Les Rémorines (REM) du groupe 1 sont des protéines spécifiques du monde végétale. Malgré leur caractère hydrophile elles sont localisées à la membrane plasmique. La phosphorylation des REM serait potentiellement impliquée dans la signalisation précoce et la défense des végétaux contre les pathogènes. Benschop et al. (2007) détecte AtREM1.3 (Arabidopsis thaliana, groupe 1b) phosphorylée en réponse au traitement par l'éliciteur générale flg22, tandis que Widjaja et al. (2008) a suggéré que la phosphorylation de AtREM1.2 est potentiellement impliquée dans la signalisation précoce à l'infection par Pseudomonas syringae. La fonction précise de la phosphorylation des protéines REM du groupe 1 reste inconnue. Des travaux antérieurs dans le laboratoire ont montré que le mouvement du virus X de la pomme de terre (PVX) est inversement corrélée à l'accumulation de StREM1.3 (Solanum tuberosum) et que StREM1.3 peut interagir physiquement avec la protéine de mouvement TRIPLE GENE BLOC Protein 1 (TGBp1) du PVX (Raffaele et al., 2009). Dans ce travail, nous avons étudié les mécanismes qui sous-tendent les interactions REM-TGBp1 et nous avons essayé de caractériser biochimiquement la kinase qui phosphoryle REM. Les conséquences physiologiques de l'interaction TGBp1 / StREM1.3 et de la phosphorylation de REM en terme de propagation des virus, d’inactivation génique post-transcriptionnelle, de régulation de l’ouverture des plasmodesmes, et d’activation de kinase ont également été étudiés. / The group 1 Remorin (REM) proteins are plant-specific oligomeric proteins that have been reported to localize to the plasma membrane despite their overall hydrophilic nature. There is evidence that the REM protein phosphorylation is potentially implicated in the early signaling and defense. Benschop et al. (2007) detected the AtREM1.3 (Arabidopsis thaliana group 1b of REM protein family) to be phosphorylated in response to treatment with the general elicitor flg22, while the Widjaja et al. (2008) suggested that the phosphorylation of AtREM1.2 is potentially implicated in early signaling upon infection with Pseudomonas syringae. The precise exact function of the group 1 REM protein phosphorylation remains unknown. Previous work in the laboratory showed that Potato virus X (PVX) movement is inversely correlated to potato StREM1.3 accumulation and that StREM1.3 can physically interacts with the movement protein TRIPLE GENE BLOCK PROTEIN 1 (TGBp1) from PVX (Raffaele et al., 2009). In this work, we studied the mechanism underlying the REM-TGBp1 interactions and we tried to characterise biochemically the kinase that phosphorylate REM. The physiological consequences of TGBp1/ StREM1.3 interaction and REM phosphorylation in terms of virus spreading, post-transcriptional gene silencing, plasmodesmata gating, kinase activation were also investigated.

De nos jours, les agents pathogènes des plantes, engendrent encore des pertes agricoles importantes. A terme, la solution la plus adaptée semble être la création de variétés de plantes cultivées présentant une résistance génétique à large spectre et durable. Créer de telles plantes nécessite la compréhension des mécanismes de résistance mis en jeu dans des pathosystèmes modèles, tels que celui impliquant le gène de résistance Rx et le virus X de la pomme de terre. Dans ce système, la protéine Rx intervient dans la perception d’un facteur d'avirulence issu du virus: sa protéine de capside (Cp). Cependant, les mécanismes moléculaires déclenchant la résistance chez la plante exprimant Rx demeurent obscurs. Cette étude s’est focalisée sur l’étape de reconnaissance de l’éliciteur viral par Rx. Une première approche a permis la mise en évidence d’un éliciteur minimal de 90 acides amine. L’étude d’un fragment légèrement plus grand par des méthodes de biochimie structurale, tend à exclure un modèle selon lequel, la différence de structuration tertiaire entre Cp élicitrice et Cp non élicitrice déterminerait la reconnaissance par Rx. Une seconde approche, de biologie moléculaire, a mis en évidence deux protéines hôte interagissant avec ce petit fragment éliciteur. L'étude s’est focalisée sur un gène codant un facteur de transcription, nbERF5, et a révélé que cette protéine interagit aussi bien avec les Cp de souches de Potexvirus avirulentes que virulentes dans la résistance liée à Rx, mais également avec la protéine de résistance Rx et l’intéracteur direct: RanGAP2. La poursuite de la caractérisation de cet ERF, permettra de déterminer son importance dans la résistance liée à Rx. / Nowadays, phytopathogenic agents are still causing significant agricultural losses. The most suitable option appears to be the creation of crop species carrying a genetic durable and broad spectrum resistance. In order to create such varieties, we need to understand the mechanisms underlying resistance, involved in model Pathosystems, Such as the one composed of the resistance gene Rx and the potato virus X. In that system, the host gene encodes a protein assimilated to a receptor implicated in the perception of an avirulence factor produced by the virus: its capside protein (Cp). Nevertheless, the molecular mechanisms triggering the resistance remain largely unknown. This study has been focused on the elicitor recognition mediated by Rx. A first approach led to the identification of a minimal elicitor containing 90 amino acids has. The structural characterization of a slightly larger protein fragment using biochemical methods suggested that the difference in the tertiary structuration of both elicitor and non-elicitor Cp would not be the determinant of Rx mediated recognition. Second, a molecular approach led to the discovery of two host proteins interacting with the small elicitor fragment. The work was focused on a transcription factor, nbERF5 and showed that this protein interacts similarly with elicitor or non-elicitor Cps of Rx mediated resistance. Interestingly, this gene product is able to directly interact with the Rx protein, but also with the direct interactor of Rx: RanGAP2, protein required for the Rx mediated resistance efficiency. Further characterization of this ethylen response factor will help us to understand its role in Rx mediated resistance.

The sequence of 3553 nucleotides corresponding to the 3'-terminal region of potato virus S (PVS) has been determined from cloned cDNA. The sequence obtained contains six open reading frames with the potential to encode proteins of Mr 10,734, Mr 32,515, Mr 7,222, Mr 11,802, Mr 25,092 and at least Mr 41,052. The amino acid sequence of the 33K ORF has been confirmed to be that of the viral coat protein gene. The nucleotide sequence of this ORF was obtained from expression plasmids which were isolated by binding with a specific monoclonal antibody to PVS, and the expression of coat protein fusion products was verified by Western blots of bacterial cell lysates. The deduced amino acid sequence of a 70 amino acid portion from the central region of the PVS coat protein was 59% identical to the analogous region of potato virus X. In addition, the 7K, 12K and 25K ORF's displayed significant sequence homology with similar sized ORF's from a number of potexviruses. The partial 41K ORF was homologous with the C-terminal portion of the viral replicase proteins of potato virus X and white clover mosaic virus. While the biological functions of the 12K and 25K non-structural proteins coded for by PVS and members of the potexvirus group remain unknown, the 12K protein displays a hydropathicity profile consistent with a membrane associated protein and the 25K protein contains a conserved sequence motif found in a number of nucleoside triphosphate binding proteins. Members of the carlavirus group are distinguished from the potexviruses by the presence of a small [11K (PVS, potato virus M) - 16K (lily symptomless virus)] 3' terminal ORF which appears to contain a sequence motif similar to the 'zinc-finger' domain found in many nucleic acid binding proteins. The coat protein gene from potato virus S (PVS) was introduced into Nicotiana debneyii tobacco as well as a commercial potato cultivar, 'Russet Burbank', by leaf disc transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral coat protein were highly resistant to subsequent infection following mechanical inoculation with the Andean or ME strains of PVS as indicated by a lack of accumulation of virus in the upper leaves. The coat protein mediated protection afforded by these transgenic plants was sufficient to prevent the accumulation of virus in the tissues of non-transformed 'Russet Burbank' shoots which had been grafted onto transgenic plants inoculated with PVS, and in reciprocal grafts, transgenic shoots accumulated less than 2% (6 weeks after grafting) of the concentration of PVS found in non-transformed shoots similarly grafted onto plants systemically infected with PVS. These transgenic plants also displayed a measure of resistance to inoculation with a related carlavirus from potato, potato virus M. In agreement with previous reports for plants expressing PVX coat protein, plants expressing PVS coat protein were also protected from inoculation with PVS RNA. These results provide further evidence that coat protein mediated protection for these two groups of viruses, which share similar genome organizations, may involve inhibition of some early event in infection, other than, or in addition to, virus uncoating. Specific monoclonal antibodies were prepared against a C-terminal derived 18 kDa portion of the 25K protein of PVS expressed as an in-frame chimeric fusion protein with the glutathione S-transferase gene. The in vivo expression of this non-structural protein in virus infected tissue, as well as tissue from transgenic tobacco (var Xanthi-nc) engineered to contain the entire 25K gene, was verified by Western immunoblot labelling. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Potato Virus Y (PVY) infection and nitrogen (N) deficiency cause similar symptoms (chlorosis and stunting) on potato foliage. While conventional methods, including ELISA and petiole testing, require destructive sampling and a longer time to diagnose, spectral analysis can be non-destructive, rapid and efficient. Spectral reflectance for potato cultivars representing three market types, chip processing, red-skinned fresh, and fresh and processing russets, were assessed in separate greenhouse trials in response to three N rates (90, 200, and 290 kg/ha) and two PVYN:O infection levels (clean and infected) at 4, 6, and 8 weeks after inoculation (WAI). Normalized Difference Vegetation Index (NDVI) was able to differentiate clean and PVYN:O infected samples of red-skinned and chip processing cultivars, at 4 and 8 WAI, respectively. Overall, cultivars differed in their spectral responses, indicating the importance of studying cultivar-specific spectral responses against PVY infection in future.

The main aims of diploma thesis were: 1) The sequence analysis of the Czech isolate of Potato virus M (PVM) VIRUBRA 4/009 and phylogenetic analysis of PVM coat proteins sequences 2) The bacterial expression of recombinant triple gene block protein 1 (TGB1) of PVM derived from the Czech isolate VIRUBRA 4/007 3) The construction of expression cassette of Potato leafroll virus (PLRV) coat protein and its transformation into A. tumefaciens for transgenic PLRV resistant plant formation In theoretical part of the thesis the taxonomic classification, morphology, genomic structure and virus transmission are discussed. Furthermore, the main rules concerning the bacterial expression of recombinant proteins and construction of transgenic plants using A. tumefaciens are described. Methodical part is devoted to description of generally used molecular biological and immunochemical methods. The following results were obtained in the thesis: The complete nucleotide sequences of open reading frames coding for three movement proteins (Triple gene block -TGB), coat protein and NA-binding protein of PVM isolate VIRUBRA 4/009; phylogenetic analysis was performed; the TGB1 protein was expressed in bacterial cells and will be used for polyclonal antibodies raising. Finally, the expression cassette containing the PLRV...

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L’utilisation de variétés de plantes porteuses de gènes majeurs de résistance a longtemps été une solution privilégiée pour lutter contre les maladies des plantes. Cependant, la capacité des agents pathogènes à s’adapter à ces variétés après seulement quelques années de culture rend nécessaire la recherche de résistances à la fois efficaces et durables. Les objectifs de cette thèse étaient (i) d’identifier chez la plante des régions génomiques contraignant l’évolution des agents pathogènes en induisant des effets de dérive génétique et (ii) d’étudier l’impact des forces évolutives induites par la plante sur la capacité d’adaptation des pathogènes aux résistances variétales, l’ambition étant par la suite d’employer au mieux ces forces pour limiter l’évolution des pathogènes. Le pathosystème piment (Capsicum annuum) – PVY (Potato virus Y) a été principalement utilisé pour mener ces travaux de recherche. Afin de répondre au premier objectif, une cartographie de QTL (quantitative trait loci) sur une population biparentale de piment et une étude de génétique d’association sur une core-collection de piments ont été réalisées. Ces deux approches ont permis de mettre en évidence des régions génomiques sur les chromosomes 6, 7 et 12 impliquées dans le contrôle de la taille efficace des populations virales lors de l’étape d’inoculation du virus dans la plante. Certains de ces QTL ont montré une action vis-à-vis du PVY et du CMV (Cucumber mosaic virus) tandis que d’autres se sont révélés être spécifiques d’une seule espèce virale. Par ailleurs,le QTL détecté sur le chromosome 6 co-localise avec un QTL précédemment identifié comme contrôlant l’accumulation virale et interagissant avec un QTL affectant la fréquence de contournement d’un gène majeur de résistance. Pour répondre au second objectif, une analyse de la corrélation entre l’intensité des forces évolutives induites par la plante et une estimation expérimentale de la durabilité du gène majeur a été réalisée. De l’évolution expérimentale de populations de PVY sur des plantes induisant des effets de dérive génétique, de sélection et d’accumulation virale contrastés a également été effectuée. Ces deux études ont démontré qu’une plante induisant une forte dérive génétique associée à une réduction de l’accumulation virale permettait de contraindre l’évolution des populations virales, voire d’entraîner leur extinction. Ces résultats ouvrent de nouvelles perspectives pour le déploiement de déterminants génétiques de la plante qui influenceraient directement le potentiel évolutif du pathogène et permettraient de préserver la durabilité des gènes majeurs de résistance. / Plants carrying major resistance genes have been widely used to fight against diseases. However, the pathogensability to overcome the resistance after a few years of usage requires the search for efficient and durable resistances.The objectives of this thesis were (i) to identify plant genomic regions limiting pathogen evolution by inducinggenetic drift effects and (ii) to study the impact of the evolutionary forces imposed by the plant on the pathogenability to adapt to resistance, the goal being to further use these forces to limit pathogen evolution. The pepper(Capsicum annuum) – PVY (Potato virus Y) pathosystem has been mainly used to conduct these researches.Regarding the first objective, quantitative trait loci (QTL) were mapped on a biparental pepper population andthrough genome-wide association on a pepper core-collection. These approaches have allowed the detection ofgenomic regions on chromosomes 6, 7 and 12 controlling viral effective population size during the inoculationstep. Some of these QTLs were common to PVY and CMV (Cucumber mosaic virus) while other were virusspecific.Moreover, the QTL detected on chromosome 6 colocalizes with a previously identified QTL controllingPVY accumulation and interacting with a QTL affecting the breakdown frequency of a major resistance gene.Regarding the second objective, a correlation analysis between the evolutionary forces imposed by the plant andan experimental estimation of the durability of a major resistance gene has been done. Experimental evolution ofPVY populations on plants contrasted for the levels of genetic drift, selection and virus accumulation they imposedhas also been performed. Both studies demonstrated that a plant inducing a strong genetic drift combined to areduction in virus accumulation limits virus evolution and could even lead to the extinction of the virus population.These results open new perspectives to deploy plant genetic factors directly controlling pathogen evolutionarypotential and could help to preserve the durability of major resistance genes.

Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: Potato virus Y (PVY) is responsible for considerable yield losses in the South African potato industry. The incidence of this virus has greatly increased over the past few years. Even more worrying is the variation of symptoms observed during PVY infection and the recent appearance of the more virulent PVYNTN strain in local fields. This project aimed to investigate the possible genetic variation within the viral genome and to establish the origin of strains. The project also aimed to establish a dependable, area specific enzyme-linked immunosorbent assay (ELISA) to replace the currently used ELISAs. Currently seed potato certification is done using ELISA kits imported from Europe. These kits were developed for the detection of overseas variants of PVY and the use thereof in South Africa has in the past lead to false negatives. Finally, this project set out to develop, optimize and establish a sensitive and reliable real-time reverse transcriptase polymerase chain reaction (qRT-PCR) detection protocol for PVY. In the first part of the study the coat protein (CP) gene of PVY isolates from plant material obtained from various parts of South Africa was amplified using RT-PCR. The resulting cDNA was then sequenced directly or cloned into a vector and then sequenced. The resulting sequences were aligned in a data matrix with international reference sequences, analyzed and grouped according to strain. Examination of the CP gene within this matrix as well as phylogenetic analysis revealed six main groups of PVY. These six groups included the traditional PVYN and PVYO groups and a recombinant group. Furthermore it also revealed variants of PVYN and PVYO. These mutants and recombinants pose a threat as they may lead to South African strains of PVY expressing coat proteins which vary from those found overseas. This may render the currently used European ELISA method of detection less effective and subsequently result in an increase in viral prevalence. This reinforced the need for a detection method based on local viral strains. Phylogenetic and Simplot analysis also confirmed that a recombinant strain between PVYN and PVYO had evolved and that PVYNTN was such a recombinant. The second part of the study aimed to develop and establish detection methods based on local variants of PVY. This included the development of ELISA and qRT-PCR detection methods of PVY. Previously amplified cDNA of the PVY CP gene was cloned into an expression vector and successfully expressed. Antibodies produced against the recombinant protein, when used in ELISA, however, failed to achieve the required levels of sensitivity. This prompted the development of qRT-PCR detection methods for PVY. Primer combinations for PVY were designed using the previously established CP gene data matrix. A reliable and sensitive SYBR® Green I based qRT-PCR assay was developed for the detection of PVY. The assay effectively detected all known South African variants of PVY. Furthermore, a Taqman® assay was developed for the detection of all variants of PVY. The Taqman® assay was 10 fold less sensitive and does not allow for amplicon verification through melting curve analysis, but it does add more specificity due to the addition of the probe. Although these qRT-PCR detection methods are still too expensive to replace the routine diagnostics done with ELISA, they do offer the opportunity to screen valuable mother material and confirm borderline cases in seed certification. / AFRIKAANSE OPSOMMING: Aartappel virus Y (PVY) is verantwoordelik vir aansienlike opbrengsverliese in die Suid-Afrikaanse aartappelindustrie. Die insidensie van infeksie deur die virus het drasties toegeneem oor die afgelope jare. Wat egter meer kommerwekkend is, is die groter variasie in simptome van PVY infeksie en die onlangse voorkoms ‘n meer virulente ras, PVYNTN. Hierdie projek poog om moontlike genetiese variasie van PVY te ondersoek en om die oorsprong van rasse op te spoor. Die projek het ook gepoog ook om ‘n bruikbare, betroubare en area spesifieke “enzyme-linked immunosorbent assay” (ELISA) toets te ontwikkel om die huidige ingevoerde ELISA te vervang. Hierdie toetse is ontwikkel om oorsese variante van PVY op te spoor en die gebruik daarvan het in die verlede gelei tot vals negatiewes. Verder is daar ook ondersoek ingestel na die ontwikkeling van ‘n sensitiewe en betroubare “real-time reverse transcriptase polymerase chain reaction” (qRT-PCR) protokol vir die opsporing van PVY. In die eerste deel van die studie is die mantelproteïen geen van PVY isolate vanuit plant materiaal geamplifiseer deur die gebruik van RT-PCR. Hierdie materiaal is vanaf verskeie streke in Suid-Afrika ontvang. ‘n Volgordebepalingsreaksie is uitgevoer op gekloneerde of ongekloneerde cDNA verkry uit die RT-PCR. DNA volgordes is in ‘n data matriks geplaas en vergelyk met internationale volgordes om die plaaslike isolate te analiseer en te groepeer. Deur vergelyking en filogenetiese ontleding kon ses hoofgroepe van PVY geïdentifiseer word, wat tradisionele PVYN en PVYO, sowel as ‘n rekombinante ras en variante binne die tradisionele PVYN en PVYO groepe ingesluit het. Rekombinante en mutante kan veroorsaak dat Suid-Afrikanse rasse van PVY mantelproteïene uitdruk wat afwyk van die oorsese rasse wat tot gevolg mag hê dat die ELISAs van oorsee minder effektief kan wees en kan lei tot verhoogde virus voorkoms. Die realiteit en gevaar versterk die gedagte dat ‘n deteksie metode gebaseer op plaaslike virusse absoluut krities is. Filogenetiese sowel as Simplot analise het bevestig dat ’n mutante ras tussen PVYN en PVYO ontstaan het en dat PVYNTN ’n rekombinante ras is. Die tweede deel van die studie was daarop gemik om deteksie metodes te ontwikkel wat gebaseer was op plaaslike variante van PVY. Dit sluit die ontwikkeling van ELISA sowel as qRT-PCR deteksie van PVY in. Voorheen geamplifiseerde cDNA is in ‘n ekspressievektor gekloneer en suksesvol uitgedruk. Teenliggaampies teen die rekombinante proteïen, indien in ELISA aangewend, kon egter nie die nodige sensitiwiteit oplewer nie. Dit het aanleiding gegee tot ontwikkeling van qRT-PCR deteksie metodes. Inleier kombinasies vir PVY was ontwikkel deur die gebruik van die bestaande mantelproteïen geen data matrikse. ‘n Betroubare en sensitiewe SYBR® Green I qRT-PCR deteksie protokol was ontwikkel vir die effektiewe deteksie van alle bekende Suid-Afrikanse rasse van PVY. Verder is ‘n sogenaamde “Taqman®” protokol ook ontwikkel vir deteksie van alle rasse. Die “Taqman®” protokol was 10 voudiglik minder gevoelig and laat nie bevestiging deur smeltkurwe analise toe nie, maar verleen meer spesifisiteit deur die toevoeging van die “Taqman® probe”. Hierdie qRT-PCR deteksie metodes is tans te duur om as roetine diagnostiese toetse te gebruik en kan dus nie ELISA vervang nie, maar hulle bied wel die geleentheid om waardevolle moeder materiaal te toets en grensgevalle in aartappelsaad sertifisering te bevestig.

Production of vaccines and pharmaceutical proteins in plants is a promising nascent technology with a great potential to provide high-quality, safe and non-expensive production and delivery platform. In this work we studied the experimental vaccine against human papillomavirus based on modified plant pathogen - Potato virus X (PVX). The experimental vaccine is based on PVX virus particles decorated with genetically fused HPV-E7 oncoprotein. These chimeric virus particles should be able to activate strong and specific cellular immune response. However the modification of the PVX coat protein with such relatively large fused protein might influence its ability to form particles. In this work we have characterized some properties of such chimeric virus particles like solubility or ability infect host plant. (In Czech)

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Potato virus Y (PVY) is responsible for considerable yield losses in the South African potato industry. The incidence of this virus has greatly increased over the past 20 years. In previous studies nonrecombinant strains of PVY, PVY N and PVY O, were detected in South African potatoes. In a recent study the occurrence of non-recombinant strains of PVY in South African potatoes was shown to have decreased while infection by more virulent recombinant strains, PVY NTN and PVY N-W, had increased dramatically. Infection of potato plants with PVY may cause stunted growth and mosaic or necrotic leaf symptoms which in turn can lead to a significant reduction in yield. Highly virulent recombinant PVY isolates as well as some of the non-recombinant strains may cause potato tuber necrotic ringspot disease (PTNRD) which may result in losses of 10% to total crop failure. For this reason investigation of infection by local recombinant isolates on local cultivars was important. To this end a representative number of isolates were selected for whole genome sequencing based on the relative occurrence of the various isolates in South Africa. A number of these sequenced isolates were subsequently used to infect local cultivars of potato in order to investigate the influence of genetic variation within the viral genome on symptom expression. In this study 27 South African isolates of PVY were sequenced through overlapping RT-PCR fragments. Seven of these isolates, six PVY NTN and one PVY N-W, were used to mechanically infect four local cultivars of potatoes under greenhouse conditions. The infected plants were monitored to establish the rate of systemic spread using a highly sensitive qRT-PCR and resulting tubers were visually screened for PTNRD. Highly variable recombinant isolates appear to be less virulent than the more conserved recombinant isolates possibly indicating molecular determinants for pathogenicity. For this reason the amino acid sequences of the South African isolates were compared to those of international isolates and scrutinized for variation and substitutions. Some South African isolates displayed amino acid substitutions unique to the specific isolate, making them unlike those found internationally. Substitution rates throughout the amino acid sequences differed greatly, with some isolates displaying hardly any changes whilst others varied a great deal from overseas isolates. Certain regions, many of which had specific functions, were more conserved than others. This study further investigated the recombination events within the PVY genome using reticulate phylogenetic analysis, molecular dating and network construction techniques. Unlike existing approaches, the one described in this study neither assumes an underlying strictly bifurcating species tree nor assumes prior knowledge of processes underlying deviations between individual gene trees. Through the use of the resulting robust time calibrated phylogeny, the patterns of diversification and recombination in PVY may be placed in the historical context of human cultivation of potatoes. Through the use of these techniques the study aimed to test whether diversification of the major strains of PVY and recombination between them occurred within the time frame of the domestication and modern cultivation of potatoes. From these analyses it can be deduced that recombinant strains of PVY were imported into South Africa. / AFRIKAANSE OPSOMMING: Aartappel virus Y (PVY) is verantwoordelik vir aansienlike opbrengs verliese in die Suid-Afrikaanse aartappelbedryf. Die voorkoms van die virus het grootliks toegeneem oor die afgelope 20 jaar. In vorige studies is nie-rekombinante rasse van PVY, PVY N en PVY O, gedokumenteer in Suid-Afrikaanse aartappels. 'n Onlangse studie het gevind dat die voorkoms van nie-rekombinante rasse van PVY in Suid- Afrikaanse aartappels aansienlik gedaal het terwyl infeksie deur virulente rekombinante rasse, PVY NTN en PVY N-W, dramaties toegeneem het. Infeksie van aartappelplante met PVY kan vertraagde groei en mosaïek- of nekrotiese blaarsimptome veroorsaak wat kan lei tot aansienlike vermindering in opbrengs. Hoogs virulente rekombinante PVY isolate, sowel as sommige nie-rekombinante rasse, kan aartappel nekrotiese ring simptome (PTNRD) veroorsaak wat verliese van 10% tot totale misoes tot gevolg kan hê. Om hierdie rede was die ondersoek van infeksie deur plaaslike rekombinante isolate op plaaslike kultivare belangrik. Vir hierdie doel is 'n verteenwoordigende aantal isolate gekies, gebaseer op die relatiewe voorkoms daarvan in Suid-Afrika, vir heelgenoom-volgordebepaling. Van die isolate is vervolgens gebruik om plaaslike kultivare te besmet ten einde die invloed van genetiese variasie binne die virale genoom op simptoom uitdrukking te ondersoek. In hierdie studie is 27 heelgenoomvolgordes van Suid-Afrikaanse PVY isolate bepaal deur oorvleuelende RT-PCR fragmente. Sewe van hierdie isolate, ses PVY NTN en een PVY N-W, is gebruik om vier plaaslike aartappel kultivare, gegroei onder kweekhuis kondisies, meganies te infekteer. Die geïnfekteerde plante is gemonitor om die tempo van sistemiese verspreiding vas te stel deur middel van 'n hoogs sensitiewe qRTPCR en knolle is visueel inspekteer vir PTNRD. Hoogs variante rekombinante isolate blyk om minder virulent te wees as die meer bewaarde rekombinante isolate wat dui op molekulêre determinante van patogenisiteit. Om hierdie rede is die aminosuurvolgordes van die Suid-Afrikaanse isolate vergelyk met die van internasionale isolate en ondersoek vir variasie en substitusies. Sommige Suid-Afrikaanse isolate vertoon aminosuur substitusies wat uniek is tot die spesifieke isolaat en maak hul dus anders as internasionale isolate. Die aantal aminosuursubstitusies in die volgordes verskil grootliks. In vergelyking met internasionale isolate toon sommige isolate skaars enige veranderinge terwyl ander ‘n aantal verskille toon. Sekere gebiede, waarvan baie spesifieke funksies het, was meer gekonserveerd as ander. Hierdie studie ondersoek ook rekombinasie gebeure binne die PVY genoom deur retikulêre filogenetiese analise, molekulêre datering en netwerk konstruksie tegnieke. In teenstelling met bestaande benaderinge, aanvaar die tegniek wat hier beskryf word nie ‘n streng bifurkeerende filogenie, wat onderliggende verdeel, of enige voorafgaande kennis van die prosesse onderliggend aan afwykings tussen individuele filogenieë nie. ‘n Robuuste, tyd gekalibreer filogenie kan diversifikasie patrone en rekombinasie van PVY plaas in die historiese konteks van menslike verbouing van aartappels. Deur gebruik te maak van hierdie tegnieke poog die studie om te toets of diversifikasie en rekombinasie van PVY rasse plaasgevind het binne die tydsbestek van die inburgering en moderne verbouing van aartappels. Van hierdie ontledinge word afgelei dat rekombinante rasse van PVY wat in Suid-Afrika voorkom, ingevoer is.