Characterization of the early response of Arabidopsis thaliana to Dickeya dadantii infection using expression profiling

AbstractTo draw a global view of plant responses to interactions with the phytopathogenic enterobacterale Dickeya dadantii, a causal agent of soft rot diseases on many plant species, we analysed the early Arabidopsis responses to D. dadantii infection. We performed a genome-wide analysis of the Arabidopsis thaliana transcriptome during D. dadantii infection and conducted a genetic study of identified responses.A limited set of genes related to plant defence or interactions with the environment were induced at an early stage of infection, with an over-representation of genes involved in both the metabolism of indole glucosinolates (IGs) and the jasmonate (JA) defence pathway. Bacterial type I and type II secretion systems are required to trigger the induction of IG and JA-related genes while the type III secretion system appears to partially inhibit these defence pathways. Using Arabidopsis mutants impaired in JA biosynthesis or perception, we showed that induction of some IG metabolism genes was COI1-dependent but, surprisingly, JA-independent. Moreover, characterisation of D. dadantii disease progression in Arabidopsis mutants impaired in JA or IG pathways showed that JA triggers an efficient plant defence response that does not involve IGs.The induction of the IG pathway by bacterial pathogens has been reported several times in vitro. This study shows for the first time, that this induction does indeed occur in planta, but also that this line of defence is ineffective against D. dadantii infection, in contrast to its role to counteract herbivorous or fungal pathogen attacks.

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Zinc phosphate protects tomato plants against Pseudomonas syringae pv. tomato

Journal of Plant Diseases and Protection ◽

10.1007/s41348-021-00444-z ◽

2021 ◽

Author(s):

Mara Quaglia ◽

Marika Bocchini ◽

Benedetta Orfei ◽

Roberto D’Amato ◽

Franco Famiani ◽

...

Keyword(s):

Disease Severity ◽

Pseudomonas Syringae ◽

Zinc Phosphate ◽

Plant Defence ◽

In Planta ◽

Tomato Plants ◽

Pathogenesis Related Protein ◽

Defence Responses ◽

Plant Defence Responses

AbstractThe purpose of this study was to determine whether zinc phosphate treatments of tomato plants (Solanum lycopersicum L.) can attenuate bacterial speck disease severity through reduction of Pseudomonas syringae pv. tomato (Pst) growth in planta and induce morphological and biochemical plant defence responses. Tomato plants were treated with 10 ppm (25.90 µM) zinc phosphate and then spray inoculated with strain DAPP-PG 215, race 0 of Pst. Disease symptoms were recorded as chlorosis and/or necrosis per leaf (%) and as numbers of necrotic spots. Soil treatments with zinc phosphate protected susceptible tomato plants against Pst, with reductions in both disease severity and pathogen growth in planta. The reduction of Pst growth in planta combined with significantly higher zinc levels in zinc-phosphate-treated plants indicated direct antimicrobial toxicity of this microelement, as also confirmed by in vitro assays. Morphological (i.e. callose apposition) and biochemical (i.e., expression of salicylic-acid-dependent pathogenesis-related protein PR1b1 gene) defence responses were induced by the zinc phosphate treatment, as demonstrated by histochemical and qPCR analyses, respectively. In conclusion, soil treatments with zinc phosphate can protect tomato plants against Pst attacks through direct antimicrobial activity and induction of morphological and biochemical plant defence responses.

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Biochemical characteristics of AtFAR2, a fatty acid reductase from Arabidopsis thaliana that reduces fatty acyl-CoA and -ACP substrates into fatty alcohols

Acta Biochimica Polonica ◽

10.18388/abp.2016_1245 ◽

2016 ◽

Vol 63 (3) ◽

Cited By ~ 3

Author(s):

Thuy T. P. Doan ◽

Anders S. Carlsson ◽

Sten Stymne ◽

Per Hofvander

Keyword(s):

Arabidopsis Thaliana ◽

Fatty Acid ◽

Fatty Acyl ◽

Fatty Alcohols ◽

Abnormal Development ◽

In Planta ◽

Alcohol Production ◽

Genes Encoding ◽

In Vitro Data

Fatty alcohols and derivatives are important for proper deposition of a functional pollen wall. Mutations in specific genes encoding fatty acid reductases (FAR) responsible for fatty alcohol production cause abnormal development of pollen. A disrupted AtFAR2 (MS2) gene in Arabidopsis thaliana results in pollen developing an abnormal exine layer and a reduced fertility phenotype. AtFAR2 has been shown to be targeted to chloroplasts and in a purified form to be specific for acyl-ACP substrates. Here, we present data on the in vitro and in planta characterizations of AtFAR2 from A. thaliana and show that this enzyme has the ability to use both, C16:0-ACP and C16:0-CoA, as substrates to produce C16:0-alcohol. Our results further show that AtFAR2 is highly similar in properties and substrate specificity to AtFAR6 for which in vitro data has been published, and which is also a chloroplast localized enzyme. This suggests that although AtFAR2 is the major enzyme responsible for exine layer functionality, AtFAR6 might provide functional redundancy to AtFAR2.

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LPIAT, a lyso-Phosphatidylinositol Acyltransferase, Modulates Seed Germination in Arabidopsis thaliana through PIP Signalling Pathways and is Involved in Hyperosmotic Response

International Journal of Molecular Sciences ◽

10.3390/ijms21051654 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1654

Author(s):

Denis Coulon ◽

Lionel Faure ◽

Magali Grison ◽

Stéphanie Pascal ◽

Valérie Wattelet-Boyer ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Seed Germination ◽

Lipid Synthesis ◽

Strong Increase ◽

In Planta ◽

Acyltransferase Activity ◽

Chain Acyl ◽

Phosphatidylinositol Phosphates ◽

Mature Seeds

Lyso-lipid acyltransferases are enzymes involved in various processes such as lipid synthesis and remodelling. Here, we characterized the activity of an acyltransferase from Arabidopsis thaliana (LPIAT). In vitro, this protein, expressed in Escherichia coli membrane, displayed a 2-lyso-phosphatidylinositol acyltransferase activity with a specificity towards saturated long chain acyl CoAs (C16:0- and C18:0-CoAs), allowing the remodelling of phosphatidylinositol. In planta, LPIAT gene was expressed in mature seeds and very transiently during seed imbibition, mostly in aleurone-like layer cells. Whereas the disruption of this gene did not alter the lipid composition of seed, its overexpression in leaves promoted a strong increase in the phosphatidylinositol phosphates (PIP) level without affecting the PIP2 content. The spatial and temporal narrow expression of this gene as well as the modification of PIP metabolism led us to investigate its role in the control of seed germination. Seeds from the lpiat mutant germinated faster and were less sensitive to abscisic acid (ABA) than wild-type or overexpressing lines. We also showed that the protective effect of ABA on young seedlings against dryness was reduced for lpiat line. In addition, germination of lpiat mutant seeds was more sensitive to hyperosmotic stress. All these results suggest a link between phosphoinositides and ABA signalling in the control of seed germination

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Immunogold Labeling of Hrp Pili of Pseudomonas syringae pv. tomato Assembled in Minimal Medium and In Planta

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.2.234 ◽

2001 ◽

Vol 14 (2) ◽

pp. 234-241 ◽

Cited By ~ 25

Author(s):

Wenqi Hu ◽

Jing Yuan ◽

Qiao-Ling Jin ◽

Patrick Hart ◽

Sheng Yang He

Keyword(s):

Arabidopsis Thaliana ◽

Pseudomonas Syringae ◽

Minimal Medium ◽

Leaf Tissue ◽

Hypersensitive Reaction ◽

Immunogold Labeling ◽

Structural Protein ◽

In Planta ◽

Hrp Genes

Hypersensitive reaction and pathogenicity (hrp) genes are required for Pseudomonas syringae pv. tomato (Pst) DC3000 to cause disease in susceptible tomato and Arabidopsis thaliana plants and to elicit the hypersensitive response in resistant plants. The hrp genes encode a type III protein secretion system known as the Hrp system, which in Pst DC3000 secretes HrpA, HrpZ, HrpW, and AvrPto and assembles a surface appendage, named the Hrp pilus, in hrp-gene-inducing minimal medium. HrpA has been suggested to be the Hrp pilus structural protein on the basis of copurification and mutational analyses. In this study, we show that an antibody against HrpA efficiently labeled Hrp pili, whereas antibodies against HrpW and HrpZ did not. Immunogold labeling of bacteria-infected Arabidopsis thaliana leaf tissue with an Hrp pilus antibody revealed a characteristic lineup of gold particles around bacteria and/or at the bacterium-plant contact site. These results confirm that HrpA is the major structural protein of the Hrp pilus and provide evidence that Hrp pili are assembled in vitro and in planta.

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Genome-wide identification of genes important for growth of Dickeya dadantii and D. dianthicola in potato (Solanum tuberosum) tubers

10.1101/2021.09.16.460530 ◽

2021 ◽

Author(s):

Tyler C. Helmann ◽

Melanie J. Filiatrault ◽

Paul V. Stodghill

Keyword(s):

Solanum Tuberosum ◽

Bacterial Growth ◽

Soft Rot ◽

Competitive Growth ◽

In Planta ◽

Bacterial Strains ◽

Dickeya Dadantii ◽

Genome Wide

AbstractDickeya species are causal agents of soft rot diseases in many economically important crops, including soft rot disease of potato (Solanum tuberosum). Using random barcode transposon-site sequencing (RB-TnSeq), we generated genome-wide mutant fitness profiles of Dickeya dadantii 3937, Dickeya dianthicola ME23, and Dickeya dianthicola 67-19 isolates collected after passage through several in vitro and in vivo conditions. Tubers from the potato cultivars “Atlantic”, “Dark Red Norland”, and “Upstate Abundance” provided highly similar conditions for bacterial growth. Using the homolog detection software PyParanoid, we matched fitness values for orthologous genes in the three bacterial strains. Direct comparison of fitness among the strains highlighted shared and variable traits important for growth. Bacterial growth in minimal medium required many metabolic traits that were also essential for competitive growth in planta, such as amino acid, carbohydrate, and nucleotide biosynthesis. Growth in tubers specifically required the pectin degradation gene kduD. Disruption in three putative DNA-binding proteins had strain-specific effects on competitive fitness in tubers. Though the Soft Rot Pectobacteriaceae can cause disease with little host specificity, it remains to be seen the extent to which strain-level variation impacts virulence.

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Negative Regulation of the Hrp Type III Secretion System in Pseudomonas syringae pv. phaseolicola

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-5-0682 ◽

2010 ◽

Vol 23 (5) ◽

pp. 682-701 ◽

Cited By ~ 26

Author(s):

Inmaculada Ortiz-Martín ◽

Richard Thwaites ◽

John W. Mansfield ◽

Carmen R. Beuzón

Keyword(s):

Gene Expression ◽

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Pathogenic Bacteria ◽

In Planta ◽

Secretion Systems ◽

Type Iii ◽

Type Iii Secretion Systems ◽

Bacterial Fitness

Many plant-pathogenic bacteria require type III secretion systems (T3SS) to cause disease in compatible hosts and to induce the hypersensitive response in resistant plants. T3SS gene expression is induced within the plant and responds to host and environmental factors. In Pseudomonas syringae, expression is downregulated by the Lon protease in rich medium and by HrpV under inducing conditions. HrpV acts as an anti-activator by binding HrpS. HrpG, which can also bind HrpV, has been reported to act as an anti-anti-activator. Previous studies have used mostly in vitro inducing conditions, different pathovars, and methodology. We have used single and double lon and hrpV mutants of P. syringae pv. phaseolicola 1448a, as well as strains ectopically expressing the regulators, to examine their role in coordinating expression of the T3SS. We applied real-time polymerase chain reaction to analyze gene expression both in vitro and in planta, and assessed bacterial fitness using competitive indices. Our results indicate that i) Lon downregulates expression of the hrp/hrc genes in all conditions, probably by constitutively degrading naturally unstable HrpR; ii) HrpV and HrpT downregulate expression of the hrp/hrc genes in all conditions; and iii) HrpG has an additional, HrpV-independent role, regulating expression of the hrpC operon.

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Chlorinated Auxins—How Does Arabidopsis Thaliana Deal with Them?

International Journal of Molecular Sciences ◽

10.3390/ijms21072567 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2567 ◽

Cited By ~ 2

Author(s):

Antje Walter ◽

Lorenzo Caputi ◽

Sarah O’Connor ◽

Karl-Heinz van Pée ◽

Jutta Ludwig-Müller

Keyword(s):

Arabidopsis Thaliana ◽

Acetic Acid ◽

Model Organism ◽

Wild Type ◽

In Planta ◽

Bacteria And Fungi ◽

Tryptophan Derivatives ◽

Indole 3 Acetic Acid ◽

Iaa Conjugates

Plant hormones have various functions in plants and play crucial roles in all developmental and differentiation stages. Auxins constitute one of the most important groups with the major representative indole-3-acetic acid (IAA). A halogenated derivate of IAA, 4-chloro-indole-3-acetic acid (4-Cl-IAA), has previously been identified in Pisum sativum and other legumes. While the enzymes responsible for the halogenation of compounds in bacteria and fungi are well studied, the metabolic pathways leading to the production of 4-Cl-IAA in plants, especially the halogenating reaction, are still unknown. Therefore, bacterial flavin-dependent tryptophan-halogenase genes were transformed into the model organism Arabidopsis thaliana. The type of chlorinated indole derivatives that could be expected was determined by incubating wild type A. thaliana with different Cl-tryptophan derivatives. We showed that, in addition to chlorinated IAA, chlorinated IAA conjugates were synthesized. Concomitantly, we found that an auxin conjugate synthetase (GH3.3 protein) from A. thaliana was able to convert chlorinated IAAs to amino acid conjugates in vitro. In addition, we showed that the production of halogenated tryptophan (Trp), indole-3-acetonitrile (IAN) and IAA is possible in transgenic A. thaliana in planta with the help of the bacterial halogenating enzymes. Furthermore, it was investigated if there is an effect (i) of exogenously applied Cl-IAA and Cl-Trp and (ii) of endogenously chlorinated substances on the growth phenotype of the plants.

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Host and Viral Determinants of Mx2 Antiretroviral Activity

Journal of Virology ◽

10.1128/jvi.00214-14 ◽

2014 ◽

Vol 88 (14) ◽

pp. 7738-7752 ◽

Cited By ~ 102

Author(s):

Idoia Busnadiego ◽

Melissa Kane ◽

Suzannah J. Rihn ◽

Hannah F. Preugschas ◽

Joseph Hughes ◽

...

Keyword(s):

Therapeutic Potential ◽

Early Stage ◽

Type I Interferons ◽

Primate Species ◽

Type I ◽

Viral Determinants ◽

Viral Target ◽

Hiv 1 ◽

Antiretroviral Activity

ABSTRACTMyxovirus resistance 2 (Mx2/MxB) has recently been uncovered as an effector of the anti-HIV-1 activity of type I interferons (IFNs) that inhibits HIV-1 at an early stage postinfection, after reverse transcription but prior to proviral integration into host DNA. The mechanistic details of Mx2 antiviral activity are not yet understood, but a few substitutions in the HIV-1 capsid have been shown to confer resistance to Mx2. Through a combination ofin vitroevolution and unbiased mutagenesis, we further map the determinants of sensitivity to Mx2 and reveal that multiple capsid (CA) surfaces define sensitivity to Mx2. Intriguingly, we reveal an unanticipated sensitivity determinant within the C-terminal domain of capsid. We also report that Mx2s derived from multiple primate species share the capacity to potently inhibit HIV-1, whereas selected nonprimate orthologs have no such activity. Like TRIM5α, another CA targeting antiretroviral protein, primate Mx2s exhibit species-dependent variation in antiviral specificity against at least one extant virus and multiple HIV-1 capsid mutants. Using a combination of chimeric Mx2 proteins and evolution-guided approaches, we reveal that a single residue close to the N terminus that has evolved under positive selection can determine antiviral specificity. Thus, the variable N-terminal region can define the spectrum of viruses inhibited by Mx2.IMPORTANCEType I interferons (IFNs) inhibit the replication of most mammalian viruses. IFN stimulation upregulates hundreds of different IFN-stimulated genes (ISGs), but it is often unclear which ISGs are responsible for inhibition of a given virus. Recently, Mx2 was identified as an ISG that contributes to the inhibition of HIV-1 replication by type I IFN. Thus, Mx2 might inhibit HIV-1 replication in patients, and this inhibitory action might have therapeutic potential. The mechanistic details of how Mx2 inhibits HIV-1 are currently unclear, but the HIV-1 capsid protein is the likely viral target. Here, we determine the regions of capsid that specify sensitivity to Mx2. We demonstrate that Mx2 from multiple primates can inhibit HIV-1, whereas Mx2 from other mammals (dogs and sheep) cannot. We also show that primate variants of Mx2 differ in the spectrum of lentiviruses they inhibit and that a single residue in Mx2 can determine this antiviral specificity.

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Characterization of the evolutionarily conserved iron–sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana

Biochemical Journal ◽

10.1042/bj20111993 ◽

2012 ◽

Vol 444 (2) ◽

pp. 227-237 ◽

Cited By ~ 10

Author(s):

Kaushik Saha ◽

Michael E. Webb ◽

Stephen E. J. Rigby ◽

Helen K. Leech ◽

Martin J. Warren ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Quaternary Structure ◽

Specific Activity ◽

Close Association ◽

Physiological Role ◽

Cysteine Residue ◽

In Planta ◽

Iron Sulfur ◽

Tetrapyrrole Metabolism

Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe–S] cluster. We determined the cluster to be a [4Fe–4S] type, which quickly oxidizes to a [2Fe–2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys135, is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe–4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe–S cluster or Cys135 retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe–S cluster in planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.

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Cell-extracellular matrix interactions in the fluidic phase direct the topology and polarity of self-organized epithelial structures

10.1101/2020.05.12.090068 ◽

2020 ◽

Author(s):

Mingxing Ouyang ◽

Jiun-Yann Yu ◽

Yenyu Chen ◽

Linhong Deng ◽

Chin-Lin Guo

Keyword(s):

Extracellular Matrix ◽

Large Scale ◽

Type I Collagen ◽

Mdck Cells ◽

Early Stage ◽

Single Cells ◽

Confocal Imaging ◽

Type I

AbstractIn vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as a large-scale scaffold to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell-ECM interactions to spatiotemporally coordinate their positioning and differentiation to different ECM at the whole-tissue scale is not fully understood. Here, using in vitro assay with engineered MDCK cells co-expressing H2B-mCherry (nucleus) and gp135 (Podocalyxin)-GFP (apical marker), we show that such spatiotemporal coordination for epithelial morphogenesis and polarization can be initiated and determined by cell-soluble ECM interaction in the fluidic phase. The coordination depends on the native topology of ECM components such as sheet-like basement membrane (BM, mimicked by Matrigel in experiments) and linear fiber-like type I collagen (COL). Two types of coordination are found: scaffold formed by BM (COL) facilitates a close-ended (open-ended) coordination that leads to the formation of lobular (tubular) epithelium, where polarity is preserved throughout the entire lobule/tubule. During lobular formation with BM, polarization of individual cells within the same cluster occurs almost simultaneously, whereas the apicobasal polarization in the presence of COL can start at local regions and proceed in a collective way along the axis of tubule, which might suggest existence of intercellular communications at the cell-population level. Further, in the fluidic phase, we found that cells can form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side. Based on reconstructions from time-lapse confocal imaging, this is likely derived from polarization occurring at early stage and being maintained through growth of the epithelial structures. Under suspension culture with COL, the polarization was impaired with formation of multi-lumens on the tubes, implying the importance of ECM microenvironment for tubulogenesis. Our results suggest a mechanism for cells to form polarity and coordinate positioning in vivo, and a strategy for engineering epithelial structures through cell-soluble ECM interaction and self-assembly in vitro.

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