constitutively active (CA) mitogen-activated protein kinases (MAPKs) were identified and used to investigate the role of Arabidopsis MPK4 in plant immunity, revealing functions of MPK4 activity in PTI and ETI, establishing that the generation of CA-MAPKs offers a powerful tool to analyze MAPK functions in plants. Plant mitogen-activated protein kinases (MAPKs) are involved in important processes, including stress signaling and development. In a functional yeast screen, we identified mutations that render Arabidopsis thaliana MAPKs constitutively active (CA). Importantly, CA-MAPKs maintain their specificity toward known activators and substrates. As a proof-of-concept, Arabidopsis MAPK4 (MPK4) function in plant immunity was investigated. In agreement with the phenotype of mpk4 mutants, CA-MPK4 plants were compromised in pathogen-induced salicylic acid accumulation and disease resistance. MPK4 activity was found to negatively regulate pathogen-associated molecular pattern-induced reactive oxygen species production but had no impact on callose deposition, indicating that CA-MPK4 allows discriminating between processes regulated by MPK4 activity from processes indirectly affected by mpk4 mutation. Finally, MPK4 activity was also found to compromise effector-triggered immunity conditioned by the Toll Interleukin-1 Receptor–nucleotide binding (NB)–Leu-rich repeat (LRR) receptors RPS4 and RPP4 but not by the coiled coil–NB-LRR receptors RPM1 and RPS2. Overall, these data reveal important insights on how MPK4 regulates plant defenses and establishes that CA-MAPKs offer a powerful tool to analyze the function of plant MAPK pathways.

Functional analysis of Arabidopsis immune-related MAPKs uncovers a role for MPK3 as negative regulator of inducible defences.

BackgroundMitogen-activated protein kinases (MAPKs) are key regulators of immune responses in animals and plants. In Arabidopsis, perception of microbe-associated molecular patterns (MAMPs) activates the MAPKs MPK3, MPK4 and MPK6. Increasing information depicts the molecular events activated by MAMPs in plants, but the specific and cooperative contributions of the MAPKs in these signalling events are largely unclear.ResultsIn this work, we analyse the behaviour of MPK3, MPK4 and MPK6 mutants in early and late immune responses triggered by the MAMP flg22 from bacterial flagellin. A genome-wide transcriptome analysis reveals that 36% of the flg22-upregulated genes and 68% of the flg22-downregulated genes are affected in at least one MAPK mutant. So far MPK4 was considered as a negative regulator of immunity, whereas MPK3 and MPK6 were believed to play partially redundant positive functions in defence. Our work reveals that MPK4 is required for the regulation of approximately 50% of flg22-induced genes and we identify a negative role for MPK3 in regulating defence gene expression, flg22-induced salicylic acid accumulation and disease resistance to Pseudomonas syringae. Among the MAPK-dependent genes, 27% of flg22-upregulated genes and 76% of flg22-downregulated genes require two or three MAPKs for their regulation. The flg22-induced MAPK activities are differentially regulated in MPK3 and MPK6 mutants, both in amplitude and duration, revealing a highly interdependent network.ConclusionsThese data reveal a new set of distinct functions for MPK3, MPK4 and MPK6 and indicate that the plant immune signalling network is choreographed through the interplay of these three interwoven MAPK pathways.

Identification and characterization of an ABA‐activated MAP kinase cascade in Arabidopsis thaliana

Abscisic acid (ABA) is a major phytohormone involved in important stress-related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA-triggered phosphoproteins as putative mitogen-activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA-activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3-1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA-dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR-SnRK2-PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA-induced MAPK pathway in plant stress signalling.

Salmonella enterica Flagellin Is Recognized via FLS2 and Activates PAMP-Triggered Immunity in Arabidopsis thaliana

Infections with Salmonella enterica belong to the most prominent causes of food poisoning and infected fruits and vegetables represent important vectors for salmonellosis. Recent evidence indicates that plants recognize S. enterica and raise defense responses. Nonetheless, the molecular mechanisms controlling the interaction of S. enterica with plants are still largely unclear. Here, we show that flagellin from S. enterica represents a prominent pathogen-associated molecular pattern (PAMP) in Arabidopsis thaliana, which induces PAMP-triggered immunity (PTI) via the recognition of the flg22 domain by the receptor kinase FLS2. The Arabidopsis fls2 mutant shows reduced though not abolished PTI activation, indicating that plants rely also on recognition of other S. enterica PAMPs. Interestingly, the S. enterica type III secretion system (T3SS) mutant prgH- induced stronger defense gene expression than wild-type bacteria in Arabidopsis, suggesting that T3SS effectors are involved in defense suppression. Furthermore, we observe that S. enterica strains show variation in the flg22 epitope, which results in proteins with reduced PTI-inducing activity. Altogether, these results show that S. enterica activates PTI in Arabidopsis and suggest that, in order to accomplish plant colonization, S. enterica evolved strategies to avoid or suppress PTI.

The Pseudomonas fluorescens Siderophore Pyoverdine Weakens Arabidopsis thaliana Defense in Favor of Growth in Iron-Deficient Conditions

Pyoverdine, a high-affinity ferric iron chelator synthesized by Pseudomonas fluorescens, impacts the growth/defense trade-off of Arabidopsis thaliana under iron deficient conditions. Pyoverdines are siderophores synthesized by fluorescent Pseudomonas spp. Under iron-limiting conditions, these high-affinity ferric iron chelators are excreted by bacteria in the soil to acquire iron. Pyoverdines produced by beneficial Pseudomonas spp. ameliorate plant growth. Here, we investigate the physiological incidence and mode of action of pyoverdine from Pseudomonas fluorescens C7R12 on Arabidopsis (Arabidopsis thaliana) plants grown under iron-sufficient or iron-deficient conditions. Pyoverdine was provided to the medium in its iron-free structure (apo-pyoverdine), thus mimicking a situation in which it is produced by bacteria. Remarkably, apo-pyoverdine abolished the iron-deficiency phenotype and restored the growth of plants maintained in the iron-deprived medium. In contrast to a P. fluorescens C7R12 strain impaired in apo-pyoverdine production, the wild-type C7R12 reduced the accumulation of anthocyanins in plants grown in iron-deficient conditions. Under this condition, apo-pyoverdine modulated the expression of around 2,000 genes. Notably, apo-pyoverdine positively regulated the expression of genes related to development and iron acquisition/redistribution while it repressed the expression of defense-related genes. Accordingly, the growth-promoting effect of apo-pyoverdine in plants grown under iron-deficient conditions was impaired in iron-regulated transporter1 and ferric chelate reductase2 knockout mutants and was prioritized over immunity, as highlighted by an increased susceptibility to Botrytis cinerea. This process was accompanied by an overexpression of the transcription factor HBI1, a key node for the cross talk between growth and immunity. This study reveals an unprecedented mode of action of pyoverdine in Arabidopsis and demonstrates that its incidence on physiological traits depends on the plant iron status.

The Salmonella effector protein SpvC, a phosphothreonine lyase is functional in plant cells

Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. We screened a range of Salmonella Typhimurium effector proteins for interference with plant immunity. Among these, the phosphothreonine lyase SpvC attenuated the induction of immunity-related genes when present in plant cells. Using in vitro and in vivo systems we show that this effector protein interacts with and dephosphorylates activated Arabidopsis Mitogen-activated Protein Kinase 6 (MPK6), thereby inhibiting defense signaling. Moreover, the requirement of Salmonella SpvC was shown by the decreased proliferation of the ΔspvC mutant in Arabidopsis plants. These results suggest that some Salmonella effector proteins could have a conserved function during proliferation in different hosts. The fact that Salmonella and other Enterobacteriaceae use plants as hosts strongly suggests that plants represent a much larger reservoir for animal pathogens than so far estimated.

GOLLUM [FeFe]-hydrogenase-like proteins are essential for plant development in normoxic conditions and modulate energy metabolism.

[FeFe]-hydrogenase-like genes encode [Fe4 S4]-containing proteins that are ubiquitous in eukaryotic cells. In humans, iron-only hydrogenase-like protein 1 (IOP1) represses hypoxia inducible factor-1α subunit (HIF1-α) at normal atmospheric partial O2 pressure (normoxia, 21 kPa O2). In yeasts, the nar1 mutant cannot grow at 21 kPa O2, but can develop at a lower O2 pressure (2 kPa O2). We show here that plant [FeFe]-hydrogenase-like GOLLUM genes are essential for plant development and cell cycle progression. The mutant phenotypes of these plants are seen in normoxic conditions, but not under conditions of mild hypoxia (5 kPa O2). Transcriptomic and metabolomic experiments showed that the mutation enhances the expression of some hypoxia-induced genes under normal atmospheric O2 conditions and changes the cellular content of metabolites related to energy metabolism. In conclusion, [FeFe]-hydrogenase-like proteins play a central role in eukaryotes including the adaptation of plants to the ambient O2 partial pressure.

Constitutively Active Arabidopsis MAP Kinase 3 Triggers Defense Responses Involving Salicylic Acid and SUMM2 Resistance Protein

The stress-activated MPK3 and the SUPPRESSOR OF MKK1 MKK2 1/2 module control a similar set of responses, which include accumulation of salicylic acid, reactive oxygen species, and phytoalexins and modulation of defense genes. Mitogen-activated protein kinases (MAPKs) are important regulators of plant immunity. Most of the knowledge about the function of these pathways is derived from loss-of-function approaches. Using a gain-of-function approach, we investigated the responses controlled by a constitutively active (CA) MPK3 in Arabidopsis thaliana. CA-MPK3 plants are dwarfed and display a massive derepression of defense genes associated with spontaneous cell death as well as the accumulation of reactive oxygen species, phytoalexins, and the stress-related hormones ethylene and salicylic acid (SA). Remarkably CA-MPK3/sid2 and CA-MPK3/ein2-50 lines, which are impaired in SA synthesis and ethylene signaling, respectively, retain most of the CA-MPK3-associated phenotypes, indicating that the constitutive activity of MPK3 can bypass SA and ethylene signaling to activate defense responses. A comparative analysis of the molecular phenotypes of CA-MPK3 and mpk4 autoimmunity suggested convergence between the MPK3- and MPK4-guarding modules. In support of this model, CA-MPK3 crosses with summ1 and summ2, two known suppressors of mpk4, resulted in a partial reversion of the CA-MPK3 phenotypes. Overall, our data unravel a novel mechanism by which the MAPK signaling network contributes to a robust defense-response system.

A Conserved Cytochrome P450 Evolved in Seed Plants Regulates Flower Maturation

Global inspection of plant genomes identifies genes maintained in low copies across taxa and under strong purifying selection, which are likely to have essential functions. Based on this rationale, we investigated the function of the low-duplicated CYP715 cytochrome P450 gene family that appeared early in seed plants and evolved under strong negative selection. Arabidopsis CYP715A1 showed a restricted tissue-specific expression in the tapetum of flower buds and in the anther filaments upon anthesis. cyp715a1 insertion lines showed a strong defect in petal development, and transient alteration of pollen intine deposition. Comparative expression analysis revealed the downregulated expression of genes involved in pollen development, cell wall biogenesis, hormone homeostasis, and floral sesquiterpene biosynthesis, especially TPS21 and several key genes regulating floral development such as MYB21, MYB24, and MYC2. Accordingly, floral sesquiterpene emission was suppressed in the cyp715a1 mutants. Flower hormone profiling, in addition, indicated a modification of gibberellin homeostasis and a strong disturbance of the turnover of jasmonic acid derivatives. Petal growth was partially restored by the active gibberellin GA3 or the functional analog of jasmonoyl-isoleucine, coronatine. CYP715 appears to function as a key regulator of flower maturation, synchronizing petal expansion and volatile emission. It is thus expected to be an important determinant of flower-insect interaction.

5' to 3' mRNA decay contributes to the regulation of Arabidopsis seed germination by dormancy

The regulation of seed germination by dormancy is controlled by the degradation of specific subsets of mRNA during imbibition through the involvement of a 5′ to 3′ decay machinery. The regulation of plant gene expression, necessary for development and adaptive responses, relies not only on RNA transcription but also on messenger RNA (mRNA) fate. To understand whether seed germination relies on the degradation of specific subsets of mRNA, we investigated whether the 5′ to 3′ RNA decay machinery participated in the regulation of this process. Arabidopsis (Arabidopsis thaliana) seeds of exoribonuclease4 (xrn4) and varicose (vcs) mutants displayed distinct dormancy phenotypes. Transcriptome analysis of xrn4-5 and vcs-8 mutant seeds allowed us to identify genes that are likely to play a role in the control of germination. Study of 5′ untranslated region features of these transcripts revealed that specific motifs, secondary energy, and GC content could play a role in their degradation by XRN4 and VCS, and Gene Ontology clustering revealed novel actors of seed dormancy and germination. Several specific transcripts identified as being putative targets of XRN4 and VCS in seeds (PECTIN LYASE-LIKE, ASPARTYL PROTEASE, DWD-HYPERSENSITIVE-TO-ABA3, and YELLOW STRIPE-LIKE5) were further studied by reverse genetics, and their functional roles in the germination process were confirmed by mutant analysis. These findings suggest that completion of germination and its regulation by dormancy also depend on the degradation of specific subsets of mRNA.