Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The importance of small antimicrobial peptides in the innate immune system of plants became increasingly apparent over the past decade. Antimicrobial peptides are unique and diverse molecules that are found in many tissue types in a variety of invertebrate, plant and animal species. Many of these peptides, such as plant defensins, have been found to be ubiquitous throughout the plant kingdom and have been isolated from flowers, leaves, roots, seeds, seedlings, pods, tubers and bark. The growing relevance of antimicrobial peptides (including plant defensins) in research can be largely attributed to their broad-spectrum antifungal activity. This makes them promising potential targets, both as therapeutic agents and for their use in crop protection and disease resistance. The continuing discovery of novel antimicrobial peptides has advanced the development of strategies to overexpress these genes in plants to attempt to enhance the plant’s natural ability to resist pathogenic attack. The first grapevine antifungal peptide, Vv-AMP1, was isolated and characterized and was shown to be tissue specific and developmentally regulated, being expressed only in berries at the onset of berry ripening. The peptide showed strong antifungal activity against a number of plant pathogenic fungi in vitro. In this study, the biological role of the Vv-AMP1 peptide was further investigated, both within its native host (Vitis vinifera) and under in vitro conditions against a panel of grapevine-specific pathogens. As a first step, recombinant production of Vv-AMP1 using an existing bacterial expression system was evaluated and the heterologous production of the Vv-AMP1 peptide improved. Specific optimizations targeting both production and purification of the peptide showed to improve the yield of Vv-AMP1. Steps in the production process targeted for improvement included induction conditions of peptide production by the bacterial culture as well as a number of purification steps, such as lysate preparation, binding conditions, column washing, elution conditions and thrombin protease cleavage. The optimized purification method produced up to 3 mg of pure Vv-AMP1 peptide from 1.6 L of overnight culture. While production was markedly improved, the resultant purified Vv-AMP1 proved biologically inactive and structurally unstable. This is uncharacteristic of the peptide, suggesting that an important aspect necessary for peptide activity, such as folding or the presence of specific co-factors might not be supported in this non-host prokaryotic production system. The study also entailed the characterization and evaluation of the Vv-AMP1 peptide against a panel of grapevine-specific pathogens that are culturable to sporulating cultures using in vitro antifungal assays and microscopy analysis. Vv-AMP1 showed strong inhibitory activity against all pathogens tested, inhibiting the growth of Diplodia seriata and Cylindrocarpon liriodendri by 50% at concentrations between 4.8 μg/ml and 9.6 μg/ml. Phaemoniella chlamydospora and Phomopsis viticola proved particularly sensitive, with IC50 values of 5.5 μg/ml and 4.0 μg/ml respectively. Microscopy analysis of the effect of the Vv-AMP1 peptide on P. viticola showed a severe inhibition on fungal germination and growth. The peptide did not induce morphological changes in fungal hyphae but compromises the fungal membranes, supporting the theory that the peptide induces membrane permeabilization. Functional analysis of a transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was included in this study to provide the opportunity to study the in planta role of the peptide in its native host. The genetic characterization of the putative population included confirming gene integration and copy number through PCR and Southern blot analysis as well as gene expression through northern blot analysis. A confirmed transgenic population was evaluated for improved disease resistance against Botrytis cinerea as a first test organism in an attempt to link the overexpression of the Vv-AMP1 gene to a disease resistance phenotype. Observations of lesion type, average lesion size and further statistical analysis concluded that the transgenic population showed a definite, albeit slight, improved resistance when compared to the untransformed control lines. In conclusion, the study determined that Vv-AMP1 had a strong antifungal action against grapevine-specific pathogenic fungi when tested in vitro. A definite link could be established between the overexpression of Vv-AMP1 and a mild resistance phenotype within its native host plant. The characterized transgenic population is important for further work to evaluate the in planta activity of the peptide against more grapevine pathogens such as the stem pathogens that were proven sensitive and specifically those that cannot be cultured and are obligate pathogens, such as the downy and powdery mildews. / AFRIKAANSE OPSOMMING: Die belang van klein antimikrobiese peptiede in die ingebore immuunstelsel van plante het tydens die afgelope dekade toenemend duidelik geraak. Antimikrobiese peptide is unieke en diverse molekules wat in verskeie weefseltipes in ‘n verskeidenheid van invertebraat-, plant- en dierspesies gevind word. Baie van hierdie peptiede, soos bv. “plant defensins”, word bevind om alomteenwoordig in die plantryk te wees en is reeds geïsoleer vanuit blomme, blare, wortels, sade, saailinge, peule, knolle en bas. Die toenemende belang van antimikrobiese peptiede (insluitend “plant defensins”) in navorsing kan grootliks toegeskryf word aan hul breë-spektrum antifungiese aktiwiteit. Hierdie eienskap maak hul belowende potensiële teikens, beide as terapeutiese middels asook vir gebruik in gewasbeskerming en siekteweerstand. Die voortdurende ontdekking van nuwe antimikrobiese peptiede bevorder tans die ontwikkeling van strategieë om hierdie gene in plante uit te druk in ‘n poging om die plant se natuurlike vermoeë om patogeniese aanval teen te staan te verbeter. Die eerste wingerd antifungale peptied, Vv-AMP1, is geïsoleer en gekarakteriseer as ‘n ontwikkelings-gereguleerde peptied wat slegs uitgedruk word in korrels, tydens die aanvang van bessie rypwording. Die peptied het tydens in vitro toetse sterk antifungale aktiwiteit getoon teen ‘n verskeidenheid plant-patogeniese swamme. In hierdie studie word die biologiese rol van die Vv-AMP1 peptied verder ondersoek, beide binne sy natuurlike gasheerplant, (Vitis vinifera) asook onder in vitro kondisies teen ‘n paneel van wingerd-spesifieke patogene. As ‘n beginpunt is rekombinante produksie van Vv-AMP1 met behulp van ‘n bakteriële ekspressie sisteem evalueer en die hetereloë produksie van die Vv-AMP1 peptied stelselmatig verbeter. Spesifieke optimerings het gefokus op beide die produksie en suiwering van die peptied en het die algehele opbrengs van Vv-AMP1 verhoog. Spesifieke stappe wat in die produksieproses vir verbetering geteiken is sluit beide induksietoestande van peptiedproduksie deur die bakteriële kultuur in sowel as ‘n aantal suiweringsstappe, soos lisaatvoorbereiding, bindingskondisies, kolom wasstappe, eluasie kondisies en “thrombin” protease snyding in. Die optimale suiweringsmetode het tot 3 mg suiwer Vv-AMP1 peptied opgelewer vanaf ‘n 1.6 L oornag bakteriële kultuur. Hoewel die produksie van die peptide noemenswaardig verbeter is, was die gesuiwerde Vv-AMP1 beide onaktief en struktureel onstabiel. Dit is buitengewoon vir hierdie peptied, wat daarop dui dat belangrike aspekte benodig vir antifungiese aktiwiteit, soos korrekte vou of die teenwoordigheid van spesifieke kofaktore, moontlik ontbreek in hierdie nie-gasheer prokariotiese produksiesisteem. Die studie het ook die karakterisering en evaluering van die Vv-AMP1 peptied teen 'n paneel van wingerd-spesifieke patogene wat kultureerbaar is en sporuleer, insluitend in vitro antifungale toetse en mikroskopiese analise, behels. Vv-AMP1 toon sterk inhiberende aktiwiteit teen alle patogene getoets. Dit inhibeer die groei van Diplodia seriata en Cylindrocarpon liriodendri met 50% teen konsentrasies tussen 4.8 μg/ml en 9.6 μg/ml. Phaemoniella chlamydospora en Phomopsis viticola was besonders sensitief, met IC50 waardes van 5.5 μg/ml en 4.0 μg/ml, onderskeidelik. Mikroskopiese analise van die effek van die Vv-AMP1 peptied op P. viticola het 'n ernstige inhibisie op swam ontkieming en groei aangedui. Die peptied het geen morfologiese veranderinge in swam hifes veroorsaak nie maar het wel die swam membraan beskadig. Hierdie bevinding ondersteun die teorie dat die peptied membraan permeabilisasie induseer. Funksionele analise van ‘n transgeniese V. vinifera (cv. Sultana) populasie wat die Vv-AMP1 geen ooruitdruk is by die studie ingesluit om ‘n geleentheid te bied om die in planta rol van die peptide binne sy natuurlike gasheerplant te bestudeer. Die genetiese karakterisering van die vermeende transgeniese bevolking het die bevestiging van beide geenintegrasie en kopiegetal deur PKR en Southern-klad analise ingesluit, sowel as geenuitdrukking d.m.v. noordelike-klad analise. ‘n Bevestigde transgeniese bevolking is evalueer vir potensiële verbeterde weerstand (in vergelyking met die wilde tipe) deur infeksie met Botrytis cinerea as ‘n eerste toetsorganisme in ‘n poging om ‘n weerstandbiedende fenotipe met die ooruitdrukking van Vv-AMP1 te assosieer. Waarnemings van letsel tipe, letsel grootte en verdere statistiese analise het tot die gevolgtrekking gelei dat die transgeniese bevolking ‘n definitiewe (dog geringe) verbeterde weerstand toon in vergelyking met die ongetransformeerde lyne. Ten slotte bepaal die studie dat Vv-AMP1 ‘n sterk antifungale effek teen wingerdspesifieke patogene toon tydens in vitro toetse. ‘n Definitiewe korrelasie is vasgestel tussen die ooruitdrukking van Vv-AMP1 in wingerd en ‘n weerstandsfenotipe in die transgeniese bevolking. Die gekarakteriseerde transgeniese bevolking is uiteraard belangrik vir toekomstige werk om die in planta aktiwiteit van die peptied te evalueer teen verdere wingerdpatogene soos bv. die stampatogene wat sensitief getoets het teen die peptide, asook patogene wat nie kultureerbaar is nie, insluitend verpligte patogene soos dons- en poeierskimmel.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The latest strategies in the protection of crops against microbial pathogens are rooted in harnessing the natural, highly complex defense mechanisms of plants through genetic engineering to ultimately reduce the application of chemical pesticides. This approach relies on an in-depth understanding of plant-pathogen interactions to develop reasonable strategies for plant improvement. Among the highly specialized defense mechanisms in the plant’s arsenal against pathogen attack, is the de novo production of proteinaceous antimicrobial peptides (AMPs) as part of the plant’s innate immunity. These AMPs are small, cysteine-rich peptides such as plant defensins that are known for their broad-spectrum of antifungal activity. These plant defensin peptides have been found to be present in most, if not all plant species and the defensin encoding genes are over-represented in plant genomes. Most of these defensins are generally the products of single genes, allowing the plant to deliver these molecules relatively rapidly and with minimal energetic expense to the plant. These factors contribute to establishing AMPs as excellent candidates for genetic engineering strategies in the pursuit of alternative crop protection mechanisms. The first antimicrobial peptide identified and isolated from grapevine, Vv-AMP1, was found to be developmentally regulated and exclusively expressed in berries from the onset of ripening. Recombinantly produced Vv-AMP1 showed strong antifungal activity against a wide range of plant pathogenic fungi at remarkably low peptide concentrations in vitro, however, no in planta defense phenotype could thus far be linked to this peptide. In this study, the antifungal activity of Vv-AMP1 constitutively overexpressed in its native host (Vitis vinifera) was evaluated against grapevine-specific necrotrophic and biotrophic fungi. Firstly, a hardened-off genetically characterised transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was generated and morphologically characterized. In order to evaluate the in planta functionality of Vv-AMP1 overexpressed in grapevine, this confirmed transgenic population was subjected to antifungal assays with the necrotrophic fungus, B. cinerea and the biotrophic powdery mildew fungus, Erysiphe necator. For the purpose of infection assays with a biotrophic fungus, a method for the cultivation and infection with E. necator was optimized to generate a reproducible pathosystem for this fungus on grapevine. Detached leaf assays according to the optimized method with E. necator revealed programmed cell death (PCD) associated resistance linked to overexpression of Vv-AMP1 that can be compared to that of the highly resistant grapevine species, Muscadinia rotundifolia. Contrastingly, whole-plant infection assays with B. cinerea revealed that Vv-AMP1 overexpression does not confer V. vinifera with elevated resistance against this necrotrophic fungus. An in silico analysis of the transcription of defensin-like (DEFL) genes previously identified in grapevine was included in this study. This analysis revealed putative co-expression of these DEFL genes and other genes in the grapevine genome driven by either tissue- or cultivar specific regulation or the plant’s response to biotic and abiotic stress stimuli. In conclusion, this study contributed to our knowledge regarding Vv-AMP1 and revealed an in planta defense phenotype for this defensin in grapevine. In silico analysis of the DEFL genes in grapevine further revealed conditions driving expression of these genes allowing for inferences to be made regarding the possible biological functions of DEFL peptides in grapevine. / AFRIKAANSE OPSOMMING: Die nuutste strategieë wat deel vorm van die beskerming van plant gewasse teen mikrobiese patogene het hul oorsprong in die inspanning van die natuurlike, hoogs gekompliseerde verdedigingsmeganismes van die plant deur middel van genetiese enginieurswese ten einde die gebruik van chemiese plaagdoders te verlaag. Hierdie benadering maak staat op ‘n in-diepte begrip van plant-patogeen interaksies om verstandige strategieë vir plantverbetering te kan ontwikkel. Van hierdie hoogs-gespesialiseerde verdedigingsmeganismses in die plant se arsenaal teen patogeen aanvalle sluit die de novo produksie van proteinagtige antimikrobiese peptiede (AMPs) in as deel van die plant se ingebore immuunstelsel. Hierdie AMPs is klein, sisteïen-ryke peptiede soos die plant “defensins” en is bekend vir hul breë-spektrum antifungiese aktiwiteit. Hierdie plant defensinpeptiede word aangetref in meeste, indien nie alle plant spesies nie en die defensin koderende gene word oor-verteenwoordig in plant genome. Meeste van hierdie defensins is gewoonlik die produkte van enkele gene wat die plant in staat stel om hierdie molekules relatief spoedig en met minimale energie verbruik in die plant te vorm. Hierdie faktore dra by tot die vestiging van AMPs as uitstekende kandidate vir genetiese ingenieursstrategieë as deel van die strewe na alternatiewe gewasbeskermingsmeganismes. Die eerste antimikrobiese peptied wat geïdentifiseer en geïsoleer is uit wingerd, Vv-AMP1, word beheer deur die ontwikkelingsstadium en word eksklusief uitgedruk in korrels vanaf die aanvang van rypwording. Rekombinant-geproduseerde Vv-AMP1 het sterk antifungiese aktiwiteit getoon teen ‘n wye reeks plantpatogeniese swamme teen merkwaardige lae peptied konsentrasies in vitro, alhoewel geen in planta verdedigingsfenotipe tot dusver gekoppel kon word aan hierdie peptied nie. In hierdie studie was die antifungiese aktiwiteit van Vv-AMP1 wat ooruitgedruk is in sy natuurlike gasheerplant (Vitis vinifera) ge-evalueer teen wingerd-spesifieke nekrotrofiese- en biotrofiese swamme. Eerstens is ‘n afgeharde geneties-gekarakteriseerde transgeniese V. vinifera (cv. Sultana) populasie wat Vv-AMP1 ooruitdruk gegenereer en morfologies gekarakteriseer. Om die in planta funksionaliteit van Vv-AMP1 ooruitgedruk in wingerd te evalueer is hierdie bevestigde transgeniese populasie blootgestel aan antifungiese toetse met die nekrotrofiese swam, B. cinerea en die biotrofiese swam, Erysiphe necator. Vir die doel om infeksiestudies uit te voer met ‘n biotrofiese swam is ‘n metode geoptimiseer vir die kweek en infeksies met E. necator wat gelei het tot ‘n herhaalbare patosisteem vir hierdie swam op wingerd. Blaarstudies, volgens die pas-verbeterde metode vir E. necator infeksies het ‘n geprogrammeerde seldood-geassosieërde weerstand, gekoppel aan die ooruitdrukking van Vv-AMP1 onthul, wat vergelyk kan word met dié van die hoogs-weerstandige wingerdspesie, Muscadinia rotundifolia. Hierteenoor het heel-plant infeksie studies met B. cinerea onthul dat Vv-AMP1 ooruitdrukking geen verhoogde weerstand teen dié nekrotrofiese swam aan V. vinifera bied nie. ‘n In silico analise van die transkripsie van defensin-agtige (DEFL) gene wat vroeër in wingerd geïdentifiseer is, is by hierdie studie ingesluit. Hierdie analise het vermeende gesamentlike uitdrukking van hierdie DEFL gene en ander gene in die wingerd genoom onthul wat aangedryf word deur weefsel- of kultivar-spesifieke regulering of die plant se reaksie tot biotiese en abiotiese stress stimuli. Ten slotte, hierdie resultate het bygedra tot ons kennis in verband met Vv-AMP1 en het ‘n in planta verdedigingsfenotipe vir hierdie defensin in wingerd onthul. In silico analiese van die DEFL gene in wingerd het verder toestande onthul wat die uitdrukking van hierdie gene aandryf wat ons toelaat om aannames te maak ten opsigte van die moontlike biologiese funksies van DEFL peptiede in wingerd en ondersteun die opstel en toets van hipoteses vir die rol en megansimes van aksie van die wingerd defensin familie.

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is a very important agricultural commodity that needs to be protected. To achieve this several in vivo tools are needed for the study of this crop and the pathogens that infect it. Recently the grapevine genome has been sequenced and the next important step will be gene annotation and function using these in vivo tools. In this study the use of Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, as transient expression and VIGS vector for heterologous protein expression and functional genomics in Nicotiana benthamiana and V. vinifera were evaluated. Full-length genomic sequences of three South African variants of the virus (GTR1-1, GTG11-1 and GTR1-2) were generated and used in a molecular sequence comparison study. Results confirmed the separation of GVA variants into three groups, with group III (mild variants) being the most distantly related. It showed the high molecular heterogeneity of the virus and that ORF 2 was the most diverse. The GVA variants GTG11-1, GTR1-2 and GTR1-1 were placed in molecular groups I, II and III respectively. A collaboration study investigating the molecular divergence of GVA variants linked to Shiraz disease (SD), described two interesting GVA variants of group II, namely GTR1-2 and P163-M5 (Goszczynski et al., 2008). The group II variants were found to be closely linked to the expression of SD. GTR1-2 was isolated from a susceptible grapevine plant that never showed SD symptoms (Goszczynski 2007). The P163-M5 variant that resulted in exceedingly severe symptoms in N. benthamiana and is that used as SD positive control by the grapevine industry, was found to contain a 119 nt insert within the native ORF2. Comparative analysis performed on the complete nt and aa sequences of group II GVA variants suggested that the components in the GVA genome that cause pathogenicity in V. vinifera are more complex (or different) to those that cause pathogenicity in N. benthamiana. The three South African variants (GTR1-1, GTG11-1 and GTR1-2) were assembled into fulllength cDNA clones under control of CaMV 35S promoters. After several strategies were attempted, including a population cloning strategy for GTR1-2, none of the clones generated were able to replicate in N. benthamiana plants. A single amino acid substitution at position 13 (Tyr/Y Cys/C) in ORF 5 of the GTR1-2 cDNA clone was shown to abolish or reduce replication of the virus to below a detectable level. Two infectious clones of Israeli variants of GVA (T7-GVA-GR5 and T7-GVA118, obtained from M. Mawassi) were brought under control of a CaMV 35S promoter (35S-GVA-GR5 and 35S-GVA118). Both clones were infectious, able to replicate, move systemically and induce typical GVA symptoms after agroinfiltration in N. benthamiana. These Israeli clones served as backbone for further experiments in characterisation of transient expression and VIGS vectors. The use of GVA as gene insertion vector (35S-GVA118) and gene exchange vector (35S-GVA-GR5- ORF2+sgMP) in N. benthamiana and V. vinifera was compared. The gene insertion vector, 35S-GVA118 was based on the full-length GVA genome. The gene exchange vector, 35SGVA- GR5- ORF2+sgMP, was constructed in this study by elimination of ORF 2 and insertion of a sgMP and unique restriction sites to facilitate transgene insertion. In N. benthamiana both vectors showed similar GUS expression levels and photobleaching symptoms upon virus-induced NbPDS silencing. In V. vinifera limited GUS expression levels and VIGS photobleaching symptoms were observed for the gene insertion vector, 35SGVA118. No GUS expression was observed for the gene exchange vector 35S-GVA-GR5- ORF2+sgMP in this host. As for silencing, one plant, agroinfiltrated with 35S-GVA-GR5- ORF2-VvPDS+sgMP, developed photobleaching symptoms in 3 systemic infected leaves after 4 months. This study showed that GVA can be used as gene insertion and gene exchange vector for expression and VIGS in N. benthamiana, but in grapevine its use is limited to expression and silencing of genes in the phloem tissue. It is also the first report that ORF 2 of GVA is not needed for long distance movement in grapevine. To investigate the possible role of the P163-M5 119 nt insertion and the GVA ORF 2 (of unknown function), in expression of symptoms in plants, ORF 2 of a 35S-GVA-GR5 cDNA clone was removed and subsequently substituted by the corresponding ORFs of four South African GVA variants. Upon agro-infiltration into N. benthamiana leaves, all chimaeric GVA constructs were able to move systemically through the plant. At this stage no correlation could be found between severity of symptoms, the presence of the P163-M5 insert and the specific GVA ORF 2 present in the chimaeras, indicating that other factors in the viral genome or the host plant probably play a crucial role. This study contributed to the pool of available in vivo tools for study and improvement of the valuable grapevine crop. It also opened several exciting research avenues to pursue in the near future. / AFRIKAANSE OPSOMMING: Wingerd (Vitis vinifera L.) is ‘n baie belangrike landboukundige gewas wat beskerm moet word. Om die rede word verskeie in vivo gereedskap vir die bestudering van die wingerdplant, en die patogene wat dit infekteer benodig. Die wingerd genoom se volgorde is bepaal en dus is die volgende logiese stap om die gene te annoteer en funksie daaraan toe te skryf. In hierdie studie is die gebruik van Grapevine virus A (GVA), genus Vitivirus, familie Flexiviridae, as tydelike uitdrukking- en virus-geinduseerde geenuitdowingsvektor vir heteroloë proteïen uitdrukking en funksionele genoomstudies in Nicotiana benthamiana en V. Vinifera getoets. Vollengte genoomvolgordes van drie Suid-Afrikaanse variante van die virus (GTR1-1, GTG11-1 en GTR1-2) is gegenereer en in ‘n molekulêre volgorde vergelyking studie gebruik. Resultate het die verdeling van GVA variante in drie groepe, waar groep III die verste verwant is, bevestig. Dit het ook gewys dat die virus ‘n baie hoë molekulêre heterogeniteit het en dat oopleesraam 2 (ORF 2) die mees divers is. ‘n Samewerking studie waar die molekulêre diversiteit van GVA variante, gekoppel aan Shiraz siekte (SD), ondersoek is, is twee interessante variante van groep II beskryf, naamlik GTR1-2 en P163-M5 (Goszczynski et al., 2008). Groep II variante is vooraf gevind om nou verwant te wees aan die ontwikkeling van SD in wingerd. Die GTR1-2 variant is uit ’n vatbare wingerd plant, wat nooit SD-simptome vertoon het nie, geïsoleer (Goszczynski et al., 2007). In die ORF 2 van die P163-M5 variant, wat simptome van die ergste graad in N. benthamiana geïnduseer het, en ook deur die industrie as betroubare SD-positiewe kontrole gebruik word, is ’n 119 nt invoeging gevind. Die vergelykende analise wat uitgevoer is, het daarop gedui dat die determinante van patogenisiteit in die GVA genoom moontlik meer kompleks kan wees in V. vinifera as in N. benthamiana. Die drie Suid-Afrikaanse variante (GTR1-1, GTG11-1 en GTR1-2) is in afsonderlike vollengte cDNA klone, onder beheer van CaMV 35S promotors, aanmekaargesit. Nadat verskeie kloneringstrategieë, insluitend ’n populasie kloneringstrategie vir die GTR1-2 kloon, gebruik is, het geen een van die cDNA klone die vermoë besit om in N. benthamiana te repliseer nie. ’n Enkele aminosuur substitusie in posisie 13 (Tyr/Y Cys/C) in ORF 5 van die GTR1-2 kloon, het die replisering van die virus tot laer as ’n opspoorbare vlak verlaag. Twee infektiewe klone van Israeliese GVA variante (T7-GVAGR5 en T7-GVA118, verkry van M. Mawassi) is onder beheer van ‘n CaMV 35S promotor geplaas (35S-GVA-GR5 and 35S-GVA118). Beide klone het na agro-infiltrasie in N. benthamiana plante gerepliseer, sistemies beweeg en tipiese GVA simptome geinduseer. Hierdie twee klone het as raamwerk gedien vir verdere eksperimente in karakterisering van tydelike uitdrukkings- en VIGS vektore. Die gebruik van GVA as geen-insvoegingsvektor (35S-GVA118) en geen-vervangingsvektor (35S-GVA-GR5- ORF2+sgMP) is in N. benthamiana en V. vinifera vergelyk. Die geen-invoegingsvektor 35S-GVA118, was op die vollengte GVA genoom gebasseer. Die geen-vervangingsvektor 35S-GVA-GR5- ORF2+sgMP, was in hierdie studie gekonstrueer. Dit is gemaak eerstens deur eliminasie van ORF 2 in die 35S-GVA-GR5 kloon, en tweedens deur die invoeging van ’n subgenomiese promotor van die beweginsproteïen (sgMP) en unieke beperkings-ensiemsetels om klonering van transgene te fasiliteer. Beide vektore het in N. benthamiana vergelykbare GUS uitdrukkingsvlakke en fotobleikende simptome getoon na virus-geinduseerde NbPDS uitdowing. In V. Vinifera is beperkte GUS uitdrukkingsvlakke en VIGS fotobleikende simptome opgemerk met die geen-invoegingsvektor, 35S-GVA118. Geen GUS uitdrukking is in hierdie gasheerplant met die geen-vervangingsvektor opgemerk nie. Slegs een wingerdplant het fotobleikende simptome, na 4 maande in 3 sistemies geïnfekteerde blare gewys, na agroinfiltrasie van die 35S-GVA-GR5- ORF2-VvPDS+sgMP konstruk. Hierdie studie het bevestig dat GVA as geen-invoeging en geen-vervangingsvektor, vir heteroloë proteïenuitdrukking en VIGS, in N. benthamiana gebruik kan word, maar dit blyk of die gebruik daarvan in wingerd meer tot die floeëm weefsel beperk is. Hierdie studie wys vir die eerste keer dat ORF 2 nie nodig is vir langafstand beweging van die virus in wingerd nie. Om die moontlike rol van die P163-M5 119 nt invoeging en die GVA ORF 2 (met onbekende funksie), in die uitdrukking van simptome in plante te ondersoek, is ORF 2 van die 35SGVA- GR5 cDNA kloon verwyder en daaropvolgens vervang met die ooreenstemmende ORFs van vier Suid-Afrikaanse GVA variante. Na agro-infiltrasie in N. benthamiana blare, het al die chimeras die vermoë gehad om te repliseer, sistemies te beweeg en simptome te induseer. Geen korrelasie kon gevind word tussen die graad van simptome, die teenwoordigheid van die P163-M5 insersie en die spesifieke GVA ORF 2 teenwoordig in die chimeras nie, wat dus daarop dui dat ander faktore in die virusgenoom of die gasheerplant `n moontlike belangrike rol kan speel. Hierdie studie het bygedrae tot die beskikbare poel van in vivo gereedskap vir die bestudering en verbetering van die kosbare wingerdgewas. Dit het ook talle interessante navorsingsgeleenthede oopgemaak om in die nabye toekoms te betree.

Thesis (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: Grapevine leafroll-associated virus-3 is one of ten members of the C/osteroviridae that are known to infect grapevine. Nine of these viruses are associated with grapevine leafroll disease, of which GLRaV-1 and GLRaV-3 are the most important and widespread. Members of the C/osteroviridae are unique amongst the viruses, as it is the only known family whose members encode a heat shock protein 70 kOa homolog (Hsp70h). The Hsp70h is a movement protein (MP) that is required for the active translocation of the virion structure through the plasmodesmata into adjacent cells. Broad-spectrum resistance to unrelated viruses can be obtained by a pathogen-derived resistance (POR) strategy that is based on the expression of a dysfunctional MP in plants. The Hsp70h has two distinct domains. The N-terminal two thirds of the protein is an ATPase domain and shares high homology with the ATPase domains of all Hsp70h proteins from the C/osteroviridae and Hsp70 proteins from the prokaryote and eukaryote kingdoms. Conserved amino acids are found in the ATPase domain and are required for the positioning of the ATP at the catalytic site for ATP hydrolysis. The C-terminal domain is variable and the function of this domain in the Closteroviridae is not known. In prokaryote and eukaryote Hsp70 proteins, the C-terminal domain is required for protein-protein interactions. The American NY-1 isolate of GLRaV-3 has been sequenced and POR strategies have been attempted with the coat protein, divergent coat protein and replicase genes, but not with a dysfunctional form of the hsp70h gene. In this study, double-stranded RNA was isolated from a commercial vineyard with unknown virus status, but with distinct grapevine leafroll symptoms, and from two grapevine sources of known virus status, one with mild and one with severe symptoms. The GLRaV-3 hsp70h gene was amplified by RT-PCR from the dsRNA and the gene sequence was analysed. The hsp70h gene from the three virus sources contained more than 94% nucleotide sequence homology to the NY-1 isolate and the conserved amino acids required for ATPase activity were present. The hsp70h gene isolated from GLRaV-3 from a commercial Stellenbosch vineyard showing clear leafroll symptoms was selected for further work and was subjected to site-directed mutagenesis to engineer four point mutations in the gene. These four mutations resulted in the substitution of Asn for Asp", Gly for Thr1O, Lys for Glu 174 and Asn for Asp 197. The wild type (WT) and mutated (Mut) forms of the hsp 70h genes were cloned into a bacterial expression vector. Expression of both the WT- and Mut-Hsp proteins was achieved, and the protein was expressed in the insoluble inclusion bodies. All attempts to refold and isolate active proteins from the inclusion bodies were unsuccessful. Attempts to increase the concentration of soluble protein within the expressing bacteria were unsuccessful. Due to the lack of active protein, biochemical tests on the ATPase activity of the WT- and Mut-Hsp proteins could not be conducted. The wt- and mut-hsp genes were cloned into a plant expression vector for transformation into tobacco plants. These transformations were successful and gave rise to 22 Km' and 18 Km' plants from the WT- and Mut-Hsp constructs respectively. Two plant lines, M5 and M10, transformed with the mut-hsp transgene construct, appeared to have a high level of resistance to the challenging potato X potexvirus, whereas all the other tested plants were susceptible to the challenging virus. It was thus shown that a dysfunctional form of the GLRaV-3 Hsp70h could provide resistance to an unrelated virus in tobacco. / AFRIKAANSE OPSOMMING: Wingerdrolblaar-geassosieerde virus 3 (GLRaV-3) is een van 10 lede van die Closteroviridae wat wingerd kan infekteer. Nege van die virusse is met wingerdrolblaar geassosieer. Die GLRaV-1 en GLRaV-3 is die belangrikste en mees wyd verspreide lede van die rolblaar-geassosieerde Closteroviridae. Lede van die Closteroviridae is uniek in die opsig dat die virusse vir 'n 70 kDa-homoloë hitteresponsproteïen (Hsp70h) kodeer. Die Hsp70 is 'n bewegingsproteïen (MP) wat belangrik is vir die translokasie van die virus deur die plasmodesmata na die naasliggende sel. Breë-spektrum weerstand teen onverwante virusse kan behaal word deur 'n patogeen-afgeleide weerstandstrategie (POR), wat op die uitdrukking van 'n disfunksionele MP wat in plante uitgedruk word, gebaseer is. Die Hsp70hproteïen het twee gebiede. Die N-terminale gebied is In ATPase-gebied en toon hoë homologie met ander ATPase-gebiede van Hsp70h-proteïene van die Closteroviridae, asook die prokariotiese en eukariotiese koninkryke. Gekonserveerde aminosure wat belangrik is vir die posisionering van ATP in die katalitiese domein vir ATP-hidrolise is in die ATPase-gebied gevind. Die C-terminale gebied is variërend en die funksie van die gebied in die Closteroviridae is onbekend. In prokariotiese en eukariotiese Hsp70h-proteïene is die C-terminale gebied belangrik vir proteïenproteïen interaksies. Die nukleotiedvolgorde van die Amerikaanse NY-1-isolaat van GLRaV-3 is al bepaal en POR-strategieë is ook op die kapsiedproteïen, uiteenlopende kapsiedproteïen en die replikasie-proteïen uitgevoer, maar nog nie op 'n disfunksionele vorm van die Hsp70h-geen nie. In hierdie studie is dubbelstring-RNA (dsRNA) van 'n kommersiële wingerd met onbekende virusstatus wat rolblaarsimptome toon, geïsoleer, asook van twee wingerde met 'n bekende virusstatus, een met ligte en een met strawwe simptome. Die GLRaV-3 hsp70h-geen is met hulp van die polimerasekettingreaksie-metode (PKR) vanaf die dsRNA geamplifiseer en die geen se nukleotiedvolgorde is bepaal. Die hsp 70-gene van drie verskillende wingerde het meer as 94% homologie met die NY-1-isolaat getoon. Die gekonserveerde aminosure wat vir ATPase-aktiwiteit belangrik is, was teenwoordig. Die hsp70h-geen van GLRaV-3, wat uit 'n kommersiële wingerd met duidelike rolblaarsimptome in die Stellenbosch-gebied geïsoleer is, is vir verdere navorsing gekies en dit is aan setel-gerigte mutagenese blootgestelom vier mutasies van die geen te bewerkstellig. Die gevolg van hierdie vier mutasies was die verandering van Asn na Asp", Gly na Thr1o, Lys na Glu174 en Asn na Asp197. Die wilde (WT) en veranderde (Mut) vorms van die hsp-gene is in 'n bakteriese uitdrukkingsvektor gekloneer. Uitdrukking van beide die WT- en die Mut-Hspproteïene is behaal, maar die proteïene was in die onoplosbare fraksie geleë. Pogings om die onoplosbare proteïene te isoleer en in 'n aktiewe oplosbare vorm te verkry, was onsuksesvol. Verdere pogings om die proteïene in die oplosbare fraksie van die bakteriese ekspressiesisteem uit te druk, was ook onsuksesvol. As gevolg van die gebrek aan aktiewe proteïen kon biochemiese toetse nie op die ATPaseaktiwiteit van die WT- en Mut-Hsp proteïne gedoen word nie. Die wt- en mut-hsp-gene is ook in In plantekspressievektor gekloneer vir transformasie in tabakplante. Hierdie transformasies was suksesvol en het aanleiding gegee tot 22 kanamisienbestande (Km') en 18 Km' plante vanaf die WT- en Mut-Hspkonstrukte onderskeidelik. Twee plantlyne, M5 en M10, wat met die mut-hsptransgene getransformeer is, het 'n hoë vlak van weerstand teen die infekterende aartappelvirus X getoon in vergelyking met ander plante wat met die virus geïnfekteer is. Daar is dus bewys gelewer dat 'n disfunksionele vorm van die GLRaV-3 Hsp70h weerstand kan bied teen 'n onverwante virus in tabak.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Grapevine fanleaf virus (GFLV) is one of the oldest, most widespread and devastating viruses infecting grapevine, and occurs globally where Vitis vinifera is grown. In South Africa (SA) GFLV is predominant in the Breede River Valley, one of the highest wine producing regions in SA. To date, only three GFLV isolates have been completely sequenced internationally, and limited sequence information is available for SA GFLV isolates. In this study, the first full-length GFLV genome sequence from a South African isolate, GFLV-SAPCS3, was determined. Full-length sequences were used for phylogenetic analysis and revealed that the SA isolates are separate from other sequenced GFLV isolates. Full-length sequences were also used to investigate putative intra- and interspecies recombination events involving GFLV-SAPCS3 RNA1 and RNA2 between GFLV and Arabis mosaic virus (ArMV) isolates. Using two different recombination analysis software packages, the most notable of the putative recombination events involving GFLV-SAPCS3 indicated that the GFLV-SAPCS3 RNA2 5’ UTR might have evolved from an interspecies recombination event between GFLVF13- type and ArMV Ta-type isolates. The presence of satellite RNAs (satRNA) associated with South African GFLV isolates was also investigated. In a collaborative study (see Chapter 4 for details), more than a 100 GFLV- infected grapevine plants were screened for satRNAs. SatRNAs were present in only two plants, containing isolates GFLV-SACH44 and GFLV-SACH47. The full-length nucleotide sequences of the GFLV-SACH44 genomic RNAs 1 and 2, and the associated satRNA were determined. No significant sequence variation could be detected between the GFLV isolates that had the presence of a satRNA and those that had not. The GFLV-SACH44 RNA2 5’ UTR also had the same conserved sequence that was found in GFLVSAPCS3, which suggests that GFLV-SACH44, like GFLV-SAPCS3, may have arisen from a common ancestor, which may have originated from an interspecies recombination event. The GFLV-SACH44 satRNA was found to be more closely related to the ArMV large satRNA than to the satRNA associated with GFLV-F13. A full-length cDNA clone of GFLV-SACH44 satRNA was constructed and its replication and systemic spread in herbaceous hosts, when mechanically co-inoculated with two GFLV isolates as helper viruses, was demonstrated. Replication of the GFLV-SACH44 satRNA cDNA clone was however abolished when co-inoculated with an ArMV helper virus, even though it is phylogenetically more closely related to ArMV satRNAs. The full-length satRNA clones were modified to be used as vectors for expression and/or silencing of foreign genes, by inserting the green fluorescence protein (GFP) full-length or partial sequences downstream of the open reading frame of the satRNA. These constructs were cloned into a binary vector to allow for agro-infiltration into plants. Full-length cDNA clones of GFLV-SAPCS3 RNA1 and RNA2 were constructed to be used in conjunction with modified GFLV-SACH44 satRNA full-length clones. The full length GFLV-SAPCS3 RNA1 and RNA2 clones were however not infectious in Nicotiana benthamiana after agro-infiltration and therefore the evaluation of the modified satRNA expression and silencing constructs had to be aborted. Attempts to understand this failure revealed that, among other point mutations, four frameshifts had occurred in the RNA1 full-length clone, rendering the transcripts untranslatable, and hence noninfectious. Strategies to correct the mutations are discussed. Once these mutations have been corrected this study can continue in evaluating the use of the satRNA component for expression and silencing analysis. / AFRIKAANSE OPSOMMING: Grapevine fanleaf virus (GFLV) is een van die oudste, mees wydverspreide en mees verwoestende virusse wat wingerd affekteer en word wêreldwyd waar Vitis vinifera verbou word, gevind. In Suid Afrika (SA) kom GFLV veral in die Breederivier vallei, een van die mees produktiewe wyn-produserende areas in SA, voor. Tot dusver is daar net drie GFLV isolate waarvan die volledige nukleïensuurvolgorde internasionaal bepaal is. Die nukleïensuurvolgorde informasie vir SA GFLV isolate is redelik beperk. In hierdie studie was die eerste volledige nukleïensuurvolgorde van ‘n SA GFLV isolaat, GFLVSAPCS3, bepaal. Die volledige nukleïensuurvolgordes was vir filogenetiese analise gebruik, en vermeende intra- en interspesie rekombinasie gebeurtenisse, wat GFLVSAPCS3 RNA1 en RNA2 betrek, tussen GFLV en Arabis mosaic virus (ArMV) isolate was ondersoek. Twee verskillende rekombinasie-analise sagteware programme was gebruik en die noemenswaardigste van die vermeende rekombinasie gebeurtenisse, met betrekking tot GFLV-SAPCS3, het aangedui dat die GFLV-SAPCS3 RNA2 5’ ontransleerde streek (UTR) waarskynlik van ‘n interspesie rekombinasie gebeurtenis tussen ‘n GFLV-F13-tipe en ‘n ArMV-Ta-tipe isolaat ontwikkel het. Die teenwoordigheid van satelliet RNAs (satRNAs), wat met SA GFLV isolate geassosieer is, was ook ondersoek. Meer as ‘n 100 GFLV ge-infekteerde wingerd plante was in ‘n samewerkingsprojek (sien Hoofstuk 4 vir besonderhede) getoets vir die teenwoordigheid van satRNAs. SatRNAs was net in twee plante teenwoordig, in isolate GFLV-SACH44 en GFLV-SACH47. Die vollengte nukleïensuurvolgordes van GFLVSACH44 RNA1, RNA2 en geassosieerde satRNA was bepaal. Geen beduidende volgorde variasie tussen die GFLV isolate wat satRNAs bevat het, en die GFLV isolate sonder satRNA was waargeneem nie. Die GFLV-SACH44 RNA2 5’ UTR het ook die gekonserveerde volgorde, wat in GFLV-SAPCS3 teenwoordig was, gehad en dit dui daarop dat GFLV-SACH44, soos GFLV-SAPCS3, van dieselfde stamvader, wat tydens ‘n vorige rekombinasie gebeurtenis ontstaan het, mag ontwikkel het. Die GFLVSACH44 satRNA was meer naverwant aan die ArMV satRNAs as aan die satRNA, wat met GFLV-F13. ‘n Vollengte cDNA kloon van die GFLV-SACH44 satRNA was ontwikkel en die replisering en sistemiese verspreiding in sagte plante, nadat dit met twee GFLV isolate as helper virusse saam ge-inokuleer was, was gedemonstreer. Replisering van die GFLV-SACH44 satRNA cDNA kloon was egter ontwrig toe dit saam met ‘n ArMV helper virus saam ge-inokuleer was, al is dit filogeneties meer verwant aan ArMV satRNAs. Die vol-lengte satRNA klone was gemodifiseer om as vektore vir uitdrukking en/of uitdowing van transgene te dien, deur om vol-lengte of gedeeltelike groen fluoressensie proteïen (GFP) nukleïensuurvolgordes aan die einde van die satRNA leesraam te koppel. Hierdie konstrukte was in ‘n binêre vektor gekloon om agroinfiltrasie in plante toe te laat. Vol-lengte cDNA klone van GFLV-SAPCS3 RNA1 en RNA2 was ontwikkel om in samewerking met die gemodifiseerde GFLV-SACH44 satRNA konstrukte gebruik te word. Die vol-lengte GFLV-SAPCS3 RNA1 en RNA2 klone het egter nie in Nicotiana benthamiana gerepliseer na agro-infiltrasie nie, daarom was die evaluasie van die gemodifiseerde satRNA konstrukte gestaak. Pogings om die mislukking te verstaan, het daarop gewys dat, behalwe punt mutasies, vier leesraam versteurings in die RNA1 vollengte kloon voorgekom het, wat ontransleerbare transkripte, en dus nie-repliserende konstrukte tot gevolg gehad het. Strategieë om die mutasies te korrigeer is bespreek. Sodra die mutasies gekorrigeer is, kan die studie voortgaan om te evalueer of die satRNA komponent vir uitdrukking en uitdowing analise gebruik kan word.

La surface des vignobles mondiaux représente environ 7,9 millions ha, ce qui correspond à une production annuelle de 67 millions de tonnes de baies. La production mondiale annuelle de vins est de l’ordre de 300 millions hl/an. La surface du vignoble français est de 843 000 ha. La viticulture moderne doit affronter trois défis majeurs interdépendants : réduire l’utilisation des produits phytosanitaires, s'adapter au changement climatique, maîtriser la qualité et la typicité pour garder ou conquérir de nouveaux marchés.En 2007, la vigne est devenue la première espèce fruitière pérenne dont le génome a été séquencé. Cette avancée scientifique ouvre de nombreuses perspectives en termes de génomique fonctionnelle (ensemble de méthodes permettant de caractériser la fonction des gènes) et de biologie intégrative (ensemble de méthodes visant à appréhender le fonctionnement global de la plante et ses réponses à l’environnement). Ces perspectives dépendent pour une bonne part de la maîtrise de quantités importantes de données qu’il convient d’organiser et de corréler grâce à des outils informatiques adaptés.Des approches fonctionnelles concernant des gènes candidats et des approches transcriptomiques à haut débit ont permis d’identifier certains gènes ou certaines familles de gènes impliqués dans le développement et la maturation de la baie de raisin, mais au moment où cette thèse a débuté, aucun travail de biologie intégrative n’avait été entrepris.Le travail présenté ici, qui décrit l’obtention et l’analyse de métadonnées transcriptomiques et biochimiques portant sur la réponse de la baie à l’environnement radiatif, s’inscrit dans ce contexte. En procédant à un effeuillage partiel après la véraison, nous avons modulé l’exposition des baies au rayonnement solaire. Ceci a permis d’étudier l’influence du rayonnement (baie exposée, non exposée), de la position de la grappe (est, ouest) et de la position de la baie (à l’extérieur ou à l’intérieur de la grappe). Des baies ont été récoltées à 5 moments différents après l’effeuillage et utilisées pour des analyses métabolomiques et transcriptomiques. Leur contenu en sucres, acides organiques, acides aminés, anthocyanes et flavonols a été analysé par des dosages enzymatiques et par chromatographie liquide à haute performance). L’expression des gènes a été étudiée avec des microarrays représentatifs de l’ensemble du génome de la vigne (29600 gènes) pour les conditions présentant les différences métaboliques les plus marquées (baies exposées, situées à l’ouest et à l’extérieur de la grappe vs baies non exposées, situées à l’est et à l’intérieur de la grappe). Des analyses statistiques et corrélatives ont été conduites pour (a) déterminer les métabolites qui répondent au traitement et les facteurs qui les influencent (b) déterminer les gènes qui répondent aux traitements et ceux qui semblent co-régulés (c) préciser les réseaux de gènes et de métabolites qui semblent reliés. L’effeuillage n’affecte pas la teneur en sucres ou en acide tartrique des baies, il affecte peu les acides aminés, mais il augmente la teneur en flavonols et diminue la teneur en acide malique. Il affecte plus particulièrement les gènes associés au stress abiotique, au métabolisme secondaire, au transport et au métabolisme hormonal. Des expériences complémentaires ont permis d’identifier divers gènes spécifiquement associés à la composante thermique de l’exposition au soleil, parmi lequels des gènes codant pour des HSP, des transporteurs ABC, et des enzymes du métabolisme flavonoïdique. Des réseaux reliant des gènes et des métabolites ont pu être construits, qui associent des métabolites secondaires à des gènes de fonctions connues, ou à de nouveaux gènes candidats dont il conviendra d’étudier la fonction précise. / The total surface of vineyards worldwide is about 7.9 millions ha, which corresponds to an annual production of 67 millions tons berries. The annual world production of wines is about 300 millions hl/year. The French wineyard occupies 843 000 ha, among which 481 000 ha are dedicated to high quality wines (VQPRD) and 362 000 ha to table wines. Modern viticulture must deal with three major and related challenges : reduce the use of organic and inorganic phytochemicals, adapt the vineyard to climatic change and control the quality and the typicity in order to keep or gain new markets.In 2007, the grapevine became the first perennial fruit species whose genome was sequenced. This scientific breakthrough opens new pespectives in terms of functional genomics (set of methods allowing to characterize the function of genes) and integrative biology (set of methods allowing to study the global functioning of the plant and its response to the environment). These perspectives mainly depend on our ability to analyze large sets of data with adequate informatic tools.Functional approaches on candidate genes, and high throughput transcriptomic approaches have allowed to identify some genes or some gene families involved in the development and ripening of the grape berry, but when this Ph. D work started, no paper based on integrative biology was published on grapevine. The present work, which describes the collection and analysis of transcriptomic and metabolomic metadata related to the response of the berry to sun exposure. The exposure of the berries to the sun was controlled through a partial defoliation after veraison. This allowed to study the effects of sun exposure (exposed or shaded berries), of the position of the cluster (east, west) and of the anatomical position of the berry (outside or inside the berry). Berries were collected at 5 different time points after defoliation and used for metabolomic and transcriptomic analysis. Their content in sugars, amino acids, organic acids, anthocyanins and flavonols was analyzed by enzymatic assays and high performance liquid chromatography. For the berries whose metabolic content differed the most (exposed, west and outside berries vs shaded, east and inside berries), gene expression was studied with microarrays bearing a set of probes covering the whole genome of grapevine (29600 genes). Correlative and statistical analysis were conducted in order to (a) determine the metabolites that are the most responsive to the treatment, and the most important factors that control them (b) determine the genes that respond to the treatment and seem to be co-regulated (c) to precise the networks of genes and metabolites which seem related. Defoliation does not affect the sugar and tartaric acid contents, hardly affects amino acids, but it increases flavonol content and decreases malic acid content. It affects more specifically genes associated with abiotic stress, secondary metabolism, transport and hormonal metabolism. Additional experiments allowed us to identify genes that are specifically associated with the thermal component of sun exposure, among which genes encoding HSP, ABC transporters, and enzymes of flavonoid metabolism. Networks relating genes and metabolites could be constructed. These networks associate secondary metabolites with genes of known function and new candidate genes for which the function will have to be precised.

This thesis set out to evaluate and compare two communism cation channels at Southland, the company magazine and the grapevine. Data were obtained through a survey in the Summer, 1975, magazine. Following an introduction, explanation of data-collection procedure of the survey, overview of communication in formal and informal organizations, and analysis of the survey data, conclusions were drawn that the magazine is an effective formal communication tool, but that it cannot serve all the formal communication needs of the company, and that expansion of the company's formal communication program is needed. Even so, the formal communication tool, The Southland Family, remains a more effective channel of.communication than the grapevine.

Fitoplasmas são procariotos sem parede celular, pertencentes à classe dos Mollicutes, que habitam os vasos de floema e causam doenças de relevância econômica em uma ampla gama de espécies vegetais. A videira (Vitis sp) é a terceira fruteira mais cultivada no mundo e apresenta elevada importância econômica no Brasil. Aspectos fitossanitários se constituem em fatores limitantes da produção, com destaque para as doenças causadas por fitoplasmas, denominadas de amarelos. Essas doenças estão amplamente disseminadas nos continentes europeu e americano, ocorrendo em tradicionais países produtores. Plantas exibindo sintomas característicos da doença têm sido também observadas em cultivos comerciais localizados em algumas áreas do território brasileiro. Visando investigar este tipo de doença, plantas sintomáticas foram amostradas em parreirais comerciais dos Estados de São Paulo e Paraná e mantidas em casa de vegetação. O presente estudo visou demonstrar a associação entre fitoplasmas e os amarelos, identificar e analisar filogeneticamente os fitoplasmas detectados, demonstrar sua transmissão para plantas sadias e determinar o melhor estádio fenológico da planta para amostragem de tecidos, para confirmação da diagnose. Para isto, durante 45 meses, amostras foram coletadas de plantas doentes envasadas sendo usado o método de PCR, análise de RFLP, sequenciamento das bases nucleotídicas e enxertia de tecidos entre plantas de videira. Os produtos amplificados em PCR evidenciaram a presença de fitoplasmas em todas as plantas portadoras de amarelos. Estes fitoplasmas foram identificados como representantes dos grupos 16SrI-B e 16SrIII, os quais ocorreram em infecções simples ou mistas. Os fitoplasmas apareceram em todas as fases de desenvolvimento da planta, porém com maior freqüência nas amostras coletadas no estádio fenológico correspondente à brotação de ramos e aparecimento de folhas novas. Esta fase se mostrou a mais indicada para as amostragens, visando à detecção de fitoplasmas para fins de diagnose. A transmissão dos fitoplasmas por enxertia foi alta, demonstrando que estes são os agentes causais da doença e que inspeções devem ser adotadas como medida de controle para evitar sua disseminação via material de propagação vegetativa. / Phytoplasmas are wall-less prokaryotes, phloem-limited plant pathogens, belonging to Mollicute class. They are agents of diseases that cause serious damage to a diversity of cultivated especies. Grapevine (Vitis sp) is the third most cultivated fruit crop in the world and it has high economical impact in Brazil. Phytossanitary aspects are important production constraint factors, especially regarding diseases caused by phytoplasmas, which are generally known as grapevine yellows. These diseases are widely spread throughout main producer countries, located in Europe and North America. Plants exhibiting typical symptoms of the disease have also been observed in crops located in some areas of Brazilian territory. In order to investigate this kind of malady, symptomatic plants were sampled from commercial vineyards located in the States of São Paulo and Paraná, Brazil and kept in greenhouse conditions. The present study aimed to: demonstrate the association between phytoplasmas and yellows; identify and analyse phylogenetically the detected phytoplasmas; demonstrate their transmission to healthy plants and determinate optimal phenologycal stage for phytoplasma detection to diagnosis confirmation. Samples were collected from diseased plotted plants grown during 45 months. PCR method, RFLP analyses, sequencing of nucleotide bases from 16S rRNA, and grafting technique were used in this study. Amplified genomic fragments revealed the presence of phytoplasmas in all plants with symptoms of yellows. Those phytoplasmas were identified as representatives of the groups 16SrI-B and 16SrIII, occurring in simple and mixed infections. Phytoplasmas were detected in all phenological stages evaluated, but they occurred with high frequences in material collected during budburst. These stage may be considered as the most indicated for sampling to confirm diagnosis based on symptomatology. Phytoplasmas transmission by grafting of healthy scion grafts onto infected rootstocks was high, demonstrating that they are the disease agents and inspection is a control practice that must be adopted to prevent their dissemination through propagative material.

La vigne (Vitis vinifera) possède un métabolisme secondaire particulièrement riche donnant naissance à une large palette de molécules dont certaines sont impliquées dans les défenses contre les pathogènes et d'autres dans la grande diversité d’arômes qui fait la renommée des vins. L’analyse de la séquence de référence du génome de la vigne a permis de mettre en évidence une remarquable expansion de certaines familles de gènes liés au métabolisme secondaire par rapport aux autres plantes. Dans ce travail, j'ai étudié les familles gènes codant pour les cytochromes P450, dont certains sont impliqués dans la production d’arômes, les gènes codant pour les stilbènes synthases (STS), les endo-β-1,3-glucanases et les gènes de résistance de type NBS impliqués dans les défenses de la vigne. Ma thèse vise à proposer des hypothèses expliquant l’organisation structurale de ces familles de gènes et ainsi à mieux comprendre pourquoi certaines familles présentent une amplification dans le génome de la vigne. Des approches bioinformatiques ont été utilisées afin d’étudier ces différentes familles de gènes. Les gènes cytochromes P450 et gènes R de type NBS ont tout d'abord été annotés de manière manuelle dans le génome de référence de la vigne. L’expression des gènes endo-β-1,3- glucanases, STS et cytochromes P450 a été analysée en utilisant une approche transcriptomique à grande échelle. Pour ce faire, un outil a été développé durant cette thèse pour estimer le niveau d’expression des gènes à partir de données RNA-Seq disponibles dans les banques de données publiques. Parallèlement, des données de reséquençage d’ADN de 56 cépages et espèces de vigne ont été analysées, afin de déterminer les variations structurales de type CNV au sein des familles de gènes à domaine NBS et de gènes STS. Ces différents travaux ont permis de montrer que l’amplification des familles de gènes étudiées n’est pas spécifique du génome de référence mais est retrouvée dans l'ensemble du genre Vitis, mais également de mettre en évidence des variations structurales au sein des différents génomes étudiés. L'analyse de la famille STS a montré que ces gènes sont organisés en blocs de duplication, et que les gènes plus conservés sont aussi les plus exprimés. Nous avons également montré que les gènes à domaine NBS sont organisés en cluster, dont certains sont particulièrement soumis à variation. Ces travaux contribuent à une meilleure connaissance de facteurs de défense efficaces et durables ainsi que des gènes impliqués dans la synthèse d’arômes dans la vigne. Ces connaissances pourront bénéficier aux programmes de création variétale mis en œuvre à l’INRA de Colmar. / Grapevine (Vitis vinifera) has a particularly rich secondary metabolism, giving rise to a wide range of molecules, some of which are involved in defences against pathogens and others in the great diversity of aromas that make wines famous. Analysis of grapevine reference genome has shown a remarkable expansion of certain families of genes linked to secondary metabolism in comparison with the other plants. In this work, I have analysed gene families coding for cytochromes P450, some of them being involved in the production of aromas, genes coding for stilbene synthases (STS), endo-β-1,3-glucanases and NBS type resistance genes involved in grapevine defences. My thesis intends to propose hypothesis to explain the structural organisation of these families and therefore better understand why some of these families are amplified in the grapevine genome. Bioinformatic approaches have been used to study these different genes families. The cytochromes P450 and R genes of NBS type were manually annotated to improve the knowledge of these families of genes. The expression of endo-β-1,3-glucanases, STS and cytochromes P450 genes has been quantified using a large-scale transcriptomic approach. To this purpose, a tool has been developed during this thesis to estimate the level of genes expression from RNA- Seq data available in public databases. In the meantime, DNA resequencing data from 56 cultivars and grapevine species have been analysed to identify structural variations of CNV types within the genes with a NBS domain and the STS genes. These works showed that the amplification of the gene families of interest was not specific to the reference genome but occurred at the scale of the Vitis genus, but also to highlighted structural variations in different genomes. Regarding the STS genes, blocks of duplication and more conserved and expressed genes were identified. For the genes with NBS domain, a clustered organisation has been highlighted with some clusters varying more than others in the studied genotypes. These works contribute to a better knowledge of gene families for efficient and durable defence against pathogens and optimal aromas synthesis in grapevine. This knowledge will benefit to breeding programs currently in progress at INRA Colmar.

Les maladies du bois de la vigne sont responsables de pertes économiques importantes pour la filière viticole. Ces maladies d'origine fongique se manifestent notamment par une dégradation de la partie boisée du matériel végétal et par l'apparition erratique de symptômes caractéristiques sur la partie foliaire. Cette thèse est dédiée à l'étude de ces maladies (principalement l'esca) à l'aide de deux capteurs imageurs en proxidétection.La question de la détection des symptômes visibles est tout d'abord abordée à l'aide d'un capteur couleur RVB permettant d'acquérir une image par pied de manière automatique ou semi-automatique. La reconnaissance des symptômes est abordée en deux étapes, d'abord en considérant la classification à l'échelle de la feuille puis la détection à l'échelle du pied. La particularité de cette étude est l'inclusion de facteurs confondants dans le problème de classification, tirant partie de l'information de forme des symptômes de l'esca pour les différencier d'autres troubles et maladies. Dans ce but, une comparaison entre approches SIFT et approches transfer learning récentes est alors conduite. Les résultats nous poussent alors à considérer une architecture deep learning simple (RetinaNet) pour la détection des symptômes sur les images, permettant d'estimer un niveau d'atteinte pour chaque pied.Le second capteur utilisé, une caméra hyperspectrale couvrant le spectre de 500 nm à 1300 nm, tente de répondre à une problématique plus expérimentale, à savoir le comportement spectral des pieds atteints par la maladie pouvant déboucher sur une détection précoce des pieds malades mais sans symptômes foliaires. Un protocole expérimental et une base de données de spectres sont alors constitués pour l'occasion. Les méthodes de réduction de la dimensionnalité permettent d'exploiter l'information hyperspectrale voire d'isoler les longueurs d'onde associées à chacune des deux classes. Les données ne permettent cependant pas, pour la plage de longueur d'onde mesurée et dans les conditions d'acquisition terrain, de réaliser une détection précoce de la maladie sur les pieds sans symptômes.Les différences et similarités entre chacune de ces deux applications, en terme de constitution de base de données, d'algorithmes, de difficultés et de potentiel d'application en conditions réelles sont discutées tout au long du manuscrit. / Grapevine wood diseases in the vineyard are responsible for significant economic losses in the wine industry. These diseases of fungal origin are caracterised by a degradation of the wooded part of the plant material and by the erratic appearance of characteristic symptoms on the leaf part. This thesis is dedicated to the study of these diseases (mainly esca disase) using two imaging sensors and proximal sensing.The issue of visible symptom detection is first addressed using an RGB color sensor to acquire an image for each plant automatically or semi-automatically. The recognition of symptoms is approached in two stages, firstly by considering the classification at leaf-scale and then the detection at the plant-scale. The particularity of this study is the inclusion of confounding factors in the classification problem, taking advantage of the shape information of esca symptoms to differentiate them from other disorders and diseases. For this purpose, a comparison between SIFT approaches and recent transfer learning approaches is then conducted. The results then lead us to consider a simple deep learning architecture (RetinaNet) for the detection of the symptoms on the images, making it possible to estimate a level of disease severity for each vineplant.The second sensor used, a hyperspectral camera covering the spectrum from 500 nm to 1300 nm, tries to tackle a more experimental problem, namely the spectral behavior of the diseased plants which may lead to early detection of diseased plants without foliar symptoms. An experimental protocol and a database of spectra are then formed for the occasion. The dimensionality reduction methods make it possible to exploit the hyperspectral information or even to isolate the wavelengths associated with each class. However, the data do not allow, for the measured wavelength range and in the field acquisition conditions, to perform early detection of the disease on the plant without symptoms.The differences and similarities between each of these two applications, in terms of database constitution, algorithms, difficulties and application potential in real conditions are discussed throughout the manuscript.