Clavibacter michiganensis downregulates photosynthesis and modifies monolignols metabolism revealing a crosstalk with tomato immune responses

The gram-positive pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial canker disease in tomato, affecting crop yield and fruit quality. To understand how tomato plants respond, the dynamic expression profile of host genes was analyzed upon Cmm infection. Symptoms of bacterial canker became evident from the third day. As the disease progressed, the bacterial population increased in planta, reaching the highest level at six days and remained constant till the twelfth day post inoculation. These two time points were selected for transcriptomics. A progressive down-regulation of key genes encoding for components of the photosynthetic apparatus was observed. Two temporally separated defense responses were observed, which were to an extent interdependent. During the primary response, genes of the phenylpropanoid pathway were diverted towards the synthesis of monolignols away from S-lignin. In dicots, lignin polymers mainly consist of G- and S-units, playing an important role in defense. The twist towards G-lignin enrichment is consistent with previous findings, highlighting a response to generate an early protective barrier and to achieve a tight interplay between lignin recomposition and the primary defense response mechanism. Upon progression of Cmm infection, the temporal deactivation of phenylpropanoids coincided with the upregulation of genes that belong in a secondary response mechanism, supporting an elegant reprogramming of the host transcriptome to establish a robust defense apparatus and suppress pathogen invasion. This high-throughput analysis reveals a dynamic reorganization of plant defense mechanisms upon bacterial infection to implement an array of barriers preventing pathogen invasion and spread

Molecular and biochemical analysis of the oleuropein β-glucosidase gene during the development and defense of olive

Oleuropein, the major secoiridoid compound in olive (Olea europaea), is involved in a sophisticated two-component defense system comprising a β-glucosidase enzyme that activates oleuropein into a toxic glutaraldehyde-like structure. Although oleuropein deglycosylation studies have been monitored extensively, an oleuropein β-glucosidase gene has not been characterized as yet. In the present study is reported the molecular characterization of OeGLU cDNA from olive encoding a β-glucosidase belonging to the defence-related group of terpenoid-specific glucosidases. Heterologous expression of OeGLU in E. coli failed to produce an active enzyme but in planta recombinant protein expression assays showed that OeGLU deglycosylated and activated oleuropein into a strong protein cross-linker. Homology and docking modelling predicted that OeGLU has a characteristic (β/α)8 TIM barrel conformation and a typical construction of a pocket-shaped substrate recognition domain composed of conserved amino acids supporting the β-glucosidase activity and non-conserved residues associated with aglycon specificity. Kinetic parameters of the purified enzyme came in agreement with reported values of purified oleuropein-specific β-glucosidases from olive. Subcellular localization studies of OeGLU proved that the enzyme is localized in the nucleus due to a Nuclear Localization Signal (NLS) located in the C-termini of the enzyme and deletion of the NLS resulted in cytoplasmic localization. The quaternary structure of OeGLU in vivo is a high molecular weight homomultimer and interestingly deletion of the NLS resulted also in a structural mutation, as the enzyme could be detected in solely monomeric form with null enzymatic activity. The C-termini of OeGLU is most likely involved in protein-protein interaction of the monomers for the assembly of the homomultimer, a key factor for the enzymatically active enzyme. Additionally it is reported for the first time that the enzymatic deglycosylation of oleuropein is a fluorogenic reaction as proved by in-gel activity assays (zymograms) and this methodology will help for highly specific screening for isoenzymes of OeGLU.Η ολευρωπαΐνη, το επικρατέστερο σεκοϊριδοειδές στην ελιά (Olea europaea), εμπλέκεται σε ένα δυαδικό σύστημα άμυνας που αποτελείται από μια β-γλυκοσιδάση η οποία απογλυκοζυλιώνει την ολευρωπαΐνη παράγοντας ένα τοξικό άγλυκο μόριο με δομή παρόμοια με την γλουταραλδεΰδη. Παρότι ο μηχανισμός άμυνας έχει μελετηθεί αρκετά, δεν έχει χαρακτηριστεί ακόμα κάποια β-γλυκοσιδάση εξειδικευμένη για την ολευρωπαΐνη. Στην παρούσα μελέτη περιγράφεται ο μοριακός χαρακτηρισμός του cDNA του OeGLU που κωδικοποιεί για μια αμυντική β-γλυκοσιδάση εξειδικευμένη για τα τερπενοειδή. Ετερόλογη έκφραση του OeGLU σε E. coli δεν οδήγησε σε ενεργό ένζυμο αλλά μέσω in planta παροδικής έκφρασης αποδείχθηκε ότι το OeGLU μπορεί να απογλυκοζυλιώσει και να ενεργοποιήσει την ολευρωπαΐνη σε έναν μεταβολίτη με ισχυρή δυνατότητα μαζικής συμπλοκοποίησης και διασύνδεσης πρωτεϊνών. Η in silico πρόβλεψη της τριτοταγούς δομής του ενζύμου και ανάλυση μοριακής προσκόλλησης της ολευρωπαΐνης σε αυτήν έδειξε ότι το OeGLU έχει την χαρακτηριστική (β/α)8 TIM barrel δομή και το ενεργό κέντρο αποτελείται από συντηρημένα αμινοξικά κατάλοιπα που σχετίζονται με την κατάλυση της αντίδρασης ενώ τα αμινοξέα που είναι υπεύθυνα για την αναγνώριση του άγλυκου τμήματος είναι μη συντηρημένα. Οι κινητικές παράμετροι του απομονωμένου ενζύμου που υπολογίστηκαν είναι σε συμφωνία με τιμές που έχουν αναφερθεί για απομονωμένες β-γλυκοσιδάσες από τη ελιά που εξειδικευμένα απογλυκοζυλιώνουν την ολευρωπαΐνη. Το ένζυμο OeGLU βρέθηκε να τοποθετείται υποκυτταρικά στον πυρήνα των κυττάρων λόγω σινιάλου πυρηνικής τοποθέτησης (NLS) που βρίσκεται στο C-άκρο και διαγραφή του οποίου οδηγεί σε κυτταροπλασματική τοποθέτηση. Η τεταρτοταγής δομή του ενζύμου βρέθηκε να είναι μεγαλομοριακή και το OeGLU ομοπολυμερίζεται. Η εξάλειψη του NLS οδήγησε επίσης σε δομική μετάλλαξη αφού το ένζυμο ανιχνεύτηκε αποκλειστικά σαν μονομερές με μη μετρήσιμη ενζυμική δραστικότητα. Το C-άκρο του OeGLU πιθανότατα εμπλέκεται στην αλληλεπίδραση των μονομερών για την δημιουργία του ομοπολυμερούς, μια βασική προϋπόθεση για την ενεργότητα του ενζύμου. Επίσης αναφέρεται για πρώτη φορά ότι η ενζυμική απογλυκοζυλίωση του OeGLU παράγει φθορισμό, όπως φάνηκε από τα ζυμογράμματα, και με αυτή την μέθοδο είναι εφικτή η εξειδικευμένη ανίχνευση ισοενζύμων του OeGLU

Beyond the semi-synthetic artemisinin: metabolic engineering of plant-derived anti-cancer drugs

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A Rapid and Efficient Vacuum-Based Agroinfiltration Protocol for Transient Gene Overexpression in Leaves of Catharanthus roseus

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Improved virus-induced gene silencing allows discovery of a serpentine synthase gene in Catharanthus roseus

International audienceAbstract Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant-specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. Catharanthus roseus is a medicinal plant well known for producing many bioactive compounds, such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase (SS). This enzyme is a cytochrome P450 (CYP) that produces the β-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants

GA-Mediated Disruption of RGA/BZR1 Complex Requires HSP90 to Promote Hypocotyl Elongation

Circuitries of signaling pathways integrate distinct hormonal and environmental signals, and influence development in plants. While a crosstalk between brassinosteroid (BR) and gibberellin (GA) signaling pathways has recently been established, little is known about other components engaged in the integration of the two pathways. Here, we provide supporting evidence for the role of HSP90 (HEAT SHOCK PROTEIN 90) in regulating the interplay of the GA and BR signaling pathways to control hypocotyl elongation of etiolated seedlings in Arabidopsis. Both pharmacological and genetic depletion of HSP90 alter the expression of GA biosynthesis and catabolism genes. Major components of the GA pathway, like RGA (REPRESSOR of ga1–3) and GAI (GA-INSENSITIVE) DELLA proteins, have been identified as physically interacting with HSP90. Interestingly, GA-promoted DELLA degradation depends on the ATPase activity of HSP90, and inhibition of HSP90 function stabilizes the DELLA/BZR1 (BRASSINAZOLE-RESISTANT 1) complex, modifying the expression of downstream transcriptional targets. Our results collectively reveal that HSP90, through physical interactions with DELLA proteins and BZR1, modulates DELLA abundance and regulates the expression of BZR1-dependent transcriptional targets to promote plant growth

New Insight into HPts as Hubs in Poplar Cytokinin and Osmosensing Multistep Phosphorelays: Cytokinin Pathway Uses Specific HPts

International audienceWe have previously identified proteins in poplar which belong to an osmosensing (OS) signaling pathway, called a multistep phosphorelay (MSP). The MSP comprises histidine-aspartate kinases (HK), which act as membrane receptors; histidine phosphotransfer (HPt) proteins, which act as phosphorelay proteins; and response regulators (RR), some of which act as transcription factors. In this study, we identified the HK proteins homologous to the Arabidopsis cytokinin (CK) receptors, which are first partners in the poplar cytokinin MSP, and focused on specificity of these two MSPs (CK and OS), which seem to share the same pool of HPt proteins. Firstly, we isolated five CK HKs from poplar which are homologous to Arabidopsis AHK2, AHK3, and AHK4, namely, HK2, HK3a, HK3b, HK4a, HK4b. These HKs were shown to be functional kinases, as observed in a functional complementation of a yeast HK deleted strain. Moreover, one of these HKs, HK4a, was shown to have kinase activity dependent on the presence of CK. Exhaustive interaction tests between these five CK HKs and the 10 HPts characterized in poplar were performed using two-hybrid and BiFC experiments. The resulting partnership was compared to that previously identified between putative osmosensors HK1a/1b and HPt proteins. Finally, in planta coexpression analysis of genes encoding these potential partners revealed that almost all HPts are coexpressed with CK HKs in four different poplar organs. Overall, these results allowed us to unravel the common and specific partnerships existing between OS and CK MSP in Populus

Identification of a second 16-hydroxytabersonine-O-methyltransferase suggests an evolutionary relationship between alkaloid and flavonoid metabolisms in Catharanthus roseus

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Identifying Genes Involved in Alkaloid Biosynthesis in Vinca minor through Transcriptomics and Gene Co-Expression Analysis

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Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle

International audienceVinblastine, a potent anticancer drug, is produced by Catharanthus roseus (Madagascar periwinkle) in small quantities, and heterologous reconstitution of vinblastine biosynthesis could provide an additional source of this drug. However, the chemistry underlying vinblastine synthesis makes identification of the biosynthetic genes challenging. Here we identify the two missing enzymes necessary for vinblastine biosynthesis in this plant: an oxidase and a reductase that isomerize stemmadenine acetate into dihydroprecondylocarpine acetate, which is then deacetoxylated and cyclized to either catharanthine or tabersonine via two hydrolases characterized herein. The pathways show how plants create chemical diversity and also enable development of heterologous platforms for generation of stemmadenine-derived bioactive compounds