Inhibition of Virulence-Related Traits in Pseudomonas syringae pv. actinidiae by Gunpowder Green Tea Extracts

Green tea is a widely-consumed healthy drink produced from the leaves of Camellia sinensis. It is renowned for its antioxidant and anticarcinogenic properties, but also displays significant antimicrobial activity against numerous human pathogens. Here we analyzed the antimicrobial activity of Gunpowder green tea against Pseudomonas syringae pv. actinidiae (Psa), the agent that causes kiwifruit bacterial canker. At the phenotypic level, tea extracts strongly inhibited Psa growth and swimming motility, suggesting it could reduce Psa epiphytic survival during plant colonization. The loss of bacterial virulence-related traits following treatment with tea extracts was also investigated by large-scale transcriptome analysis, which confirmed the in vitro phenotypes and revealed the induction of adaptive responses in the treated bacteria allowing them to cope with iron deficiency and oxidative stress. Such molecular changes may account for the ability of Gunpowder green tea to protect kiwifruit against Psa infection

Plant and fungus transcriptomic data from grapevine berries undergoing artificially-induced noble rot caused by Botrytis cinerea

Noble rot is a latent infection of grape berries caused by the necrotrophic fungus Botrytis cinerea, which develops under specific climatic conditions. The infected berries undergo biochemical and metabolic changes, associated with a rapid withering, which altogether offer interesting organoleptic features to sweet white wines. In this paper, we provide RNAseq datasets (raw and normalized counts as well as differentially expressed genes lists) of the transcriptome profiles of both grapevine berries (Vitis vinifera cv. Garganega) and B. cinerea during the establishment of noble rot, artificially induced in controlled conditions. The sequencing data are available in the NCBI GEO database under accession number GSE116741. These data were exploited in a comprehensive meta-analysis of gene expression during noble rot infection, gray mold and post-harvest withering. This highlighted an important common transcriptional reprogramming in different botrytized grape berry varieties and led to the identification of key genes specifically modulated during noble rot infection, which are described in the article entitled \u201cSpecific molecular interactions between Vitis vinifera and Botrytis cinerea are required for noble rot development in grape berries\u201

General and species-specific transcriptional responses to downy mildew infection in a susceptible (Vitis vinifera) and a resistant (V. riparia) grapevine species

<p>Abstract</p> <p>Background</p> <p>Downy mildew is a destructive grapevine disease caused by <it>Plasmopara viticola </it>(Berk. and Curt.) Berl. and de Toni, which can only be controlled by intensive fungicide treatments. Natural sources of resistance from wild grapevine (<it>Vitis</it>) species are used in conventional breeding approaches, but the signals and effectors involved in resistance in this important crop species are not well understood.</p> <p>Results</p> <p>Early transcriptional changes associated with <it>P. viticola </it>infection in susceptible <it>V. vinifera </it>and resistant <it>V. riparia </it>plants were analyzed using the Combimatrix microarray platform. Transcript levels were measured 12 and 24 h post-inoculation, reflecting the time points immediately preceding the onset of resistance in <it>V. riparia</it>, as determined by microscopic analysis. Our data indicate that resistance in <it>V. riparia </it>is induced after infection, and is not based on differences in basal gene expression between the two species. The strong and rapid transcriptional reprogramming involves the induction of pathogenesis-related proteins and enzymes required for the synthesis of phenylpropanoid-derived compounds, many of which are also induced, albeit to a lesser extent, in <it>V. vinifera</it>. More interestingly, resistance in <it>V. riparia </it>also involves the specific modulation of numerous transcripts encoding components of signal transduction cascades, hypersensitive reaction markers and genes involved in jasmonate biosynthesis. The limited transcriptional modulation in <it>V. vinifera </it>represents a weak attempted defense response rather than the activation of compatibility-specific pathways.</p> <p>Conclusions</p> <p>Several candidate resistance genes were identified that could be exploited in future biotechnological approaches to increase disease resistance in susceptible grapevine species. Measurements of jasmonic acid and methyl jasmonate in infected leaves suggest that this hormone may also be involved in <it>V. riparia </it>resistance to <it>P. viticola</it>.</p

Genome-wide characterisation and expression profile of the grapevine ATL ubiquitin ligase family reveal biotic and abiotic stress-responsive and development-related members

The Arabidopsis T\uf3xicos en Levadura (ATL) protein family is a class of E3 ubiquitin ligases with a characteristic RING-H2 Zn-finger structure that mediates diverse physiological processes and stress responses in plants. We carried out a genome-wide survey of grapevine (Vitis vinifera L.) ATL genes and retrieved 96 sequences containing the canonical ATL RING-H2 domain. We analysed their genomic organisation, gene structure and evolution, protein domains and phylogenetic relationships. Clustering revealed several clades, as already reported in Arabidopsis thaliana and rice (Oryza sativa), with an expanded subgroup of grapevine-specific genes. Most of the grapevine ATL genes lacked introns and were scattered among the 19 chromosomes, with a high level of duplication retention. Expression profiling revealed that some ATL genes are expressed specifically during early or late development and may participate in the juvenile to mature plant transition, whereas others may play a role in pathogen and/or abiotic stress responses, making them key candidates for further functional analysis. Our data offer the first genome-wide overview and annotation of the grapevine ATL family, and provide a basis for investigating the roles of specific family members in grapevine physiology and stress responses, as well as potential biotechnological applications

Genome-wide identification and analysis of mitogen activated protein kinase kinase kinase gene family in grapevine (Vitis vinifera)

Mitogen-activated protein kinase kinase kinases (MAPKKKs; MAP3Ks) are important components of MAPK cascades, which are highly conserved signal transduction pathways in animals, yeast and plants, play important roles in plant growth and development. MAPKKKs have been investigated on their evolution and expression patterns in limited plants including Arabidopsis, rice and maize

The role of Glycerol-3-phosfate biosynthesis in the regeneration of cytosolic NAD+ as a possible therapeutic approach against mitochondrial diseases

reservedLe malattie mitocondriali sono disturbi clinici caratterizzati da difetti biochimici nel funzionamento dei complessi della catena respiratoria degli elettroni (electron transfer chain, ETC). Le cause di queste alterazioni sono mutazioni a carico di un ampio gruppo di geni del DNA nucleare o mitocondriale che codificano proteine necessarie per la formazione dei complessi del ETC. L’effetto comune rispetto alle cellule WT, è una modificazione del rapporto NAD+/NADH citosolico, il cui sbilanciamento comporta manifestazioni eterogenee. Non è ancora disponibile una vera e propria cura per le malattie mitocondriali. In questo elaborato si affronta la biosintesi del Glicerolo-3-fosfato (Gro3P) come pathway conservato in lievito, C.elegans, cellule di fegato di topo e cellule tumorali umane, per rigenerare il NAD+ citosolico in seguito all’inibizione/malfunzionamento dei complessi respiratori. L’inibizione farmacologica e genetica dei complessi I e III dell’ETC, ha permesso di studiare nei vari organismi i difetti metabolici che causano un accumulo di NADH citosolico, che diventa tossico. Per comprendere i cambiamenti nel metabolismo in seguito a inibizione dei geni coinvolti e seguire il destino del glucosio in presenza di disfunzioni dell’ ETC, è stata utilizzata una strategia di marcatura del glucosio in vivo attraverso un isotopo [U-13C]. L’obbiettivo che si pone nello studio analizzato è di comprendere se sia possibile promuovere l’espressione degli enzimi coinvolti nella biosintesi del Gro3P, come possibile strategia terapeutica per trattare le malattie mitocondriali.Mitochondrial diseases are clinical disorders characterized by biochemical defects in the functioning of electron transfer chain (ETC) complexes. The causes of these alterations are mutations in a large group of nuclear or mitochondrial DNA genes that encode proteins necessary for the formation of ETC complexes. The common effect with respect to WT cells, is a change in the cytosolic NAD+/NADH ratio, the imbalance of which results in heterogeneous manifestations. No real cure for mitochondrial diseases is yet available. In this thesis, we address the biosynthesis of Glycerol-3-phosphate (Gro3P) as a conserved pathway in yeast, C.elegans, mouse liver cells, and human cancer cells to regenerate cytosolic NAD+ following inhibition/malfunctioning of respiratory complexes. Pharmacological and genetic inhibition of ETC complexes I and III has enabled the study in various organisms of metabolic defects that cause cytosolic NADH to accumulate and become toxic. To understand the changes in metabolism following inhibition of the genes involved and to follow the fate of glucose in the presence of ETC dysfunction, an in vivo glucose-tagging strategy using an isotope [U-13C] was used. The goal in the analyzed study is to understand whether it is possible to promote the expression of enzymes involved in Gro3P biosynthesis as a possible therapeutic strategy to treat mitochondrial diseases

Development of transient gene silencing tools in grapevine

A causa della mancanza di tecniche efficienti per la trasformazione di vite, non sono ancora stati sviluppati protocolli per studi su larga scala di genomica funzionale in Vitis ssp. I primi tentativi di trasformazione stabile in vite sono stati effettuati da alcuni gruppi di ricerca attraverso co-coltivazione con Agrobacterium tumefaciens e successiva rigenerazione, sia per embriogenesi somatica che per organogenesi. Tuttavia, questi approcci mostrano, nella maggior parte dei casi, variabilità nell’efficienza di trasformazione e di rigenerazione; inoltre, richiedendo tempi troppo lunghi, non risultano compatibili con le esigenze dell'analisi genomica funzionale. All’interno dei nostri studi, abbiamo quindi tentato lo sviluppo di quattro diverse tecniche basate sul silenziamento genico post-trascrizionale per lo studio di funzione genica in Vitis riparia cv. Gloire de Montpellier. Essendo questa specie resistente a molti patogeni (tra cui Plasmopara viticola) queste tecniche potrebbero consentire di individuare e caratterizzare in modo rapido ed efficace i geni associati alle risposte di difesa della vite contro i patogeni. La prima strategia sviluppata è basata sulla somministrazione diretta di piccoli RNA interferenti (siRNAs) di lunghezza diversa e di RNA a doppio filamento (dsRNA) a foglie di V. riparia e N. benthamiana per valutare la loro efficacia nella repressione dei geni di interesse. In particolare, popolazioni diverse di piccoli RNA (sRNAs) disegnati contro la famiglia genica che codifica per le proteine di patogenesi PR-10 sono stati somministrati a foglie di V. riparia mentre altri, disegnati contro il gene che codifica per l’enzima fitoene desaturasi (PDS), sono stati somministrati a foglie di N. benthamiana. Inoltre, siRNA oppure siRNA sintetici (lunghi 21 nucleotidi e che presentano una 2’-O-metilazione) disegnati contro il gene che codifica per la GFP sono stati somministrati a piante transgeniche di N. benthamiana che esprimono stabilmente il gene marcatore GFP (GFP16C), per valutare la loro efficacia nell’induzione del silenziamento genico. Sfortunatamente, la somministrazione diretta di diverse popolazioni di RNA a piante di V. riparia e N. benthamiana ha fornito risultati contrastanti e non facilmente interpretabili. Saranno necessari ulteriori esperimenti per determinare quali debbano essere le caratteristiche dei siRNA, la loro concentrazione e la strategia adatta di somministrazione, nell’induzione del silenziamento genico post-trascrizionale in pianta. Lo scopo del secondo approccio è di verificare se il segnale di silenziamento è in grado di diffondere sistemicamente dalle radici di vite trasformate con A. rhizogenes e causare il silenziamento del gene in esame nelle parti aeree della pianta. La diffusione sistemica del segnale di silenziamento agli apici non transgenici di piante trasformate è stata osservata in v Lotus japonica, Medicago truncatula e Arabidopsis thaliana sebbene il silenziamento osservato nelle foglie non sia stato completo. Radici trasformate di Vitis vinifera sono state ottenute ma non sono ancora stati effettuati esperimenti volti a delucidare se la diffusione sistemica del segnale di silenziamento avvenga anche in vite. Nei nostri studi, foglie, talee e plantule di Vitis riparia sono state infettate con A. rhizogenes ARqua1 trasformato con il vettore pRedRoot che contiene un costrutto “hairpin” per il silenziamento del gene codificante la PDS di vite per valutare l’insorgenza di eventuali alterazioni fenotipiche causate dall’induzione di un meccanismo di silenziamento genico contro questo gene. Il terzo approccio è basato sulla trasformazione transiente di foglie di V. riparia attraverso l’applicazione del vuoto mediato da siringa. Esperimenti preliminari sono stati condotti utilizzando il ceppo EHA105 di A. tumefaciens trasformato con un vettore pBin19 che porta una sequenza “hairpin” contro il gene codificante la PDS di Vitis riparia oppure con un vettore pBin19 vuoto, come controllo negativo. Dopo il trattamento si sono osservati gravi danni meccanici in tutte le foglie infiltrate; per valutare se il danno era correlato al genotipo di vite utilizzato oppure alla tecnica, si sono agroinfiltrate diverse varietà di V. vinifera applicando il vuoto con una siringa oppure con una pompa. Sono stati usati due ceppi diversi di A. tumefaciens (EHA105 and C58C1) trasformati con un vettore pBin61 che porta una cassetta di espressione transiente per il gene GUS. Questo esperimento ha permesso di valutare le condizioni ottimali di trasformazione, la diversa efficienza utilizzando ceppi diversi e il grado di suscettibilità di diverse Vitis. L’ultimo approccio consiste nello sviluppo di un sistema VIGS utilizzando un vettore basato sul GALV (Grapevine algerian latent virus). Infezioni preliminari condotte utilizzando RNA virali prodotti in vitro hanno rivelato che trascritti infettivi di GALV sembrano in grado di infettare e di diffondere sistemicamente in vite. Considerando che la procedura di infezione con trascritti prodotti in vitro non è risultata affidabile per scarsa efficienza di infezione, abbiamo deciso di standardizzare il modo di trasferimento virale alla pianta producendo un vero vettore VIGS che verrà fornito attraverso agroinfezioni. La strategia di manipolazione molecolare per la produzione di un vettore VIGS basato su GALV è stata quindi sviluppata. Tutte queste strategie rappresentano un contributo alle tecniche già disponibili per lo studio genico funzionale di una coltura preziosa come la vite. Inoltre, esse aprono la strada a nuovi possibili studi ed approfondimenti che possono essere svolti in futuro.Establishment of functional genomics in Vitis ssp. is still challenging, due to the lack of reliable high-throughput tools for grapevine transformation. For instance, A. tumefaciens-mediated stable transformation is possible, but it is time consuming and the transformation efficiency is rather low. The aim of this work is the development of four different post-transcriptional gene silencing strategies for functional genomics in Vitis riparia cv. Gloire de Montpellier for future studies on the function of genes putatively related to pathogen resistance, being this species resistant to many pathogens such as Plasmopara viticola. The first strategy is based on the direct administration of small interfering RNAs (siRNAs), different in length, and dsRNAs to V. riparia and N. benthamiana leaves, to test their effectiveness in downregulation of target genes. In particular, different populations of short RNAs (sRNAs) against the PR-10 gene family were supplied to V. riparia leaves while others designed against the phytoene desaturase gene (PDS) were administered to N. benthamiana leaves. In addition, siRNAs or 2’-O-methylated synthetic siRNAs against the Green Fluorescent Protein (GFP) gene were delivered to N. benthamiana plants stably transformed with the GFP marker gene (GFP16C plants), to test their activity in gene silencing induction against this gene. Unfortunately, results obtained by direct delivery of different small RNAs to V. riparia and N. benthamiana plants were controversial and not easily interpretable. More evidences are still needed to determine the role of siRNA characteristics and concentration, and to find the best delivery strategy for the induction of post-transcriptional gene silencing in plants. The second approach aimed to test whether the silencing signal can spread systemically from grapevine hairy roots to the aerial parts of the plant, resulting in the downregulation of the target gene. Some systemic spreading of the silencing signal to the non-transgenic shoots of transformed plants was already observed in Lotus japonica, Medicago truncatula and Arabidopsis thaliana plants, though the silencing in the leaves was not complete. V. vinifera hairy roots have been obtained by A. rhizogenes infection; the ability of the silencing signal to spread to shoots has not been investigated in grape. In our study, V. riparia leaves, cuttings and plantlets were infected with A. rhizogenes ARqua1, transformed with a pRedRoot vector harbouring a marker gene encoding the red fluorescence protein (DsRed1), to find the optimal transformation conditions and to evaluate V. riparia susceptibility. Then, V. riparia infections were carried out using A. rhizogenes strain ARqua1 transformed with a pRedRoot vector containing a 35S hairpin construct against the grapevine PDS gene to evaluate possible plant phenotypic alterations. iii The third approach is based on transient transformation of V. riparia leaves through syringemediated vacuum application. Preliminary experiments were conducted using the A. tumefaciens EHA105 strain transformed either with a pBin19 vector harbouring a hairpin sequence against the Vitis riparia PDS gene (35S:PDShp) or with an empty pBin19 vector, as a negative control. Severe mechanical damage was observed in all infiltrated leaves; to evaluate if this damage was related to the Vitis genotype or to the technique, agroinfiltrations were performed in leaves and plantlets of different V. vinifera cultivars. Two different A. tumefaciens strains (EHA105 and C58C1) transformed with pBin61 vector carrying a GUSi expression cassette (35S:GUSi) were used for transient transformation by syringe or pump-mediated vacuum application. This experiment permitted the evaluation of the optimal transformation conditions, difference in efficiency between the bacterial strains and Vitis susceptibility. The last approach consists in the development of a VIGS system using a GALV (Grapevine algerian latent virus)-based vector. Preliminary infections carried out with in vitro-produced viral RNAs revealed that GALV infective transcripts could infect and spread systemically. Considering that the procedure of infection with in vitro transcripts was not reliable enough due to low infection efficiency we decided to consider the standardization of viral delivery to the plants by agroinfection, producing a true VIGS vector. The molecular manipulation strategy for the production of a GALV-based VIGS vector was then developed. All these four strategies contributed to the pool of available in vivo tools for functional genomics of the valuable grapevine crop. They also opened several exciting research avenues to pursue in the near future

Approcci di \u2018Biologia dei Sistemi\u2019 per la caratterizzazione del processo di appassimento delle uve utilizzate nella produzione di vini di qualit\ue0 nel Veronese. Approcci di \u2018Biologia dei Sistemi\u2019 per la caratterizzazione dei microrganismi coinvolti nel processo di appassimento delle uve utilizzate nella produzione di vini di qualit\ue0 nel Veronese

In questo lavoro si sono valutate le condizioni ambientali che favoriscono lo sviluppo di Botrytis cinerea e si sono ottenute per la prima volta indicazioni sperimentali riguardo ai meccanismi molecolari di colonizzazione del substrato da parte del microrganismo, in forma larvata e non.In this work, the environmental condition for the development of the noble rot were evaluated. Moreover, sperimental indication about the molecular mechanisms of fungal colonization were obtained