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The disertation thesis was focused on the analysis of variability of two nepoviruses genomes, Grapevine fanleaf virus -- GFLV and Arabis mosaic virus -- ArMV. The thesis described single isolates of these nepoviruses and their until recently non-explored genome portions coded by RNA1 strand, the coding regions for 1BHel and 1EPol proteins precisely. The main topic of this thesis was the identification of infected plants that were infected by nepoviruses GFLV and ArMV. Based on design of specific primers there was established sequence homology of coding sequences. Another topic was the development and optimalization of diagnostic system for the efficient GFLV and ArMV detection. This work was based on hypothesis of very high level GFLV and ArMV genomes variability. The results were recieved mainly from sequencing analyses performed by capillary automatic sequencer ABI-PRISM 310 (Applied Biosystems, Carlsbad, USA). Obtained results were assessed with CLC Main Workbench 5 software (CLC Bio, Aarhus, Denmark). Real-time PCR TaqMan multiplex was also designed using this software. This real-time tool is able to detect and quantify both nepoviruses within one PCR reaction simultaneously. There were sequenced and analysed the genetic codes at the nucleotide level for MP 2B , 2CCP, 1BHel and 1EPol proteins in this work. The first one was assessed because of the development of real-time PCR TaqMan multiplex. In this case were sequenced 290 bp from 14 GFLV and ArMV isolates, the variability level was established in frame of nucleotides 71.7-97.6%. The partial genetic code for 2CCP was assessed in frame of 6 GFLV isolates and the variability was established in 935 bp fragment to 83-86% at the nucleotide level and 81-91% at the amino acid level. The partial genetic code for 1BHel was sequenced in frame of 6 isolates GFLV and the variability was established in 379 bp in frame of all accessible GFLV and ArMV isolates, the variability was established 86.4-98.9% at the nucleotide level and 96-100% at the aminoacid level. The partial genetic code for 1EPol protein was sequenced in 6 GFLV isolates in frame of 365 bp and the variability was established for all accessible sequences in GenBank/NCBI. The variability was established at the nucleotide level 79.5-100% and at the aminoacid level 91.4-100%. All received sequences longer than 300 bp were submitted to GenBank/NCBI. The obtained partial genetic codes for 1BHel a 1EPol proteins coded in RNA1 were unique at the time of publication. This work provided the important results which were used subsequently like source information for construction of vectors intended for the preparation of transgenic plants. Then the incurred grapevine would show resistance against GFLV. Moreover, based on results of this work there was created certified methodology of the real-time PCR TaqMan multiplex method which can be standartly used as an efficient diagnostic tool for simultaneous detection of GFLV and ArMV. This real-time system was succesfully compared with ELISA what would be interesting mainly for diagnostic laboratories engaged in the certification processes of propagation material.

La maladie du court-noué est principalement causée en Europe par le Grapevine fanleaf virus (GFLV) et l’Arabis mosaic virus (ArMV). Le principal moyen de lutte contre sa dispersion consiste à certifier l’absence de ces virus dans les vignes commercialisées par des méthodes sérologiques tels que le DAS-ELISA. De par leurs propriétés biophysique et structurale exceptionnelles, les Nanobodies (Nb) issus des domaines variables d’immunoglobulines (Ig) composées uniquement de chaînes lourdes, se distinguent avantageusement des Ig conventionnelles. L’objectif majeur de ma thèse était d’établir un test de diagnostic du court-noué à base de Nb. À partir de deux collections de Nb contre le GFLV et l’ArMV, j’ai identifié des Nb reconnaissant un large spectre d’isolats viraux. Leur fusion à une protéine fluorescente ou à la phosphatase alcaline a conduit à l’obtention de réactifs de détection performants du GFLV et de l’ArMV par DAS-ELISA. La structure atomique d’un complexe Nb/GFLV résolue à 2.8 Å par cryomicroscopie électronique a permis de cartographier l’épitope en surface du virus et a révélé une couverture maximale de la particule virale par le Nb. La comparaison des tests Nb à des réactifs sérologiques commerciaux a révélé leur supériorité en terme de sensibilité et de spécificité, ouvrant ainsi la voie à la commercialisation d’un nouveau test de diagnostic des virus du court-noué de la vigne. / The grapevine fanleaf disease is mainly caused in Europe by the Grapevine fanleaf virus (GFLV) and the Arabis mosaic virus (ArMV). The principal mean to limit their spread, is to certify their absence in marketed grapevines by serological methods such as DAS-ELISA. Their unique biophysical and structural properties make the variable domains of heavy chain-only immunoglobulin, called Nanobodies (Nb) a real asset for the development of a diagnostic test against fanleaf disease viruses. I identified Nb able to detect a broad spectrum of viral isolates from two Nb collections against GFLV and ArMV. Their fusion to a fluorescent protein or to a bacterial alkaline phosphatase resulted in the production of efficient DAS-ELISA detection reagents. The atomic structure of a Nb/GFLV complex was solved at 2.8 Å by cryoelectron microscopy, allowing the precise mapping of the viral epitope. This result showed a maximum coverage of the viral particle by the Nb, leading to a maximal signal in DAS-ELISA. The full Nb tests against GFLV and ArMV were compared to commercial reagents and showed the superiority of the former in both sensitivity and specificity, opening the way for the development and commercialization of a new type of serological kits for the detection of grapevine viruses.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The nepoviruses are a group of nematode-transmitted plant viruses that are distributed worldwide and infect a wide range of plant species, including grapevine. Most of the nepoviruses are foreign to South Africa and to date, only Grapevine fanleaf virus (GFLV) is present. The Department of Agriculture, Forestry and Fisheries (DAFF), as the official National Plant Protection Organisation (NPPO) of South Africa, is committed to prevent the importation and spread of plant pathogens by administering the Agricultural Pests Act, 1983 (Act No. 36 of 1983). Effective measures are implemented by which the introduction of agricultural pests may be prohibited to safeguard the agricultural environment. One of the core functions of DAFF is to render a routine plant health diagnostic service for imported plants and plant products to prevent exotic pathogens from entering the country. The objective of this study was to develop a diagnostic assay for the detection of nepoviruses in grapevine. The project aimed to produce antibodies by recombinant DNA technology against bacterially expressed viral coat protein of a specific nepovirus [Tomato ringspot virus (ToRSV)] and subsequently develop a DAS-ELISA (Double Antibody Sandwich Enzyme-linked Immunosorbent) assay for the detection of the virus. The coat protein (CP) was successfully isolated from imported ToRSV-infected grapevine material. Two expression systems were utilised for expression of the ToRSV-CP, the GST gene fusion system and an Agrobacterium-mediated expression system. The GST gene fusion system was unsuccessful as insufficient soluble protein expression prevented the production of antibodies and thus the development of the DAS-ELISA assay. Tissue print immunoassay (TPIA) initially showed positive results for transient expression of the fusion protein in tobacco plants, but further confirmation proved to be inconclusive. The project also aimed to develop a real-time PCR assay for the specific detection and relative quantification of GFLV, based on a conserved region of the RNA-2 genome. A partial GFLV-RNA-2 from a South African isolate of grapevine was sequenced and used for the design of specific primers. The quantitative real-time PCR assay based on SYBR green technology proved to be sensitive in detecting levels as low as 0.11ng/reaction in infected plants, making it a highly effective diagnostic tool for the detection of GFLV. / AFRIKAANSE OPSOMMING: Die nepovirusse is 'n groep van nematode-oordraagbare plant virusse wat wêreldwyd versprei word en 'n wye verskeidenheid van plantspesies infekteer, insluitend wingerd. Die meeste van die nepovirusse is uitheems aan Suid-Afrika en tot op datum is net Wingerd netelblaar virus (GFLV) teenwoordig. Die Departement van Landbou, Bosbou en Visserye (DAFF), as die amptelike Nasionale Plant Beskermings Organisasie (NPBO) van Suid-Afrika, is daartoe verbind om die invoer en verspreiding van plantpatogene te voorkom deur administrasie van die Wet op Landbouplae, 1983 (Wet No. 36 van 1983). Doeltreffende maatreëls word geïmplementeer waardeur die invoer van landbouplae verbied word om sodoende die landbou-omgewing te beskerm. Een van die kernfunksies van DAFF is om 'n roetine plant gesondheid diagnostiese diens vir ingevoerde plante en plantprodukte te lewer om te verhoed dat eksotiese patogene die land binnedring. Die doel van hierdie studie was om 'n diagnostiese toets vir die opsporing van nepovirusse in wingerd te ontwikkel. Die projek was daarop gemik om antiliggame te vervaardig deur rekombinante DNA-tegnologie teen bakterieël-uitgedrukte virale mantelproteïen van 'n spesifieke nepovirus [Tomato ringspot virus (ToRSV)] en vervolgens ‘n DASELISA (Double Antibody Sandwich Enzyme-linked Immunosorbent) toets vir die opsporing van die virus te ontwikkel. Die mantelproteïen (CP) is met sukses geïsoleer vanaf ingevoerde ToRSV-besmette wingerdmateriaal. Twee uitdrukking stelsels is gebruik vir uitdrukking van die ToRSV-CP, die “GST gene fusion” stelsel en 'n Agrobacterium-bemiddelde uitdrukking stelsel. Die “GST gene fusion” stelsel was egter onsuksesvol aangesien onvoldoende oplosbare proteïen uitdrukking die produksie van antiliggame en dus die ontwikkeling van die DAS-ELISA toets verhoed het. “Tissue print immunoassay” (TPIA) het aanvanklik positiewe resultate getoon vir tydelike uitdrukking van die fusie proteïen in tabakplante, maar verdere bevestiging was onoortuigend. Die projek was ook daarop gemik om ‘n in-tyd polimerase ketting reaksie (PKR) toets vir die spesifieke opsporing en relatiewe kwantifisering van GFLV, gebaseer op 'n gekonserveerde volgorde van die RNA-2 genoom, te ontwikkel. 'n Gedeeltelike GFLV-RNA-2 nukleïensuurvolgorde van 'n Suid-Afrikaanse wingerd isolaat is bepaal en gebruik vir die ontwerp van spesifieke inleiers. Die kwantitatiewe in-tyd PKR toets gebaseer op SYBR groen tegnologie was sensitief genoeg om vlakke van so laag as 0.11ng/reaksie in geïnfekteerde plante op te spoor, wat dit 'n hoogs effektiewe diagnostiese hulpmiddel vir die opsporing van GFLV maak.

Le Grapevine fanleaf virus (genre Nepovirus) est l'agent principal du court-noué de la vigne. Il induit des symptômes très variables. Ce travail présente une étude mécanistique de la symptomatologie du GFLV sur un hôte herbacé et sur l'hôte d’intérêt agronomique. Par détection de marqueurs biochimiques et moléculaires j'ai montré que le GFLV-F13 induit une réponse hypersensible (HR) sur N. occidentalis et une restriction partielle du virus. J'ai identifié puis cartographié le déterminant viral de cette HR en utilisant des réassortants, des recombinants et des variants naturels du virus. Sur vigne, sur un dispositif expérimental unique en son genre, j'ai mené une approche sans a priori d’étude transcriptomique par RNA-Seq. J'ai comparé des vignes du cépage gewurztraminer mono-infectées par une souche sévère induisant des symptômes de rabougrissement et par une souche plus modérée. 1 023 gènes sont spécifiquement dérégulés par la souche sévère parmi lesquels des gènes impliqués dans la régulation de la HR. Ce résultat permet de proposer pour la première fois qu'une HR pourrait être mise en place dans la vigne en réponse à une infection virale. / Grapevine fanleaf virus (genus Nepovirus) the causative agent of fanleaf degeneration, induces variable symptoms. This manuscript presents a mechanistic study of GFLV symptomatology on both an herbaceous model plant and an agronomically important crop plant.On N. occidentalis, I demonstrated that GFLV-F13 induces a reaction exhibiting hallmarks of a hypersensitive response (HR), partially restricting virus spread. Using reassortants, recombinants and natural variants of the virus, I could identify and map the viral determinant of this HR. On grapevine, I took advantage of a unique experimental set-up and used RNA-Seq to compare the transcriptoms of Gewurztraminer plants infected with two different GFLV strains, one of which induced stunting symptoms and the other mild symptoms. 1,023 genes among which genes involved in the regulation of HR, were specifically regulated by the more severe strain This is the first hint of a HR taking place in grapevine in response to a virus infection.

Par leur stabilité, leur petite taille et leur nature monomérique, les domaines variables des immunoglobulines à chaînes lourdes, ou Nanobodies (Nb), sont incontournables en diagnostic et recherche médicale. Pourtant, leur utilisation en agro-biotechnologies demeure confidentielle.Dans l'idée de les utiliser pour étudier et combattre le Grapevine fanleaf virus (GFLV), responsable de la maladie du court-noué très préjudiciable à l'économie viticole mondiale, j'ai produit une collection de Nb spécifiques du GFLV.Fusionné à une protéine fluorescente et exprimé en plante de façon stable, un de ces Nb (alors appelé Chromobody, Cb) a conféré une haute résistance au GFLV inoculé mécaniquement ou transmis par nématodes.Le potentiel du Cb comme biocapteur a été validé par le suivi in vivo d’un isolat contournant la résistance mais toujours reconnu par le Cb. La structure du complexe Nb/GFLV a été résolue à 2,8 Å et révèle la zone occupée par le Nb à la surface de la capside.Ces résultats ouvrent des perspectives innovantes pour la compréhension du cycle infectieux d'un phytovirus et l'élaboration de nouvelles stratégies de lutte antivirale. / Due to their small size, high stability and strict monomeric nature, Nanobodies (Nbs) deriving from camelids heavy chain only antibodies have proven very valuable as diagnostic and therapeutic tools. However their use in agro biotechnology remains limited.In order to apply Nbs to the study and the control of grapevine fanleaf degeneration, I produced acollection of Nbs against Grapevine fanleaf virus (GFLV), the causal agent of this devastating disease worldwide.When fused to a fluorescent protein and stably expressed in plants, one of these Nbs (calledChromobody, Cb) conferred high resistance to GFLV, whether inoculated mechanically or by vector-mediated transmission.The identification of an isolate overcoming the resistance but still bound by the Cb allowed real-time tracking of the infection showing the high potential of Cbs as biosensors.The cryoEM structure of the Nb/GFLV complex was obtained at 2,8 Å and provides a clear picture of the footprint of the Nb on the surface of the GFLV capsid.These results pave the way for the innovative use of Nbs to unravel viral life cycle and to counter viral diseases.

Dans cette étude, nous avons dans un premier temps adapte le protocole d'embryogenèse somatique primaire a différentes variétés d'hybrides porte-greffes (3309C, 110R, Fercal, 41B et SO4) en nous appuyant sur l'expérience acquise au laboratoire sur Vitis vinifera cv Chardonnay. Les résultats montrent que le génotype, le type d'explant (étamine, fleur ou nœud), le type et la dose d'auxine utilisés dans le milieu d’induction (2,4-D ou 2,4,5-T) ont une influence sur les efficacités d'embryogenèse somatique. En effet, pour le 3309C, l'utilisation du 2,4,5-T dans le milieu d'induction a montré une efficacité embryogène supérieure à partir de nœuds par rapport à celle obtenue à partir d'étamines. Cependant la meilleure efficacité a été obtenue à partir de fleurs de cette variété, sur un milieu d'induction contenant du 2,4-D. De plus, le protocole d'embryogenèse somatique secondaire utilise de manière récurrente au laboratoire nous a permis d'obtenir des masses embryogènes ainsi que des embryons somatiques secondaires de ces porte-greffes. Le protocole de conversion des embryons en plantes, en présence de 4,5 uM de cytokinine (BAP) s'est avère efficace pour le 11OR et le 41B. Dans un second temps, nous avons co-cultivé le matériel embryogène obtenu pour quatre de ces génotypes (110R, 3309C, Fercal et 41B), avec Agrobacterium tumefaciens contenant trois constructions génétiques : (i) une copie d'une séquence partielle (1020 pb) du gène de la coque protéique du virus en orientation sens; (ii) une partie courte en sens et en anti-sens (280 pb) de cette même séquence formant une structure en épingle a cheveux (hpRNA = hairpin RNA) ; (iii) un amiRNA ciblant une séquence virale. Le gène bactérien codant la néomycine phosphotransférase et conférant la résistance à un antibiotique, la kanamycine, a été utilisé comme gène de sélection. Les conditions de sélection a la kanamycine ont nécessité des adaptations expérimentales telles que l’ajustement de la concentration en antibiotique puisque la sélection avec 75 mg.L-1 de kanamycine s'avère insuffisamment drastique dans Ia plupart de nos expériences de co-cullture. Les résultats d'analyse moléculaire par PCR ont montré l'amplification probable des fragments d'intérêt (CPGFLV et amiRI1TA-71) dans des échantillons de 11OR et de 41B résistants à la kanamycine. Cependant des analyses moléculaires supplémentaires par AL-PCR ne nous ont pas renseignées sur une éventuelle intégration du transgène amiRATA-71 dans des masses embryogènes de 41B. / In this study, we initially adapted the protocol of primary somatic embryogenesis in different varieties of hybrid rootstocks (3309C, 110R, Fercal, 41B and SO4) building on the experience gained in the laboratory on Vitis vinifera cv Chardonnay. The results show that the genotype, the explant type (stamen, flower or node), the type and the dose of auxin used in the induction medium (2,4-D or 2,4,5-T) influence the efficiency of somatic embryogenesis. Indeed, for the 3309C, the use of 2,4,5-T in the induction medium showed a higher efficiency from embryogenic nodes compared to that obtained from stamens. However, the better efficiency was obtained from the flowers of this variety on an induction medium containing 2,4-D. In addition, a protocol used in the laboratory for secondary somatic embryogenesis allowed us to obtain embryogenic masses as well as secondary somatic embryos from these rootstocks. The protocol conversion of embryos into plants, in the presence of 4.5 [tM of cytokinin (BAP), was effective for the 110R and 41B. In a second step, we co-cultivated embryogenic material obtained for four of these genotypes (110R, 3309C, Fercal and 41B), with Agrobacteriwn tumefaciens containing three genetic constructs: (i) a copy of a partial sequence (1020 bp) of the coat protein gene of the virus in the sense orientation, (ii) a short part-way and antisense (280 bp) of the same sequence forming a hairpin structure (hairpin RNA = hpRNA) (iii) one amiRNA targeting a viral sequence. The nptll bacterial gene encoding neomycin phosphotransferase and conferring resistance to the antibiotic kanamycin, was used as the selection gene. The selection conditions to kanamycin have required experimental adaptations such as adjusting the concentration of antibiotic because the selection with 75 mg.L-1 of kanamycin was not enough drastic in most of our experiments of co-culture. The results of molecular analysis by PCR showed probable amplification of fragments of interest (CPGFLV and amiRNA-71) in samples of 11OR and 41B resistant to kanamycin. However, additional molecular analysis by AL-PCR did not inform us about a possible integration of the transgene amiRNA-71 in embryogenic masses of 41B.

Thesis (MSc (Genetics))--Stellenbosch University, 2008. / South Africa is one of the top ten wine producing countries in the world. The South African wine industry contributes approximately R16.3 billion to South Africa’s annual gross domestic product with 42.8% of wine being exported. To compete with the top wine producing countries and to ensure a viable export market, South Africa needs to ensure that healthy, virus free propagation material is produced and sold. One of the viruses that need to be tested for is Grapevine fanleaf virus (GFLV). Grapevine fanleaf virus causes degeneration and malformation of berries, leaves and canes and is responsible for significant economic losses by reducing crop yields by as much as 80%, reducing the longevity of the vines and affecting fruit quality. It is widespread in the Breede River Valley of the Western Cape where the nematode vector, Xiphinema index, is prevalent. The Breede River Valley contributes approximately 30% of the total production of the local wine industry, and severe losses in this region could threaten the viticulture. The Plant Improvement Act states that all propagation material sold must be tested for GFLV by a reputable scientific technique. The technique commonly used in South Africa is the Double Antibody Sandwich - Enzyme-linked Immunosorbent Assay (DAS-ELISA) and the kits are imported from Europe at a significant cost to the South African viticulture industry. The objective of this study was to produce a reliable and sensitive diagnostic assay specific for the South African strains of GFLV. This project aimed to develop and optimize a DAS-ELISA, by using recombinant DNA technology to produce antibodies against bacterially expressed viral coat protein. Total RNA was extracted from GFLV infected grapevine material and the viral coat protein (CP) amplified. The CP was cloned into the pGex-6P-2 expression vector, fusing a Glutathione STransferase (GST) partner to the viral coat protein enhancing solubility and protein purification. Insufficient amounts of the soluble protein were expressed and purified, preventing the production of antibodies and thus the development of the DAS-ELISA. An alternative diagnostic rapid-direct-one-tube-RT-PCR assay was developed. This rapid-directone- tube-RT-PCR assay was compared to commercially available DAS-ELISA and ImmunoStrip tests (Agdia) to assess the reliability, sensitivity and specificity of the rapid-direct-one-tube-RTPCR assay. Twelve GFLV isolates from South Africa were sequenced to investigate the variability between the isolates as well as the variability between the South African isolates and GFLV sequences available in Genbank. Sequence identities between clones from different GFLV isolates from South Africa were between 86-99% and 94-99% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the coat protein gene sequences showed that the South African isolates form two distinct clades or sub-populations. No significant correlation was found between geographical origin and symptoms, nor between geographical origin and sequence variability or between grapevine cultivar and symptom expression. Of the 23 samples tested with all three tests, 21 tested positive with rapid-direct-one-tube-RT-PCR, 19 with the ImmunoStrips and 17 with an imported DAS-ELISA kit (Agdia). Rapid-direct-one-tube-RT-PCR was found to be the most reliable technique for GFLV detection. Although the establishment of a DAS-ELISA directed to the South African strain(s) of GFLV was not successful, an alternative PCR based diagnostic system was developed, and proved to be sensitive and reliable. RT-PCR based diagnostic assays are generally accepted to be more sensitive than DAS-ELISA, but the latter is still used as the diagnostic assay of choice for routine testing due to ease of use. This rapid-direct-one-tube-RT-PCR assay is a rapid, sensitive and reliable diagnostic test, reducing the prevalence of false negatives, contributing to a virus free viticulture industry. The rapid-direct-one-tube-RT-PCR assay is as easy to use as DAS-ELISA, faster and can be performed by semi skilled workers, thus providing all the advantages associated with DAS-ELISA.