The Pseudomonas fluorescens siderophore pyoverdine weakens arabidopsis thaliana defense in favor of growth in 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

In vitro inhibition of shikimate hydroxycinnamoyltransferase by acibenzolar acid, the first metabolite of the plant defence inducer acibenzolar-S-methyl

International audienceAcibenzolar acid, the first metabolite formed in planta from the defence inducer acibenzolar-S-methyl (ASM), has been shown to be an inhibitor of the enzyme shikimate hydroxycinnamoyltransferase (HST), extracted from grapevine or tobacco cell suspension cultures. Using a purified recombinant Arabidopsis thaliana HST, the inhibition was found to be competitive, acibenzolar acid binding reversibly to the shikimate binding site of the HST:p-coumaroyl-CoA complex, with a Ki value of 250 μM. The other hydroxycinnamoyltransferases tested in the course of this study, using either hydroxypalmitic acid, putrescine, tyramine, or quinic acid as acyl acceptors were not, or only slightly, inhibited by acibenzolar acid. To understand the specificity of the interaction of acibenzolar acid with HST, we analyzed the structure-activity relationship of a series of benzoic or acibenzolar acid analogues, tested either as AtHST substrates or as inhibitors. This analysis confirmed previously published data on the substrate flexibility of HST and demonstrated that both the carboxyl group and the thiadiazole moiety of acibenzolar acid are playing an important role in the interaction with the shikimate binding site. Acibenzolar acid, which cannot form an ester bond with p-coumaric acid, was however a less potent inhibitor than protocatechuic or 3-hydroxybenzoic acids, which are used as acyl acceptors by HST. Our results show that the interaction of acibenzolar acid with HST, which is probably directly linked to the substrate promiscuity of HST, is unlikely to play a direct role in the defence-inducing properties of ASM in plants

Role of nitric oxide synthases from Klebsormidium nitens: structural characterization and identification of protein partners

National audienceNitric oxide (NO) is an important cellular signalling molecule regulating various physiological processes, in both animals andplants. In animals, NO synthesis is mainly catalysed by NO synthase (NOS) enzymes. During biotic or abiotic stresses, NOSlike activities that are sensitive to mammalian NOS inhibitors have been detected in plants, although no sequences encodingthe well described mammalian NOS are highlighted in land plants. Interestingly, we identified NOS-like sequences in 20 algaespecies. Among them, NOSs are found in Klebsormidium nitens the model alga to study the early transition step from aquaticalgae to land plants.As mechanisms governing NO synthesis and signalling in green lineage remain unclear, the study of NOSs from K. nitens(KnNOS) through (i) characterization of their nucleotide and protein sequences, (ii) their expression levels, and (iii) theidentification of partners proteins might be an important breakthrough knowledge. To achieve this goal, nucleotide sequenceswere verified by RACE-PCR and sequencing, and after cloning, tagged-proteins were produced. Then the identification ofprotein partners will be performed by pull-down experiments followed by mass spectrometry analysis.Currently, after re-annotating the nucleotide sequence of KnNOSs, a yeast His-tagged protein production strategy has beenadopted. While waiting for the final results of the KnNOSs interactome, an in sillico study was conducted using the BioGriddatabase and human NOS interaction data. This finding opens the way for a deeper characterization of KnNOSs and its proteinpartners and will facilitate further investigation of NO signalling in green lineage

Study of the stress-related signalling of endoplasmic reticulum in grapevine immunity associated to LysM receptor kinases (VvLYKs)

National audienceIn the actual situation of decreasing the use of chemicals in agriculture, enhancing our understanding of plant immunity is a critical task to develop more sustainable plant health protection methods. An interesting strategy is to study how plants, and in particular grapevine, perceives and responds to different microorganisms. Microorganisms are notably recognizedby LysM Receptor-like Kinase (LYKs) and previous works have identified 16 LYKs encoded by the grapevine genome (VvLYKs) (Roudaire et al. 2023). Among them, VvLYK1-1, VvLYK1-2 and VvLYK5-1 are involved in chitin perception and thus play a role in the plant immunity. Interestingly, immune responses are also involved during symbiotic interaction but the receptors involved in this process are still unknown. In addition, it has been demonstrated that the Unfolded Protein Response (UPR), which is activated when unfolded proteins accumulate in the endoplasmic reticulum, is also involved during the plant immune response.It’s therefore important to improve our knowledge about the role of the different grapevine LYK receptors involved in the balance between immunity and symbiosis and in addition to determine the role of stress-related signalling of endoplasmic reticulum and the consequent activation of the UPR in these two different pathways.In this context, we aim to characterize new VvLYKs involved in the perception of Myc-factors which allow the down-regulation of the plant immunity during the establishment of mycorrhizal symbiosis. In addition we also intend to characterize the UPR signalling pathways in grapevinewhich is still unknown and finally ho

Role of nitric oxide synthase (NOS) of Klebsormidium nitens: Identification and characterization of partners

National audienc

Role of nitric oxide synthases from Klebsormidium nitens: first structural characterization and partners identification

International audienceObjectives: Nitric oxide (NO) is an important cellular signaling molecule regulating various physiologicalprocesses, in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase(NOS) enzymes. In plants, NOS-like activities sensitive to mammalian NOS inhibitors have beenmeasured, although no sequences encoding mammalian NOSs have been found in land plants.Interestingly, we identified NOS-like sequences in 20 algae species. These latter include thefilamentous charophyte green algae Klebsormidium nitens, a biological model to study the earlytransition step from aquatic algae to land plants. In order to understand the mechanisms governingNO synthesis and signaling in green lineage we initiated the functional characterization of K. nitensNOSs (KnNOS) by analyzing their primary sequences as well as their expression levels in response toabiotic stresses.Methods: KnNOSs nucleotide sequences were verified by RACE-PCR and sequencing, and their mRNAlevel were monitored by RT-qPCR and protein abundance by western blot. Protein partners werestudied, firstly in sillico using the BioGrid database and human NOS interaction data, and secondly invivo by immunoprecipitation experiments followed by mass spectrometry analysis.Results: Currently, two NOSs were identified in K. nitens genome: the KnNOS1 which possessesclassical mammalian NOS architecture consisting of oxygenase and reductase domains with somespecificities as lack of conserved residues in binding domain of BH4 cofactors; and the KnNOS2displaying a large C-ter extension containing an ANK motif and a globin domain. The two KnNOSs seemto be regulated in different ways. KnNOS1 exhibited constitutive expression during the conditionstested, whereas KnNOS2 appeared to be transcriptionally regulated during stress. In parallel studies,we also built the in silico protein–protein interaction network of human NOSs. Interestingly, genesencoding orthologs of several of these candidates were found in K. nitens genome. Some of theseconserved partners are known to be involved in mammalian NOSs regulation and represent interestingcandidates for further investigation.Conclusions: Overall these findings open the way for a deeper characterization of KnNOSs and itsprotein partners and will facilitate further investigation of NO signaling in green lineage.Relevant references:Chatelain, P., Astier, J., Wendehenne, D., Rosnoblet, C., and Jeandroz, S. (2021). Identification of Partner Proteinsof the Algae Klebsormidium nitens NO Synthases: Toward a Better Understanding of NO Signaling in EukaryoticPhotosynthetic Organisms. Front. Plant Sci. 12, 3068. doi: 10.3389/fpls.2021.797451.Jeandroz, S., Wipf, D., Stuehr, D. J., Lamattina, L., Melkonian, M., Tian, Z., et al. (2016). Occurrence, structure,and evolution of nitric oxide synthase–like proteins in the plant kingdom. Sci. Signal. 9, re2–re2. doi:10.1126/scisignal.aad4403

Role of nitric oxide synthases from klebsormidium nitens: first structural characterization and partners identification

International audienceNitric oxide (NO) is an important cellular signaling molecule regulating various physiological processes, in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase (NOS) enzymes. In plants, NOS-like activities sensitive to mammalian NOS inhibitors have been measured, although no sequences encoding mammalian NOSs have been found in land plants. Interestingly, we identified NOS-like sequences in 20 algae species. These latter include the filamentous charophyte green algae Klebsormidium nitens, a biological model to study the early transition step from aquatic algae to land plants. In order to understand the mechanisms governing NO synthesis and signaling in green lineage we initiated the functional characterization of K. nitens NOSs (KnNOS) by analyzing their primary sequences as well as their expression levels in response to abiotic stresses. Currently, two NOSs were identified in K. nitens genome: the KnNOS1 which possesses classical mammalian NOS architecture consisting of oxygenase and reductase domains with some specificities as lack of conserved residues in binding domain of BH4 cofactors; and the KnNOS2 displaying a large C-ter extension containing an ANK motif and a globin domain. The two KnNOSs seem to be regulated in different ways. KnNOS1 exhibited constitutive expression during the conditions tested, whereas KnNOS2 appeared to be transcriptionally regulated during stress.In parallel studies, we also built the in silico protein–protein interaction network of human NOSs using the BioGRID database and human NOS interaction data. Interestingly, genes encoding orthologs of several of these candidates were found in K. nitens genome. Some of these conserved partners are known to be involved in mammalian NOSs regulation and represent interesting candidates for further investigation. Overall these findings open the way for a deeper characterization of KnNOSs and its protein partners and will facilitate further investigation of NO signaling in green lineage

Study of the stress-related signalling of endoplasmic reticulum in grapevine immunity associated to LysM receptor kinases (VvLYKs)

National audienceEnhancing our understanding of plant immunity is a critical task to develop more sustainableplant health protection methods. An interesting strategy is to study how plants, and in particulargrapevine, perceives and responds to different microorganisms. Microorganisms are notably recognized by LysM Receptor-like Kinase (LYKs) and previous works have identified 16 LYKs encoded by the grapevine genome (VvLYKs) (Roudaire et al. 2023). Among them, VvLYK1-1, VvLYK1-2 and VvLYK5-1 are involved in chitin perception and thus play a role in the plant immunity. Interestingly, immune responses are also involved during symbiotic interaction but the receptors involved in this process are still unknown. In addition, it has been demonstrated that the Unfolded Protein Response (UPR), which is activated when unfoldedproteins accumulate in the endoplasmic reticulum, is also involved during the plant immune response. It’s therefore important to improve our knowledge about the role of the different grapevine LYK receptors involved in the balance between immunity and symbiosis and in addition to determine the role of stress-related signalling of endoplasmic reticulum and the consequent activation of the UPR in these two different pathways. In this context, we aim to characterize new VvLYKs involved in the perception of Myc-factors which allow the downregulation of the plant immunity during the establishment of mycorrhizal symbiosis. In addition, we also intend to characterize the UPR signalling pathways in grapevine which is still unknown and finally how different biotic factors are involved in the stress-related signalling of endoplasmic reticulum

Nitric oxide and glutathione impact the expression of iron uptake- and iron transport-related genes as well as the content of metals in <em>A. thaliana</em> plants grown under iron deficiency

International audienceMounting evidence indicate that nitric oxide (NO) acts as a signaling molecule mediating iron deficiency responses through the upregulation of the expression of iron uptake-related genes. Accordingly, NO donors such as nitrosoglutathione (GSNO) were reported to improve the fitness of plants grown under iron deficiency. Here, we showed that glutathione, a by-product of GSNO, triggered the upregulation of the expression of iron uptake- and transport-related gene and an increase of iron concentration in Arabidopsis thaliana seedlings facing iron deficiency. Furthermore, we provided evidence that under iron deficiency, NO released by GSNO did not improve the root iron concentration but impacted the content of copper. Collectively, our data highlight the complexity of interpreting data based on the use of NO donors when investigating the role of NO in iron homeostas