Remorins are a family of multigenic phosphoproteins of the plasma membrane, involved in biotic an... more Remorins are a family of multigenic phosphoproteins of the plasma membrane, involved in biotic and abiotic plant interaction mechanisms, partnering in molecular signaling cascades. Signaling activity of remorins depends on their phosphorylation states and subsequent clustering into nano-sized membrane domains. The presence of a coiled-coil domain and a C-terminal domain is crucial to anchor remorins to negatively charged membrane domains, however the exact role of the N-terminal intrinsically disordered domain (IDD) on protein clustering and lipid interactions is largely unknown. Here we combine chemical biology and imaging approaches to study the partitioning of group 1 remorin into anionic model membranes mimicking the inner leaflet of the plant plasma membrane. Using reconstituted membranes containing a mix of saturated and unsaturated PhosphatidylCholine (PC), PhosphatidylInositol Phosphates (PIPs), and sterol, we investigate the clustering of remorins to the membrane and monito...
In Arabidopsis cell suspension, hyperosmotic stresses (mannitol and NaCl) were previously shown t... more In Arabidopsis cell suspension, hyperosmotic stresses (mannitol and NaCl) were previously shown to activate nine sucrose non-fermenting 1 related protein kinases 2 (SnRK2s) whereas only five of them were also activated by abscisic acid (ABA) treatment. Here, the possible activation by phosphorylation/ dephosphorylation of each kinase was investigated by studying their phosphorylation state after osmotic stress, using the Pro-Q Diamond, a specific dye for phosphoproteins. All the activated kinases were phosphorylated after osmotic stress but the induced phosphorylation changes were clearly different depending on the kinase. In addition, the increase of the global phosphorylation level induced by ABA application was lower, suggesting that different mechanisms may be involved in SnRK2 activation by hyperosmolarity and ABA. On the other hand, SnRK2 kinases remain activated by hyperosmotic stress in ABA-deficient and ABA-insensitive mutants, indicating that SnRK2 osmotic activation is independent of ABA. Moreover, using a mutant form of SnRK2s, a specific serine in the activation loop was shown to be phosphorylated after stress treatments and essential for activity and/or activation. Finally, SnRK2 activity was sensitive to staurosporine, whereas SnRK2 activation by hyperosmolarity or ABA was not, indicating that SnRK2 activation by phosphorylation is mediated by an upstream staurosporine-insensitive kinase, in both signalling pathways. All together, these results indicate that different phosphorylation mechanisms and at least three signalling pathways are involved in the activation of SnRK2 proteins in response to osmotic stress and ABA.
Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has ... more Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure
Dans des cultures cellulaires et des plantules d'Arabidopsis thaliana, l'hyperosmoiarite ... more Dans des cultures cellulaires et des plantules d'Arabidopsis thaliana, l'hyperosmoiarite active 4 proteines kinases calcium-independantes n'appartenant pas a la famille MAPK. L'identification des familles de kinases impliquees a ete entreprise en utilisant 2 anticorps famille-specifiques diriges chacun contre l'une des 2 familles candidates : les AtSK (Arabidopsis thaliana Shaggy-like kinase) et les SnRK2 (sucrose non-fermenting 1-related protein kinase 2). Des analyses de western-blot ont montre que les 2 anticorps reconnaissaient specifiquement les 10 proteines de la famille contre laquelle ils ont ete diriges. Des experiences d'immunoprecipitation suivies d'activite kinase ont montre que les stress hyperosmotiques activaient au moins une AtSK et 4 SnRK2. Les poids moleculaires des SnRK2 immunoprecipitees correspondent a ceux des kinases activees et visualisees dans les extraits bruts, indiquant qu'il s'agit des memes proteines. L'identifica...
Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathway... more Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathways. To date, how the plant plasma membrane is involved in responses to viruses is mostly unknown. Here, we show that plant cells sense the Potato virus X (PVX) COAT PROTEIN and TRIPLE GENE BLOCK 1 proteins and subsequently trigger the activation of a membrane-bound calcium-dependent kinase. We show that the Arabidopsis thaliana CALCIUM-DEPENDENT PROTEIN KINASE 3-interacts with group 1 REMORINs in vivo, phosphorylates the intrinsically disordered N-terminal domain of the Group 1 REMORIN REM1.3, and restricts PVX cell-to-cell movement. REM1.3's phospho-status defines its plasma membrane nanodomain organization and is crucial for REM1.3-dependent restriction of PVX cell-to-cell movement by regulation of callose deposition at plasmodesmata. This study unveils plasma membrane nanodomain-associated molecular events underlying the plant immune response to viruses.
REMORINs (REMs) are a plant-specific protein family, proposed regulators of membrane-associated m... more REMORINs (REMs) are a plant-specific protein family, proposed regulators of membrane-associated molecular assemblies and well-established markers of plasma membrane nanodomains. REMs play a diverse set of functions in plant interactions with pathogens and symbionts, responses to abiotic stresses, hormone signaling and cell-to-cell communication. In this review, we highlight the established and more putative roles of REMs throughout the literature. We discuss the physiological functions of REMs, the mechanisms underlying their nanodomain-organization and their putative role as regulators of nanodomain-associated molecular assemblies. Furthermore, we discuss how REM phosphorylation may regulate their functional versatility. Overall, through data-mining and comparative analysis of the literature, we suggest how to further study the molecular mechanisms underpinning the functions of REMs.
Wounding is caused by abiotic and biotic factors and triggers complex short- and long-term respon... more Wounding is caused by abiotic and biotic factors and triggers complex short- and long-term responses at the local and systemic level. These responses are under the control of complex signaling pathways, which are still poorly understood. Here, we show that the rapid activation of MKK4/5-MPK3/6 by wounding is independent of jasmonic acid (JA) signaling and that, contrary to what happens in tobacco, this fast module does not control wound-triggered JA accumulation in Arabidopsis. We also demonstrate that a second MAPK module, constituted by MKK3 and the clade-C MAPKs MPK1/2/7, is activated by wounding in an independent manner. We provide evidence that the activation of this MKK3-MPK1/2/7 module occurs mainly through wound-induced JA production via the transcriptional regulation of upstream clade-III MAP3Ks and particularly MAP3K14. We show that mkk3 mutant plants are more susceptible to the larvae of the generalist lepidopteran herbivore Spodoptera littoralis, indicating that the MKK3...
Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathway... more Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathways. To date, how the plant plasma membrane is involved in responses to viruses is mostly unknown. Here, we show that plant cells sense the Potato virus X (PVX) COAT PROTEIN and TRIPLE GENE BLOCK 1 proteins and subsequently trigger the activation of a membrane-bound calcium-dependent kinase. We show that the Arabidopsis thaliana CALCIUM-DEPENDENT PROTEIN KINASE 3-interacts with group 1 REMORINs in vivo, phosphorylates the intrinsically disordered N-terminal domain of the Group 1 REMORIN REM1.3, and restricts PVX cell-to-cell movement. REM1.3's phospho-status defines its plasma membrane nanodomain organization and is crucial for REM1.3-dependent restriction of PVX cell-to-cell movement by regulation of callose deposition at plasmodesmata. This study unveils plasma membrane nanodomain-associated molecular events underlying the plant immune response to viruses.
Plant surface-localized pathogen recognition receptors (PRRs) perceive conserved microbial featur... more Plant surface-localized pathogen recognition receptors (PRRs) perceive conserved microbial features, termed pathogen-associated molecular patterns (PAMPs), resulting in disease resistance. PAMP perception leads to calcium influx, mitogen-activated protein kinase (MAPK) activation, a burst of reactive oxygen species (ROS) mediated by RbohD, accumulation of the defense hormone salicylic acid (SA) and callose deposition. Lectin receptor-like kinases (LecRKs) belong to a specific PRR family and are important players in plant innate immunity. Here, we report that LecRK-IX.2 is a positive regulator of PRR-triggered immunity. Pathogen infection activated the transcription of Arabidopsis LecRK-IX.2 and the LecRK-IX.2 knockout lines exhibited enhanced susceptibility to virulent Pseudomonas syringae pv. tomato DC3000 (Pst). In addition, LecRK-IX.2 is capable of inducing RbohD phosphorylation, likely by recruiting calcium dependent protein kinases (CPKs) to trigger ROS production in Arabidopsi...
Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, i... more Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report unique Ca(2+) signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca(2+)-sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, r...
Calcium-dependent protein kinases undergo a rapid biochemical activation in response to an intrac... more Calcium-dependent protein kinases undergo a rapid biochemical activation in response to an intracellular Ca increase induced by the PRR-dependent perception of a pathogen-related stimulus. Based on SDS gel resolution, the in-gel kinase assay allows the analysis of multiple in vivo protein samples in parallel, combining the advantage of protein separation according to molecular mass with the activity read-out of a protein kinase assay. It thus enables to follow the transient CDPK activation and inactivation in response to in vivo elicitation with a time-wise resolution. In addition, changes of CDPK phosphorylation activity often correlate with slight shifts in the enzyme's apparent molecular mass, indicating posttranslational modifications and a conformational change of the active enzyme compared to its inactive resting form. These band shifts can be detected by a simple immunoblotting to monitor CDPK activation.
Remorins are a family of multigenic phosphoproteins of the plasma membrane, involved in biotic an... more Remorins are a family of multigenic phosphoproteins of the plasma membrane, involved in biotic and abiotic plant interaction mechanisms, partnering in molecular signaling cascades. Signaling activity of remorins depends on their phosphorylation states and subsequent clustering into nano-sized membrane domains. The presence of a coiled-coil domain and a C-terminal domain is crucial to anchor remorins to negatively charged membrane domains, however the exact role of the N-terminal intrinsically disordered domain (IDD) on protein clustering and lipid interactions is largely unknown. Here we combine chemical biology and imaging approaches to study the partitioning of group 1 remorin into anionic model membranes mimicking the inner leaflet of the plant plasma membrane. Using reconstituted membranes containing a mix of saturated and unsaturated PhosphatidylCholine (PC), PhosphatidylInositol Phosphates (PIPs), and sterol, we investigate the clustering of remorins to the membrane and monito...
In Arabidopsis cell suspension, hyperosmotic stresses (mannitol and NaCl) were previously shown t... more In Arabidopsis cell suspension, hyperosmotic stresses (mannitol and NaCl) were previously shown to activate nine sucrose non-fermenting 1 related protein kinases 2 (SnRK2s) whereas only five of them were also activated by abscisic acid (ABA) treatment. Here, the possible activation by phosphorylation/ dephosphorylation of each kinase was investigated by studying their phosphorylation state after osmotic stress, using the Pro-Q Diamond, a specific dye for phosphoproteins. All the activated kinases were phosphorylated after osmotic stress but the induced phosphorylation changes were clearly different depending on the kinase. In addition, the increase of the global phosphorylation level induced by ABA application was lower, suggesting that different mechanisms may be involved in SnRK2 activation by hyperosmolarity and ABA. On the other hand, SnRK2 kinases remain activated by hyperosmotic stress in ABA-deficient and ABA-insensitive mutants, indicating that SnRK2 osmotic activation is independent of ABA. Moreover, using a mutant form of SnRK2s, a specific serine in the activation loop was shown to be phosphorylated after stress treatments and essential for activity and/or activation. Finally, SnRK2 activity was sensitive to staurosporine, whereas SnRK2 activation by hyperosmolarity or ABA was not, indicating that SnRK2 activation by phosphorylation is mediated by an upstream staurosporine-insensitive kinase, in both signalling pathways. All together, these results indicate that different phosphorylation mechanisms and at least three signalling pathways are involved in the activation of SnRK2 proteins in response to osmotic stress and ABA.
Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has ... more Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure
Dans des cultures cellulaires et des plantules d'Arabidopsis thaliana, l'hyperosmoiarite ... more Dans des cultures cellulaires et des plantules d'Arabidopsis thaliana, l'hyperosmoiarite active 4 proteines kinases calcium-independantes n'appartenant pas a la famille MAPK. L'identification des familles de kinases impliquees a ete entreprise en utilisant 2 anticorps famille-specifiques diriges chacun contre l'une des 2 familles candidates : les AtSK (Arabidopsis thaliana Shaggy-like kinase) et les SnRK2 (sucrose non-fermenting 1-related protein kinase 2). Des analyses de western-blot ont montre que les 2 anticorps reconnaissaient specifiquement les 10 proteines de la famille contre laquelle ils ont ete diriges. Des experiences d'immunoprecipitation suivies d'activite kinase ont montre que les stress hyperosmotiques activaient au moins une AtSK et 4 SnRK2. Les poids moleculaires des SnRK2 immunoprecipitees correspondent a ceux des kinases activees et visualisees dans les extraits bruts, indiquant qu'il s'agit des memes proteines. L'identifica...
Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathway... more Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathways. To date, how the plant plasma membrane is involved in responses to viruses is mostly unknown. Here, we show that plant cells sense the Potato virus X (PVX) COAT PROTEIN and TRIPLE GENE BLOCK 1 proteins and subsequently trigger the activation of a membrane-bound calcium-dependent kinase. We show that the Arabidopsis thaliana CALCIUM-DEPENDENT PROTEIN KINASE 3-interacts with group 1 REMORINs in vivo, phosphorylates the intrinsically disordered N-terminal domain of the Group 1 REMORIN REM1.3, and restricts PVX cell-to-cell movement. REM1.3's phospho-status defines its plasma membrane nanodomain organization and is crucial for REM1.3-dependent restriction of PVX cell-to-cell movement by regulation of callose deposition at plasmodesmata. This study unveils plasma membrane nanodomain-associated molecular events underlying the plant immune response to viruses.
REMORINs (REMs) are a plant-specific protein family, proposed regulators of membrane-associated m... more REMORINs (REMs) are a plant-specific protein family, proposed regulators of membrane-associated molecular assemblies and well-established markers of plasma membrane nanodomains. REMs play a diverse set of functions in plant interactions with pathogens and symbionts, responses to abiotic stresses, hormone signaling and cell-to-cell communication. In this review, we highlight the established and more putative roles of REMs throughout the literature. We discuss the physiological functions of REMs, the mechanisms underlying their nanodomain-organization and their putative role as regulators of nanodomain-associated molecular assemblies. Furthermore, we discuss how REM phosphorylation may regulate their functional versatility. Overall, through data-mining and comparative analysis of the literature, we suggest how to further study the molecular mechanisms underpinning the functions of REMs.
Wounding is caused by abiotic and biotic factors and triggers complex short- and long-term respon... more Wounding is caused by abiotic and biotic factors and triggers complex short- and long-term responses at the local and systemic level. These responses are under the control of complex signaling pathways, which are still poorly understood. Here, we show that the rapid activation of MKK4/5-MPK3/6 by wounding is independent of jasmonic acid (JA) signaling and that, contrary to what happens in tobacco, this fast module does not control wound-triggered JA accumulation in Arabidopsis. We also demonstrate that a second MAPK module, constituted by MKK3 and the clade-C MAPKs MPK1/2/7, is activated by wounding in an independent manner. We provide evidence that the activation of this MKK3-MPK1/2/7 module occurs mainly through wound-induced JA production via the transcriptional regulation of upstream clade-III MAP3Ks and particularly MAP3K14. We show that mkk3 mutant plants are more susceptible to the larvae of the generalist lepidopteran herbivore Spodoptera littoralis, indicating that the MKK3...
Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathway... more Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathways. To date, how the plant plasma membrane is involved in responses to viruses is mostly unknown. Here, we show that plant cells sense the Potato virus X (PVX) COAT PROTEIN and TRIPLE GENE BLOCK 1 proteins and subsequently trigger the activation of a membrane-bound calcium-dependent kinase. We show that the Arabidopsis thaliana CALCIUM-DEPENDENT PROTEIN KINASE 3-interacts with group 1 REMORINs in vivo, phosphorylates the intrinsically disordered N-terminal domain of the Group 1 REMORIN REM1.3, and restricts PVX cell-to-cell movement. REM1.3's phospho-status defines its plasma membrane nanodomain organization and is crucial for REM1.3-dependent restriction of PVX cell-to-cell movement by regulation of callose deposition at plasmodesmata. This study unveils plasma membrane nanodomain-associated molecular events underlying the plant immune response to viruses.
Plant surface-localized pathogen recognition receptors (PRRs) perceive conserved microbial featur... more Plant surface-localized pathogen recognition receptors (PRRs) perceive conserved microbial features, termed pathogen-associated molecular patterns (PAMPs), resulting in disease resistance. PAMP perception leads to calcium influx, mitogen-activated protein kinase (MAPK) activation, a burst of reactive oxygen species (ROS) mediated by RbohD, accumulation of the defense hormone salicylic acid (SA) and callose deposition. Lectin receptor-like kinases (LecRKs) belong to a specific PRR family and are important players in plant innate immunity. Here, we report that LecRK-IX.2 is a positive regulator of PRR-triggered immunity. Pathogen infection activated the transcription of Arabidopsis LecRK-IX.2 and the LecRK-IX.2 knockout lines exhibited enhanced susceptibility to virulent Pseudomonas syringae pv. tomato DC3000 (Pst). In addition, LecRK-IX.2 is capable of inducing RbohD phosphorylation, likely by recruiting calcium dependent protein kinases (CPKs) to trigger ROS production in Arabidopsi...
Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, i... more Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report unique Ca(2+) signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca(2+)-sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, r...
Calcium-dependent protein kinases undergo a rapid biochemical activation in response to an intrac... more Calcium-dependent protein kinases undergo a rapid biochemical activation in response to an intracellular Ca increase induced by the PRR-dependent perception of a pathogen-related stimulus. Based on SDS gel resolution, the in-gel kinase assay allows the analysis of multiple in vivo protein samples in parallel, combining the advantage of protein separation according to molecular mass with the activity read-out of a protein kinase assay. It thus enables to follow the transient CDPK activation and inactivation in response to in vivo elicitation with a time-wise resolution. In addition, changes of CDPK phosphorylation activity often correlate with slight shifts in the enzyme's apparent molecular mass, indicating posttranslational modifications and a conformational change of the active enzyme compared to its inactive resting form. These band shifts can be detected by a simple immunoblotting to monitor CDPK activation.
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Papers by Marie Boudsocq