Dissertationen zum Thema „Plant viruses Genetics“

Bibliography: leaves 127-138. This thesis studies the minor double stranded RNAs (dsRNA) and single stranded RNAs (ssRNA) which are consistently associated with plants infected with Q strain of cucumber mosaic virus (Q-CMV). The investigations are focused on the structural elucidation of new RNAs which have been observed in single stranded and double stranded RNA profiles of Q strain of CMV.

Corrigendum inserted at the back. Includes bibliographical references (leaves 133-158). Ch. 1. General introduction -- ch. 2. General materials and methods -- ch. 3. Biological characterisation of Australian PSbMV isolates -- ch. 4. Developing nucleic acid based diagnostics for PSbMV -- ch. 5. Detection of PSbMV isolates by RT-PCR and RFLP analysis -- ch. 6. Developing an internal control for PSbMV RT-PCR -- ch. 7. Molecular analysis of the PSbMV VPG -- ch. 8. PSbMV sequence and phylogenetic analysis -- ch. 9. General discussion Sixteen pea seed borne mosaic virus (PSbMV) isolates were collected between 1995 and 1998. These isolates were biologically distinct yet serologically indistinguishable. The conclusion is that PSbMV is widespread and occurs at a low incidence in Australia. Reports sequence information on new isolates of PSbMV which has allowed genomic regions to be identified which distinguish PSbMV pathotypes and isolates; and, to the development of PSbMV nucleic acid hybridisation and RT-PCR assays.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination.
AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: Ribosome inactivating proteins (RIPs) are currently classified as rRNA N-glycosidases, but also have polynucleotide: adenosine glycosidase activity. RIPs are believed to have anti-viral and anti-fungal properties, but the exact mechanism of these proteins still need to be elucidated.The mechanism of resistance however, appears to be independent of the pathogen. For resistance the RIP terminates virus infected plant cells and stops the reproduction and spread of the virus. Transgenic plants containing RIPs should thus be resistant to a wide range of viruses. The ultimate goal of the larger project of which this forms part is the development of virus resistant plants. To monitor the expression of a RIP in a transgenic plant a detection method had to be developed. Antibody detection of the RIP was decided upon as the most cost effective method. The RIP, Dianthin 30 from Dianthus caryophyllus (carnation), was used and expressed in bacterial and insect expression systems. The bacterial expression experiments were done using the pET expression system in BL21(DE3)pLysS cells. The expression in this system yielded recombinant protein at a very low concentration. Expression experiments were also performed in insect tissue culture with the baculovirus vector BAC-TO-BAC™.With this system the expression was also too low to be used for the production of antibodies. A Dianthin 30 specific peptide was then designed and then produced by Bio-Synthesis. This peptide was then used to raise antibodies to detect Dianthin 30. These antibodies were tested on Dianthus caryophyllus proteins. To establish if this detection method was effective to monitor the expression in plants, tobacco plants were transformed with Agrobacterium tumefaciens containing Dianthin 30 in the pART27 plant expression vector. The putative transformed plants were analysed with peR and Southern blots.
AFRIKAANSE OPSOMMING: Tans word Ribosomale-inaktiverende proteïene (RIPs) geklassifiseer as rRNA N-glikosidase wat ook polinukleotied: adenosien glikosidase aktiwiteit bevat. Daar word geglo dat RIPs anti-virale en anti-fungus eienskappe bevat, maar die meganisme van beskerming word nog nie ten volle verstaan nie. Dit is wel bewys dat die meganisme van weerstand onafhanklik is van die patogeen. Virus geinfekteerde plantselle word deur die RIP gedood om die voortplanting en verspreiding te bekamp en sodoende word weerstand bewerkstellig. Transgeniese plante wat dan 'n RIP bevat sal dus weerstandbiedend wees teen 'n wye spektrum virusse. Die hoofdoel van die breër projek, waarvan die projek deel uitmaak: is die ontwikkeling van virusbestande plante. Om die uitdrukking van die RIP in die transgeniese plante te kontroleer, moes 'n deteksie metode ontwikkel word. Die mees koste effektiewe deteksie metode is met teenliggame. Die RIP, Dianthin 30 from Dianthus caryophyllus (angelier) was gebruik vir uitdrukking in bakteriele- en insekweefselkultuur. Die bakteriele uitdrukkingseksperimente was gedoen met die pET uitdrukkings sisteem III BL21(DE3)pLysS selle. Die uitdrukking in die sisteem het slegs rekombinante proteïene gelewer in uiters lae konsentrasies. Uitdrukkingseksperimente was ook gedoen in insekweefselkultuur met die baculovirus vektor BAC-To- BACTM. Met die sisteem was die uitdrukking ook veels te laag om bruikbaar te wees vir die produksie van teenliggame. Daar is toe 'n peptied ontwerp wat Dianthin 30 kan verteenwoordig vir die produksie van teenliggame. Die teenliggame is getoets teen Dianthus caryophyllus proteïene. Om vas te stel of die deteksiemetode wel die uitdrukking van Dianthin 30 sal kan monitor, is tabak ook getransformeer met Dianthin 30. Die transformasies is gedoen met die hulp van Agrobacterium tumefaciens en die pART27 plant uitdrukkings vektor. Die plante is getoets met die polimerase ketting reaksie en Southern klad tegnieke.

Includes appendix containing journal publications co-authored by the author. Includes bibliographical references (leaves 130-151). Eighteen strains of Cucumber mosaic virus, including forteen from Australia, two from the USA, and two from Japan were used in this study.

The cacao swollen shoot disease of Theobroma cacao L. (cacao) is caused by Cacao swollen shoot virus (CSSV; genus, Badnavirus, family, Caulimoviridae). The virus is endemic to West Africa, where it poses a serious threat to cocoa production. Despite efforts to control CSSV spread by replacement of infected trees with tolerant cultivars and mealybug vector management, the disease is widespread in West Africa. In Trinidad, leaf mosaic and vein-banding symptoms have been observed in cacao plants in the field since the 1940s, and recently at the International Cocoa Genebank (ICGT), a custodian of cacao germplasm resources. The strains A and B of the suspect Cacao Trinidad virus (CTV) caused the symptoms, and were thought to be related to CSSV, however, viral causality was not demonstrated, until now. To develop molecular detection methods for CSSV in infected plants, polymerase chain reaction (PCR) amplification of eight regions of the CSSV genome was implemented. The PCR results showed variable amplification frequencies of 19 - 42% at each region, for 124 isolates collected in Cote d'Ivoire and Ghana. Pairwise nucleotide (nt) analyses of the eight regions showed 66-99% shared identities, indicating that CSSV isolates exhibit extensive variability with respect to primer design. The results provided preliminary evidence for the existence of a CSSV complex consisting of four divergent species. The full length genome of 14 CSSV isolates from cacao determined using the Illumina HiSeq platform showed 70-99% shared nt identities. The pairwise nt identities placed CSSV sequences into a group of four distinct species, one of which represented a previously undescribed species. Moreover, the full-length genomes grouped phylogenetically with other badnaviruses and revealed two CSSV subclades with three types of genome arrangements; four, five or six open reading frames (ORFs). Predicted functional protein domains were conserved on each ORF. Two distinct, full-length genome sequences were determined using the Illumina HiSeq platform, from DNA isolated from cacao leaves exhibiting distinct symptoms in Trinidad. The sequences were validated by PCR-amplification and sequencing of overlapping viral genome fragments. Pairwise nt analysis indicated that each genome shared 52-62% nt identities with CSSV and other badnaviruses, suggesting that the two are distinct species. Phylogenetic analysis indicated that the two sequences are not strains of the same virus, as supposed, but they represent two previously undescribed species in the genus, Badnavirus, and they have been named Cacao mild mosaic virus (CaMMV) and Cacao yellow-vein-banding virus (CYVBV). Despite sharing the same host and causing similar symptoms in cacao, CSSV, CaMMV, and CYVBV are phylogenetically-distinct species. The discovery of a CSSV species complex and the identification of three new cacao-infecting badnavirus species will support the development of molecular detection tools using the partial and complete genome sequences determined in this study. The ability to develop validated molecular tools for the detection of CSSV and related viruses, CaMMV and CYVBV, in cacao will aid quarantine efforts and safe movement of germplasm from the ICGT in Trinidad to cacao-growing countries, worldwide. Also, molecular diagnostics tools are expected to be useful in efforts underway to develop CSSV-resistant planting material for countries in West Africa, which are currently experiencing continued or new disease outbreaks.

Macrophages are one of the three main human cell types infected by HIV-1. They are highly plastic cells requiring a dynamic actin cytoskeleton for their role in development, homeostasis, tissue repair and immunity. For HIV-1, disrupting actin in macrophages is detrimental in that it leads to a complete block of viral uptake and reduces reverse transcription but, significantly, not fusion. Rho GTPases (Rac1, RhoA and Cdc42) regulate many aspects of actin dynamics including those required for endocytosis. Using a pharmacological approach, it was shown that Rac1 along with Rho GTPase effectors Pak1 and N-WASP are important for productive HIV-1 entry in macrophages. However, pharmacological inhibitors aren’t available for many host factors and may have off-target effects. To overcome this, expression of dominant negative (DN) Rho GTPases was attempted in human stem cell-derived macrophages (esMDMs). While DN Rac1 expressing esMDMs were successfully generated, this was not possible for the other two. DN Rac1 expressing esMDMs, as expected, had less filamentous actin and reduced dextran uptake compared to control esMDMs. In contrast to the pharmacological studies, HIV-1 infection studies in Rac1 DN esMDMs revealed a significant increase in HIV-1 fusion, reverse transcription and nuclear import, which could be due to reduced filamentous actin leading to a slower rate of endocytosis thereby allowing more time for viral fusion within endocytic vesicles. Surprisingly, reduced HIV-1 gene expression was observed in Rac1 DN esMDMs. This was corroborated by transfection studies implicating Rho GTPases in LTR driven gene expression. To overcome the ineffectiveness of RhoA and Cdc42 DN constitutive gene expression in esMDMs, an inducible lentiviral gene expression system based on the use of a constitutive promoter and a FLEx switch mediating irreversible DNA inversions was generated. The novel FLEx vector was the first system shown to induce transgene expression in esMDMs albeit at a very low efficiency.

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

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: The aim of this study was to combine two common diagnostic tools: serological kits and genetic fingerprinting to identify cherry leaf-roll nepovirus (CLRV), and to establish a marker system to characterize walnut germplasm. The detection of plant viruses is difficult. Restrictions are imposed for quarantine purposes on the importation of plant material from foreign countries. Modern techniques such as a PCR based screening method for CLRV are required to ensure material do not harbour viruses. A primer pair was designed to amplify a 430 bp non-coding homologous region. For the choice of primers, consensus sequences were considered and areas where the sequence data shared 98.5% homology, were chosen. The sensitivity of this detection method was 100-fold higher when compared to the ELISA. The PCR fragment was verified by nucleotide sequencing. AFLP technology was used to identify polymorphic fragments for 6 walnut cultivars and a rootstock, and SCARs were developed from AFLP specific bands. The AFLP technique distinguished all the walnut cultivars and the rootstock. However, conversion of AFLP fragments to SCAR markers for the development of a simple robust technique for cultivar discrimination, was not successful. Using 27 AFLP primer combinations, polymorphic fragments as high as 47.8% were scored. The reason for the lack of efficient conversion was as the result of the AFLP technique. The SCAR primers were generated from sequences internal to the AFLP primers but the specificity of the markers was in the AFLP primers not the internal sequence. In this study using AFLP, walnut cultivars were found to be closely related. The AFLP primer pairs used, provided polymorphic fragments. From these fragments, 7 SCAR markers were developed. It was expected that these SCARs derived from the AFLP markers would detect slight differences between cultivars. The Paradox SCAR marker was the only one that could divide the cultivars into two groups. When Chandler SCAR products were digested with the restriction enzyme Rsal, the same banding pattern as that of Paradox SCAR products was observed.
AFRIKAANSE OPSOMMING: Die doel van hierdie studie was om twee algemene opsporingstegnieke te kombineer: serologiese toetsstelle en genetiese vingerafdrukke om cherry leaf-roll nepovirus (CLRV) te eien en om In merkersisteem te ontwikkel wat okkerneut kiemplasma kan karakteriseer. Die opsporing van plant virusse is baie moeilik. As gevolg van kwarantyn vereistes, word daar beperkinge geplaas word op die invoer van plant materiaal vanuit die buiteland. Moderne tegnieke soos hierdie een wat op PKR berus, word benodig om te verseker dat CLRV nie in plantmateriaal teenwoordig is nie. In Stel inleiers is ontwerp wat In 430 bp nie-koderende homoloë area amplifiseer. Hiervoor is konsensus volgordes bestudeer en slegs die volgordes wat 98,5% homologie getoon het, is gekies. In vergelyking met ELISA was die sensitiwiteit van hierdie deteksie metode 100 maal beter. DNA volgordebepaling is op die resulterende fragment gedoen om die PKR produk te verifieer. AFLP tegnologie is gebruik om polimorfiese fraqmente vir 6 okkerneut kultivars en 'n onderstok te identifiseer en SCARs is uit hierdie fragmente ontwikkel. Die AFLP tegniek kon tussen al die okkerneut kultivars en die onderstok onderskei. Die omskakeling van die AFLP fragmente in SCAR merkers om sodoende In eenvoudige kragtige tegniek vir kultivar onderskeiding te ontwikkel, was egter nie suksesvol nie. Met die gebruik van 27 AFLP inleier kombinasies, kon polimorfiese fragmente van so hoog as 47.8% verkry word. Die rede hoekom omskakeling onsuksesvol was lê by die aard van die AFLP tegniek. Die SCAR inleiers is ontwikkel uit volyordes intern tot die AFLP inleiers, maar die spesifisiteit van die merkers het juis in die AFLP inleiers gelê en nie in die interne volgordes nie. In hierdie studie, met die gebruik van AFLP, is gevind dat okkerneut kultivars baie naby verwant is. Die AFLP inleierstelle wat gebruik is, het polimorfiese fragmente gelewer. Uit hierdie fragmente is 7 SCAR merkers ontwikkel. Daar is verwag dat die SCARs wat uit die AFLP merkers ontwikkel is, klein verskille tussen kultivars sou opspoor. Dit was egter net die Paradox SCAR merker wat die kultivars in twee groepe kon verdeel. Restriksie ensiem vertering met Rsalop die Chandler SCAR produkte het dieselfde bandpatrone as die van die Paradox SCAR produkte gelewer.

Cassava brown streak disease (CBSD) threatens cassava production in eastern and southern African countries. Diagnostic protocols currently available for the causal agents of CBSD, Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV), were unreliable but were urgently needed. In this study, sampling procedures and diagnostic protocols were developed for accurate and reliable detection of both CBSV and CBSUV. The cetyltrimethylammonium bromide (CTAB) method of RNA extraction was optimized for sample preparation from infected cassava plants and compared with the commercial kit RNeasy (Qiagen) for sensitivity and reproducibility. Results showed that both protocols were reliable but CTAB was more cost-effective and ideal for resource-poor laboratories. Mixed infections of cassava mosaic begomoviruses (CMBs) that cause cassava mosaic disease (CMD), CBSV and CBSUV have become more common with the recent spread of CBSD at mid-altitudes. A multiplex PCR for the simultaneous detection of viruses that cause both diseases, the first of its kind for cassava, was therefore developed to detect CBSV and CBSUV along with the three commonly occurring CMBs (African cassava mosaic virus (ACMV)), East African cassava mosaic virus (EACMV), and East African cassava mosaic virus-Uganda (EACMVUG) in eastern Africa. Similarly, a duplex PCR was developed for the simultaneous detection of CBSV and CBSUV, both viruses being detected in field-collected samples from Tanzania and Kenya. The genetic diversity of more than 40 CBSD isolates from Kenya, Tanzania, Uganda, and Mozambique was further examined by sequencing the coat protein (CP) gene and partial HAM1 gene sequences. The phylogenetic tree clustered the CBSD isolates into two groups reflecting the two virus species causing CBSD. In this study, various strategies were carried out for generating infectious clones of CBSV; gateway cloning, in vivo and in vitro transcription methods, and amplification of the viral genome in three fragments. Although 3 overlapping CBSV fragments were successfully cloned, the presence of an unexpected mutation at one of the cloning sites unfortunately did not allow reassembling of the fragments to construct the full-length cDNA.

A major therapeutic challenge in musculoskeletal regenerative medicine is how to effectively replenish bone tissue lost due to pathological conditions such as fracture, osteoporosis, or rheumatoid arthritis. Mesenchymal stem cells are currently investigated for applications in bone-tissue engineering and human bone marrow-derived mesenchymal stem cells (hMSCs) could be a promising source for generation of tissue-engineered bone. However, the therapeutic potential of MSCs has not been fully exploited due to a lack of knowledge regarding the identity, nature, and differentiation of hMSCs. Epigenetic mechanisms regulating the chromatin structure as well as specific gene transcription are crucial in determination of stem cell differentiation. With the aim to systematically identify epigenetic factors that modulate MSC differentiation, the work in this thesis encompasses an approach to identify epigenetic mechanisms underlying, initiating, and promoting osteoblast differentiation, and the investigation of individual epigenetic modulators. Various osteogenic inducers were validated for differentiation of MSCs and an assay allowing assessment of differentiation outcome was developed. This assay was subsequently employed in knockdown experiments with lentiviral short hairpin RNAs and inhibitor screens with small molecules targeting putative druggable epigenetic modulator classes. This approach identified around 100 epigenetic modulator candidates involved in osteoblast differentiation, of these candidates approximately 2/3 downregulated and 1/3 upregulated alkaline phosphatase (ALP) activity. Serving as a proof-of-concept, orthogonal validation experiments employing locked nucleic acid (LNA) knockdown were performed to validate a subset of candidates. Two identified target genes were selected for further investigation. Bromodomain-containing protein 4 (BRD4) was identified as one component of epigenetic regulation; its inhibition led to a decrease in ALP expression, downregulation of key osteoblast transcription factors Runx2 and Osterix, as well as impaired bone matrix formation. Knockdown of lysine (K)-specific demethylase 1A (KDM1A/LSD1) upregulated ALP activity and treatment with a small molecule inhibitor targeting KDM1A led to an increase in ALP, RUNX2, and bone sialoprotein expression. Intriguingly, in a transgenic mouse model overexpressing Kdm1a a decrease in bone volume and bone mineral density was observed, thus supporting the hypothesis that KDM1A is a central regulator of osteoblast differentiation.

Thesis (PhD)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Apple stem grooving virus (ASGV) is globally associated with latent infection in commercial apple trees. Little is known about this plant-­‐virus interaction. This study made use of next-­‐generation sequencing to investigate the effect of virus-­‐infection on the expression of the different small RNA (sRNA) species namely, miRNAs, nat-­‐siRNAs, phasiRNAs, rasiRNAs, tRNA-­‐derived sRNAs and vsiRNAs. Broad and narrow size-­‐range datasets were generated using sRNA libraries prepared from total and size-­‐selected RNA, respectively. Through bioinformatic data analyses, 130 genomic loci were predicted to give rise to miRNAs, 85 of which were novel MIR genes. Targets were predicted for the majority of miRNAs, a few of which could be validated with a publicly available degradome dataset. Cis-­‐ and trans-­‐natural antisense transcripts (NATs) were identified, of which only the latter were highly enriched for sRNAs in their overlapping regions. Transcript as well as genomic regions were also identified that can give rise to phasiRNAs. For 25 of these loci an in-­‐phase miRNA target site was identified, half of which could be validated with the degradome dataset. Nearly all apple repeat sequences in Repbase were associated with sRNA synthesis. sRNAs derived from both ends of mature tRNAs were identified. These sRNAs corresponded to tRFs and tRNA-­‐halves. Reads associated with tRNA-­‐halves were prominent in the broad range datasets. sRNAs, originating from the central regions of tRNAs, were also observed. Analysis of the vsiRNAs suggested the presence of two ASGV genetic variants in two of the samples, while the third sample was infected with only one variant. Comparison of the vsiRNA profiles generated from the two datasets highlighted the influence of library preparation on the interpretation of results. Differential expression analysis of the identified apple sRNA species showed no variation between healthy and infected plants, except for the tRNA-­‐derived sRNAs, which did show altered expression levels. Taken together, the various sRNA species characterised in this study significantly extended the existing knowledge of apple sRNAs and provide a broad platform for future functional studies in apple. This study also presents the first and most comprehensive report on sRNAs involved in ASGV infection in apple.
AFRIKAANSE OPSOMMING: Appel gleufstam virus (ASGV) word wêreldwyd geassosieer met latente infeksie in kommersiële appelbome. Min inligting oor hierdie plant-­‐virus interaksie is beskikbaar. Hierdie studie het van volgende-­‐generasie volgordebepaling gebruik gemaak om die effek van virusinfeksie op die uitdrukking van verskillende klein RNA (sRNA) spesies, nl. miRNAs, nat-­‐siRNAs, phasiRNAs, rasiRNAs, tRNA-­‐afkomstige sRNAs en vsiRNAs, te ondersoek. Datastelle met breë en smal grootte-­‐verspreiding is gegenereer m.b.v. sRNA biblioteke wat onderskeidelik voorberei is vanaf totale RNA en RNA van ‘n bepaalde grootte. Deur middel van bioinformatiese data-­‐ontleding is 130 genomiese loci voorspel wat aanleiding kan gee tot miRNAs, waarvan 85 nuwe MIR gene is. Teikens is voorspel vir die meerderheid van die miRNAs en 'n aantal daarvan kon bevestig word m.b.v. 'n publiek-­‐beskikbare degradoom datastel. Cis-­‐ en trans-­‐natuurlike antisense transkripte (NATs) is geïdentifiseer, waarvan slegs die laasgenoemde verryk was vir sRNAs in hul oorvleuelende areas. Transkrip sowel as genomiese areas, wat aanleiding kan gee tot phasiRNAs, is ook geïdentifiseer. Vir 25 van hierdie loci is 'n in-­‐fase miRNA teiken geïdentifiseer, waarvan die helfte bevestig kon word met die degradoom datastel. Byna al die appel herhalende volgordes in Repbase was geassosieer met sRNA sintese. sRNAs afkomstig van beide kante van volwasse tRNAs is geïdentifiseer. Hierdie sRNAs het ooreengestem met tRFs en tRNA-­‐helftes. Volgordes geassosieer met tRNA-­‐helftes was prominent in die breë grootte-­‐verspreiding datastelle. sRNAs, afkomstig van die sentrale dele van tRNAs, is ook waargeneem. Ontleding van die vsiRNAs het die teenwoordigheid van twee ASGV genetiese variante in twee van die monsters aangetoon, terwyl die derde monster met slegs een variant geïnfekteer was. Die vergelyking van vsiRNA profiele, gegenereer vanaf die twee datasteltipes, beklemtoon die invloed van biblioteek voorbereiding op die interpretasie van resultate. Ontleding van die differensiële uitdrukking van die geïdentifiseerde appel sRNA spesies het geen verskil tussen gesonde en geïnfekteerde plante getoon nie, behalwe vir die tRNA-­‐afkomstige sRNAs, wat wel verandering die vlak van uitdrukking getoon het. Die verskillende sRNA spesies wat in hierdie studie geïdentifiseer is, het die bestaande kennis van appel sRNAs aansienlik uitgebrei en bied 'n breë platform vir toekomstige funksionele studies in appel. Hierdie studie bied ook die eerste, en mees omvattende verslag oor sRNAs betrokke in ASGV infeksie in appel.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
Grapevine Vitivirus A (GVA), genus Vitivirus, family Flexiviridae is a well characterised single-stranded RNA virus that has been implicated in the grapevine diseases, Kober stem grooving and Shiraz disease. The virus infects both its host, Vitis vinifera and the experimental model plant, Nicotiana spp.. Biological studies performed on the virus in its herbaceous host, Nicotiana benthami- ana, revealed that many divergent variants of the virus exists in South Africa and can induce di erent symptoms in the model plant. Further molecular analysis divided the variants into three molecular groups based on molecular heterogeneity and nucleotide identity. The establishment of an infectious full-length cDNA clone of GVA contributed towards the elucidation of gene functions for 4 of the 5 open reading frames (ORF's), and indicated ORF5 as the pathogenicity determinant within the genome. Further studies also showed that ORF5 encodes for a nucleic acid binding protein that exhibits suppression activity of a plants' natural virus silencing mechanism. Many proteins that have previously been identi ed as the pathogenicity determinant within a viral genome have been found to encode for suppression activity. Although suppression activity has been elucidated within the ORF5 of the Italian cDNA clone of GVA, IS 151, no such study has yet been performed on the divergent South African variants of GVA. Three variants, GTR1-1, GTR1- 2 and GTG11-1, which represent each of the molecular groups (Group III, II and I), were selected for this study. The aim of this study was to visually elucidate suppression activity of RNA transgene silencing by the ORF5's of GTR1-1, GTR1-2 and GTG11-1 in a transient expression assays in transgenic N. benthamiana (line 16c). Pathogenicity studies for these variants were also performed. The ORF5 of the infectious full-length clone, GVA118, which can also serve as an expression vector, was deleted and provided with restriction enzyme sites into which the respective ORF5s and the marker genes, GFP and GUS could be cloned directionally. Infectivity, symptom development and systemic movement were compared between the di erent full length clones after co-in ltration in N. benthamiana. Preliminary results obtained in this study failed to visually indicate any suppression activity encoded by the ORF5 of GTR1-1, GTR1-2 and GTG11-1. The deletion of ORF5 within GVA118 was successful and rendered the infectious full length clone asymptomatic. Directional cloning of the ORF5 of GTR1-1 into the unique restriction enzymes provided previously, resulted in much milder symptoms than those observe for GTR1-2 and GTG11-1. No GFP and GUS accumulation could be detected. This study has established an infectious full-length cDNA clone, pBINSN-e35SGVA118 ORF5-1-1-pA, that can possibly induce much milder symptoms in the herbaceous host, N. benthamiana. This construct can be further characterised as a possible expression vector of foreign proteins in herbaceous hosts and grapevine.

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies.
AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.

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Fundação de Amparo a Pesquisa do Estado de Minas Gerais
Receptores cinases (RLKs) compõem uma grande famíla de proteínas transmembrânicas que possuem funções importantes na propagação e percepção de sinais celulares nas plantas. Em Arabidopsis thaliana, a superfamília de RLK é composta de mais de 600 membros e vários destes, principalmente aqueles que possuem repetições ricas em leucina (LRR), são considerados excelentes alvos para manipulação molecular em cultivares superiores no intuito de aumentar a produtividade e a resistência contra estresses bióticos e abióticos. A subfamília LRRII é particularmente relevante neste aspecto uma vez que seus membros apresentam funções duplas tanto no desenvolvimento quanto na resposta de defesa da planta. Apesar da relevância desta superfamília e da recente finalização do sequenciamento do genoma de tomateiro, a superfamília de RLK de tomate ainda não se encontra caracterizada e são poucos os trabalhos que analisaram a função biológica de seus membros. Neste trabalho, foi construído um inventário completo dos membros da superfamília de RLK de tomate. Para identificar os membros da superfamília RLK em tomate, foi realizado uma análise filogenética utilizando a superfamília de RLK de Arabidopsis como modelo. Um total de 647 RLKs foram recuperados do genoma de tomate e estes encontravam- se organizados no mesmo clado das subfamílias de RLKs de Arabidopsis. Apenas oito das 58 subfamílias exibiram expansão/redução específica no número de menbros comparado com Arabidopsis e apenas seis RLKs foram específicos em tomate, indicando que os RLKs de tomate compartilham aspectos funcionais e estruturais com os RLKs de Arabidopsis. Também foi caracterizado a subfamília LRRII através de análises filogenéticos, genômico, expressão gênica e interação com o fator de virulência de begomovírus, o nuclear shuttle protein (NSP). Os membros da subfamília LRRII de tomate e Arabidopsis demonstraram-se altamente conservados tanto em sequência quanto em estrutura. No entanto, a maioria dos pares ortólogos não mostraram conservados em relação à expressão gênica, indicando que estes ortólogos tenham se divergido na função após a especiação do ancestral comum entre o tomate e Arabidopsis. Baseado no fato de que membros de RLKs de Arabidopsis (NIK1, NIK2, NIK3 e NsAK) interagem com o NSP de begomovirus, foi verificado se ortólogos de NIKs, BAK1 e NsAK interagem com o NSP de Tomato Yellow Spot Virus (ToYSV). Os ortólogos dos genes que interagem com o NSP em tomate, SlNIKs e SlNsAK, interagiram especificamente com NSP na levedura e demonstraram um padrão de expressão consistente com o padrão de infecção de geminivírus. Além de sugerir uma analogia funcional entre estes ortólogos, estes resultados confirmam a observação anterior de que as interações NSP-NIK não são específicos para um vírus ou para um hospedeiro. Portanto, a sinalização antiviral mediado por NIK provavelmente ocorre em tomate, sugerindo que NIKs de tomate sejam alvos potenciais para manipular a resistência contra begomovírus que infectam esta planta.
Receptor-like kinases (RLKs) represent a large family of transmembrane proteins that play important roles in cellular signaling perception and propagation in plants. In Arabidopsis thaliana, the RLK superfamily is made-up of over 600 proteins and many of these RLKs, mainly those bearing leucine-rich repeats (LRR), have been considered as excellent targets for engineering superior crops with enhancement of yield and resistance to biotic and abiotic stresses. The LRRII-RLK subfamily is particularly relevant due to the dual function of its members in both development and defense. In spite of the relevance of the RLK family and the completion of the tomato genome sequencing, the tomato RLK family has not been characterized and a framework for functional predictions of the members of the family is lacking. In this investigation we disclosed a complete inventory of the members of the tomato RLK family. To generate a complete list of all members of the tomato RLK superfamily, we performed a phylogenetic analysis using the Arabidopsis RLKs as a template. A total of 647 RLKs were identified in the tomato genome, which were organized into the same RLK subfamily clades as Arabidopsis. Only eight of 58 RLK subfamilies exhibited specific expansion/reduction compared to their Arabidopsis counterparts and only six proteins were lineage-specific in tomato, indicating that the tomato RLKs share functional and structural conservation with Arabidopsis. We also characterized the LRRII-RLK family by phylogeny, genomic analysis, expression profile and interaction with the virulence factor from begomoviruses, the nuclear shuttle protein (NSP). The LRRII subfamily members from tomato and Arabidopsis were highly conserved in both sequence and structure. Nevertheless, the majority of the orthologous pairs did not display similar conservation in the gene expression profile, indicating that these orthologs may have diverged in function after speciation of tomato and Arabidopsis common ancestor. Based on the fact that members of the Arabidopsis RLK superfamily (NIK1, NIK2, NIK3 and NsAK) interact with the begomovirus nuclear shuttle protein (NSP), we examined whether the tomato orthologs of NIK, BAK1 and NsAK genes interacted with NSP of Tomato Yellow Spot Virus (ToYSV). The tomato orthologs of NSP interactors, SlNIKs and SlNsAK, interacted specifically with NSP in yeast and displayed an expression pattern consistent with the pattern of geminivirus infection. In addition to suggesting a functional analogy between these phylogenetically classified orthologs, these results expand our previous observation that NSP-NIK interactions are neither virus-specific nor host-specific. Therefore, NIK-mediated antiviral signalling is also likely to operate in tomato, suggesting that tomato NIKs may be good targets for engineering resistance against tomato-infecting begomoviruses.

Infection of the resistant Arabidopsis ecotype Di-17 with Turnip Crinkle Virus (TCV) elicits hypersensitive response (HR), accompanied by increased expression of defense genes. HR to TCV is conferred by HRT, which encodes a coiled-coil (CC)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class of resistance (R) protein. In contrast to HR, resistance requires HRT and a recessive locus designated rrt. Unlike most CC-NBS-LRR R proteins, HRT-mediated resistance is dependent on EDS1 and independent of NDR1. Resistance is also dependent on salicylic acid (SA) pathway and light. A dark treatment, immediately following TCV inoculation, suppresses HR, resistance and activation of a majority of the TCV-induced genes. To determine the genetic, molecular and biochemical basis of light-dependent defense pathway, we studied the role of various photoreceptors in HRT-mediated resistance to TCV, HRT protein levels and its localization. Interestingly, mutation in blue-light photoreceptors led to degradation of HRT via a proteasome-dependent pathway and resulted in susceptibility to TCV. Exogenous application of SA induced transcription of HRT, which restored HRT levels in some, but not all, mutant backgrounds. These results show that different photoreceptors function distinctly in maintaining post-transcriptional stability of HRT. In addition to photoreceptors, HRT also forms a complex with several other proteins, many of which participate in the RNA silencing pathway and are required for HRT-mediated resistance. Together, our results suggest that HRT forms a multi-protein complex and that HRT-mediated signaling involves reconstitution of this complex.

Os vírus possuem potencial de variabilidade genética muito alto, isso porque necessitam divergir seu material genético suficientemente para se adaptar às inúmeras mudanças às quais são submetidos. Portanto, a variabilidade genética é essencial para a sobrevivência desses organismos; é o primeiro passo para a adaptação em um novo hospedeiro, quebra de resistência, alterações nos sintomas e virulência, o que justifica o interesse em estudos nessa área. Os estudos de variabilidade consistem numa excelente ferramenta para a compreensão da evolução dos vírus e busca pelo manejo adequado de doenças virais. Por isso objetivou-se estudar a variabilidade genética da ORF p29 do CiLV-C, a fim de gerar informações relevantes acerca do patossistema e da preponderância de isolados, com possíveis implicações na epidemiologia da doença e seu manejo no campo, além de uma melhor compreensão sobre a evolução desse vírus, que até então nunca havia sido explorada. Neste trabalho foram avaliadas plantas de citros e outras hospedeiras potenciais do CiLV-C. Os resultados sugerem que as plantas de tangerina Cravo, Tardia da Sicília, Cleópatra, Vermelha, tangor Ortanique, laranja Azeda e trapoeraba são suscetíveis à doença e também podem servir como fontes de inóculo do vírus para citros. Já as plantas de limão Siciliano e Cravo, e limas ácidas Tahiti e Galego e Mimosa caesalpiniaefolia mostraram-se resistentes à doença, mas não à colonização do ácaro vetor. As plantas de Malvaviscus arboreus e Solanum violaefolium não apresentaram sintomas, mas mostraram-se possíveis fontes de inóculo do vírus para plantas de citros. Além disso, foram avaliadas as respostas de 62 genótipos de tangerinas e seus híbridos à doença, sendo que 15 mostraram-se resistentes e podem, posteriormente, ser utilizados em programas de melhoramento genético, que é uma das alternativas para reduzir o uso de pesticidas para o controle do vetor. Foi identificada baixa variabilidade genética entre os isolados do CiLV-C, independentemente do hospedeiro ou localidade, entretanto, o isolado de São José do Rio Preto pareceu ser o mais divergente e capaz de passar suas alterações durante sua transmissão a outros hospedeiros. Mais estudos devem ser feitos para que conclusões inquestionáveis sejam tiradas desse assunto, mas os resultados obtidos abriram um novo leque de possibilidades para futuros estudos nessa área até então pouco explorada.
Viruses have, potentially, broad genetic variability because of their need to adapt to several changes that they are exposed to. Therefore, genetic variability is essential for their survival; it is the first step to adapt to a new host, to break resistance down, to change symptoms and virulence, which justifies the interest in studies in this area. These studies consist in a great tool for a better understanding on the virus evolution and the search for a proper management of viral diseases. Hence, it was aimed to study the genetic variability of ORF p29 from CiLV-C in order to generate relevant information about the pathosystem and the predominance of isolates with possible implications on the epidemiology of the disease and its management in the field, besides a better understanding on the evolution of this virus, which has never been explored before. In this work, we evaluated citrus plants and potential hosts for CiLV-C. The results suggest that the plants of Cravo, Tardia da Sicília, Cleopatra, and Vermelha mandarin, Ortanique tangor, Sour orange and spiderwort are susceptible to the disease and can also serve as sources of inoculum of the virus to citrus. Siciliano lemon, Rangpur, Tahiti, and Mexican limes, and Mimosa caesalpiniaefolia were resistant to the disease, but not to the colonization of the mite vector. Malvaviscus arboreus and Solanum violaefolium plants did not present symptoms, but can be considered possible sources of CiLV-C inoculum to citrus plants. In addition, we evaluated the response of 62 mandarin genotypes and their hybrids to the disease. Fifteen of them were considered resistant and could be used in breeding programs with the objective to reduce the use of pesticides to control the vector. Low genetic variability was found amongst CiLV-C isolates, regardless of the host or geographic region; however, the São José do Rio Preto isolate was the most divergent and the changes in nucleotides were transmitted to the other hosts. Further studies should be conducted before unquestionable conclusions can be drawn from this issue, but the results obtained here have opened a new range of possibilities for future studies in this area so far almost unexplored.

Thesis (MSc)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Grapevine leafroll is one of the most damaging viral diseases that affect many viticultural regions of the world. Numerous reports over the last few years have associated closterovirus-like particles with leafroll disease. To date, eight serologically distinct closteroviruses have been isolated from leafroll infected vines, of which grapevine leafroll associated closterovirus-3 (GLRaV-3) is the best characterized. Virus resistance in transgenic plants based on the expression of a virusderived gene is known as pathogen-derived resistance. The viral coat protein (CP) gene, which expresses a structural protein responsible for coating the virus particles, was used in the first demonstration of virus-derived resistance. Coat protein-mediated resistance is currently the most feasible and most widely used method to obtain virus resistance in crop plants. The CP gene of a South African isolate of GLRaV-3 infected grapevine was isolated, cloned and sequenced. Double stranded RNA (dsRNA) was extracted from GLRaV-3 infected material and a high molecular weight band, of -18 kb was identified from infected vines. The dsRNA was used as a template in a reverse transcription PCR together with GLRaV-3 CP gene specific primers for the amplification of the GLRaV-3 CP gene (975 bp). The GLRaV-3 CP gene was cloned into the pGem®-T Easy vector. Clones hosting the CP gene in the sense (pLR3CP+) and antisense (pLR3CP-) orientations respectively were obtained. The sequence obtained from these two clones showed 99.26 % similarity to the only other GLRaV-3 CP nucleotide sequence available. The GLRaV-3 CP gene was excised from pLR3CP+ and pLR3CP- and subcloned into a plant expression vector, pCAMBIA 3301 in the sense (pCamBLR3CP+) and antisense (pCamBLR3CP-) orientations respectively, therefore enabling sense and antisense gene expression in transgenic plants. The GLRaV-3 CP gene was also subcloned from pCamBLR3CP+ into another plant expression vector, pCAMBIA 2301 in the sense orientation and designated as pCVSLR3CP+. These three constructs were given to Dr. M. Vivier (Institute for Wine Biotechnology, Stellenbosch) for grapevine transformation experiments. Two of these constructs, pCamBLR3CP+ and pCamBLR3CP- as well as pCAMBIA 3301 were used to transform Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation. Plants were selected for their ability to withstand the herbicide, Basta. This resistance is due to the presence of a plant selectable marker gene on each of these constructs, known as the bar gene. PCR with GLRaV-3 CP gene specific primers showed no amplification of the GLRaV-3 CP gene in the plants transformed with pCamBLR3CP+ and pCamBLR3CP-. Southern blot analysis with the GLRaV-3 CP gene as hybridization probe showed no signal for these plants, thus confirming the PCR results. PCR with bar gene specific primers showed no amplification of the bar gene in the plants infected with pCAMBIA 3301. The plants transformed with pCamBLR3CP+ and pCamBLR3CP- were also screened for the presence of the bar gene. Three of the eight plants tested showed amplification of the -560 bp bar gene. This result suggests that these plants were transformed with pCAMBIA 3301 (vector without the ligated GLRaV-3 CP gene) and not pCamBLR3CP+ or pCamBLR3CP- as had been expected. This project provides preliminary work for the subsequent transformation of grapevine with the GLRaV-3 CP gene, in an attempt to impart virus resistance.
AFRIKAANSE OPSOMMING: Wingerd rolblaar is een van die mees beskadigende virale siektes wat baie wingerd areas in die wêreld aantas. In Aantal verslae oor die afgelope jare het closterovirus partikels met wingerd rolblaar geassosieer. Tot hede, is agt serologiese onderskeibare closterovirusse geïsoleer vanuit geaffekteerde wingerde, waarvan wingerd rolblaar geassosieerde closterovirus-3 (GLRaV-3) die beste gekarakteriseerd is. Virus bestandheid in transgeniese plante gebaseer op die uitdrukking van gene afkomstig vanaf virusse, staan bekend as patogeen-afgeleide weerstand. Die virale kapsule protein (CP) geen vervaardig In strukturele protein wat verantwoordelik is vir die bedekking van die virus partikel. Dié geen was gebruik in die eerste demonstrasie van patogeen-afgeleide weerstand. Kapsuul protein-bemiddelde weerstand is tans die mees praktiese en algemene gebruikte metode om virus weerstand in plant gewasse te verkry. Die CP geen van In Suid Afrikaanse isolaat van GLRaV-3 geïnfekteerde wingerde is geïsoleer, gekloneer en die volgorde is bepaal. Dubbelstring RNA (dsRNA) was uit GLRaV-3 geïnfekteerde materiaal geëkstraheer en In hoë molekulêre gewig band van -18 kb is geïdentifiseer. Die dsRNA is gebruik as In templaat vir In omgekeerde transkripsie PKR saam met GLRaV-3 CP geen spesifieke inleiers vir die amplifikasie van die GLRaV-3 CP geen (975 bp). Die GLRaV-3 CP geen is gekloneer in die pGem®-T Easy vektor. Klone met die CP geen in die sin (pLR3CP+) en teensin (pLR3CP-) oriëntasies respektiewelik is verkry. Die volgorde wat verkry is vanuit hierdie twee klone dui op In 99.26 % ooreenstemming met die enigste ander GLRaV-3 CP geen volgorde wat beskikbaar is. Die GLRaV-3 CP geen is uit pLR3CP+ en pLR3CP- gesny en is gesubkloneer in In plant ekspressie vektor, pCAMBIA 3301 in die sin (pCamBLR3CP+) en teensin (pCamBLR3CP-) oriëntasies respektiewelik, wat die sin en teensin geen ekspressie in transgeniese plante in staat stel. Die GLRaV-3 CP geen was ook gesubkloneer vanaf pCamBLR3CP+ in In ander plant ekspressie vektor, pCAMBIA 2301 in die sin orientasie en is as pCVSLR3CP+ benoem. Hierdie drie konstruksies is aan Dr. M. Vivier (Instituut vir Wyn Biotegnologie, Stellenbosch) gegee vir wingerd transformasie eksperimente. Twee van hierdie konstruksies, pCamBLR3CP+ en pCamBLR3CP- asook pCAMBIA 3301 is gebruik om Nicotiana tabacum deur middel van Agrobacterium tumefaciens-bemiddelde transformasie te transformeer. Plante is geselekteer vir hul vermoë om die onkruiddoder, Basta, te weerstaan. Die teenwoordigheid van die plant selekteerbare merker geen, bar, op elke konstruksie lui tot dié weerstand. Die plante wat getransformeer is met pCamBLR3CP+ en pCamBLR3CP- is deur PKR saam met die GLRaV-3 CP geen spesifieke inleiers getoets, en geen amplifikasie van die GLRaV-3 CP geen is getoon nie. Southern blot analise met die GLRaV-3 CP geen as hibridisasie peiler het geen sein gewys vir hierdie plante nie, wat die PKR resultate bevestig. Die plante wat getransformeer is met pCAMBIA 3301 is deur PKR saam met die bar geen spesifieke inleiers getoets, en geen amplifikasie van die bar geen is getoon nie. Die plante wat getransformeer is met pCamBLR3CP+ en pCamBLR3CP- is ook getoets vir die teenwoordigheid vir die bar geen. Drie van die agt plante wat getoets is, het amplifikasie van die -560 bp bar geen getoon. Hierdie onverwagte resultate stel voor dat dié plante met pCAMBIA 3301 (vektor sonder die geligeerde GLRaV-3 CP geen) en nie met pCamBLR3CP+ en pCamBLR3CPgetransformeer is nie. Hierdie projek verskaf voorlopige werk vir die daaropvolgende transformasie van wingerd met die GLRaV-3 CP geen in 'n poging om virus bestandheid te verskaf.

Tesis por compendio
[ES] Los virus de plantas poseen la capacidad de parasitar células vegetales y poner a su disposición la maquinaria celular de la planta para la síntesis de sus propias proteínas. Aprovechando esta capacidad, la ingeniería genética ha dirigido muchos de sus esfuerzos en la modificación del genoma viral para utilizar los virus de plantas como vectores de expresión de proteínas de interés humano. Los potyvirus son un grupo de virus de plantas ampliamente estudiado y utilizado en biotecnología. Su genoma está formado por un RNA de cadena sencilla que codifica, principalmente, una poliproteína que se procesa para dar aproximadamente 10 proteínas maduras. En diferentes posiciones intercistrónicas se pueden insertar cDNAs que codifican proteínas de interés, las cuales se producen junto al resto de productos de la poliproteína. Si además estos cDNAs se flanquean por secuencias que codifican los sitios de procesamiento específicos de las proteasas virales, las proteínas heterólogas se liberan eficientemente de la poliproteína viral. Así, la inserción de un cDNA correspondiente al factor de transcripción Rosea1 de la ruta de las antocianinas en distintos potyvirus resulta en la biosíntesis de estos compuestos en las células vegetales infectadas. Las antocianinas son compuestos flavonoides coloreados que se pueden observar a simple vista, por lo que la expresión de Rosea1 es una herramienta biotecnológica muy útil para seguir la infección de virus de plantas. En esta Tesis se han investigado diferentes aplicaciones biotecnológicas basadas en la expresión del factor de transcripción Rosea1 y la consecuente acumulación de antocianinas en el contexto de la biología de los potyvirus. Primero, se construyó un clon del virus del mosaico amarillo del calabacín (ZYMV) etiquetado con Rosea1 (ZYMV-Ros1). El ZYMV es capaz de replicarse a nivel local pero no de moverse a larga distancia en plantas de Nicotiana benthamiana. Un análisis de la infección por ZYMV-Ros1 en una serie de plantas transgénicas de N. benthamiana silenciadas en distintas RNasas de tipo Dicer (DCL) permitió profundizar en el conocimiento de los mecanismos defensivos de la planta frente a este virus. Los resultados indicaron que DCL4 está implicada en restringir el movimiento sistémico del virus, ya que en plantas con este gen silenciado el virus es capaz de moverse a larga distancia. Además, las antocianinas tienen un gran interés nutricional, farmacéutico e incluso industrial, por su gran actividad antioxidante. Un clon viral derivado del virus Y de la patata (PVY) que expresaba Rosea1 (PVY-Ros1) indujo la acumulación de más antocianinas que las que contienen frutas y verduras que son fuente rica de estos valiosos antioxidantes. Este mismo clon PVY-Ros1 también se utilizó para estudiar la transmisión del virus mediante áfidos vectores, observándose como estos provocan frecuentemente el inicio de la infección en el tejido vascular. Este mismo clon viral permitió mostrar visualmente el efecto antiviral que tienen las nanopartículas de plata en plantas. Por último, en esta Tesis se combinó el factor de transcripción Rosea1 con distintas proteasas de los potyvirus para crear circuitos lógicos de regulación génica en plantas. Se observó que la fusión de distintas proteínas virales, como la proteína de inclusión nuclear b (NIb) de los potyvirus o fragmentos de ella, al extremo carboxilo terminal de Rosea1 inhibía la actividad del factor de transcripción. Sin embargo, la acumulación de antocianinas se pudo restablecer insertando sitios de reconocimiento de proteasas virales entre ambas partes y coexpresando tales proteasas. La especificidad de corte y la eficiente actividad catalítica de las proteasas de inclusión nuclear a (NIaPro) del virus del grabado del tabaco (TEV) y del virus de las venas moteadas del tabaco (TVMV) permitió construir circuitos genéticos basados en regulación postranscripcional que son capaces de realizar algunas operaciones lógicas básicas (YES, OR y AND) en tejidos vegetales.
[CA] Els virus de plantes posseeixen la capacitat de parasitar cèl·lules vegetals i posar a la seva disposició la maquinària cel·lular de la planta per a la síntesi de les seves pròpies proteïnes. Aprofitant aquesta capacitat, l'enginyeria genètica ha dirigit molts dels seus esforços a la modificació del genoma viral per utilitzar els virus de plantes com a vectors d'expressió de proteïnes d'interès humà. Els potyvirus són un grup de virus de plantes àmpliament estudiat i utilitzat en biotecnologia. El seu genoma està format per un RNA de cadena senzilla que codifica, principalment, una poliproteïna que es processa per donar aproximadament 10 proteïnes madures. En diferents posicions intercistróniques es poden inserir cDNAs que codifiquen proteïnes d'interès, les quals es produeixen alhora amb la resta de productes de la poliproteïna. Si a més aquests cDNAs es flanquegen per seqüències que codifiquen els llocs de processament específics de les proteases virals, les proteïnes heteròlogues s'alliberen eficientment de la poliproteïna viral. Així, la inserció d'un cDNA corresponent al factor de transcripció Rosea1 de la ruta de les antocianines en diferents potyvirus resulta en la biosíntesi d'aquests compostos en les cèl·lules vegetals infectades. Les antocianines són compostos flavonoides colorits que es poden observar a simple vista, de manera que l'expressió de Rosea1 és una eina biotecnològica molt útil per seguir la infecció de virus de plantes. En aquesta Tesi s'han investigat diferents aplicacions biotecnològiques basades en l'expressió del factor de transcripció Rosea1 i la conseqüent acumulació d'antocianines en el context de la biologia dels potyvirus. Primer, es va construir un clon del virus del mosaic groc del carbassó (ZYMV) etiquetat amb Rosea1 (ZYMV-Ros1). El ZYMV és capaç de replicar-se a nivell local però no de moure's a llarga distància en plantes de Nicotiana benthamiana. Un anàlisi de la infecció per ZYMV-Ros1 en una sèrie de plantes transgèniques de N. benthamiana silenciades en diferents RNases de tipus Dicer (DCL) va permetre aprofundir en el coneixement dels mecanismes defensius de la planta front aquest virus. Els resultats van indicar que DCL4 està implicada en restringir el moviment sistèmic del virus, ja que en plantes amb aquest gen silenciat el virus és capaç de moure's a llarga distància. A més, les antocianines són objecte d'un gran interès nutricional, farmacèutic i fins i tot industrial, per la seva gran activitat antioxidant. Un clon viral derivat del virus Y de la patata (PVY) que expressava Rosea1 (PVY-Ros1) va induir l'acumulació de més antocianines que les que contenen fruites i verdures que són font rica d'aquests valuosos antioxidants. Aquest mateix clon PVY-Ros1 es va utilitzar per estudiar la transmissió del virus mitjançant àfids vectors, observant com aquests provoquen freqüentment l'inici de la infecció en el teixit vascular. Aquest mateix clon viral també va permetre mostrar visualment l'efecte antiviral que tenen les nanopartícules de plata en plantes. Finalment, en aquesta Tesi es va combinar el factor de transcripció Rosea1 amb diferents proteases dels potyvirus per crear circuits lògics de regulació gènica en plantes. Es va observar que la fusió de diferents proteïnes virals, com la proteïna d'inclusió nuclear b (NIb) dels potyvirus o fragments d'ella, a l'extrem carboxil terminal de Rosea1 inhibia l'activitat del factor de transcripció. No obstant això, l'acumulació d'antocianines es va poder restablir inserint llocs de reconeixement de les proteases virals entre les dues parts i coexpressant aquestes proteases. L'especificitat de tall i l'eficient activitat catalítica de les proteases d'inclusió nuclear a (NIaPro) del virus del gravat del tabac (TEV) i del virus de les venes clapejades del tabac (TVMV) va permetre construir circuits genètics basats en regulació postranscripcional que són capaços de realitzar algunes operacions lògiques bàsiques (YES, OR i AND) en teixits vegetals.
[EN] Plant viruses have the ability to parasitize plant cells and use the plant cellular machinery for the synthesis of their own proteins. Taking advantage of this capacity, genetic engineering has focused many of its efforts in modifying the viral genome in order to use modified plant viruses as expression vectors of proteins of human interest. These proteins are stored in host plants that act as low-cost and highly secure biofactories. Potyviruses are a group of plant viruses widely studied and used in biotechnology. Their genome consist of a single-stranded RNA that mainly encodes a polyprotein that is processed in approximately 10 mature proteins. cDNAs flanked by protease specific processing sequences can be inserted in different intercistronic positions and efficiently processed by the viral proteases to produce proteins of interest together with the rest of the viral polyprotein products. Thus, the insertion of the cDNA corresponding to the transcription factor Rosea1 of the anthocyanin pathway in different potyviruses results in the biosynthesis of these compounds in infected plant cells. Anthocyanins are colored flavonoid compounds that can be observed with the naked eye, and thus, the expression of Rosea1 is a very useful biotechnological tool to follow the infection of plant viruses. In this work, we provided different biotechnological applications based on the expression of this transcription factor and the consequent accumulation of anthocyanins in the context of potyvirus biology. First, a zucchini yellow mosaic virus (ZYMV, genus Potyvirus) clone tagged with Rosea1 (ZYMV-Ros1) was constructed. ZYMV is able to replicate locally but not to move long distances in Nicotiana benthamiana plants. We analyzed the infection by ZYMV-Ros1 in a series of N. benthamiana transgenic plants in which the different Dicer-like (DCL) RNases were slilenced. The results showed that DCL4 is involved in restricting the systemic movement of the virus in N. benthamiana plants. This study allowed to deepen the knowledge of the defense mechanisms of the plant against this virus. Besides their biotechnological potential as markers of biological activities, anthocyanins have great nutritional, pharmaceutical and even industrial interest due to their high antioxidant activity. We found that a potato virus Y clone (PVY; genus Potyvirus) expressing Rosea1 (PVY-Ros1) induced the accumulation of a higher amount of anthocyanins than those contained in fruits and vegetables that are a rich source of these valuable antioxidants. The PVY-Ros1 clone was also used to study the transmission of the virus by aphid vectors. We observed that aphids frequently initiate infection in vascular tissue. This viral clone also allowed to visually show the antiviral effect of silver nanoparticles in plants. Finally, in this work, the Rosea1 transcription factor was combined with different potyvirus proteases to create genetic circuits in plants. We observed that the fusion of the nuclear inclusion protein b (NIb) of potyviruses, or fragments of it, to the carboxyl terminal end of Rosea1 inhibited the activity of the transcription factor. However, the accumulation of anthocyanins could be restored by inserting viral protease recognition sites in between NIb and Rosea1 and co-expressing those proteases. The cleavage specificity and the efficient catalytic activity of nuclear inclusion a proteases (NIaPro) of tobacco etch virus (TEV; genus Potyvirus) and tobacco spotted vein virus (TVMV; genus Potyvirus) allowed the construction of genetic circuits based on post-transcriptional regulation that are capable of performing some basic logical operations (YES, OR and AND) in plant tissues.
This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) through grants BIO2014-54269-R and AGL2013-49919-EXP, and by the Greek Ministry for Education and Religious Affairs (Program Aristeia II, 4499, ViroidmiR; ESPA 2007-2013). This research was supported by the Ministerio de Ciencia, Innovación y Universidades (Spain) grants AGL2013-49919-EXP, BIO2014-54269-R, BFU2015-66894-P and BIO2017-83184-R (co-financed FEDER funds) and by the Engineering and Physical Sciences Research Council and the Biotechnology and Biological Sciences Research Council (UK) grant BB/M017982/1.
Cordero Cucart, MT. (2021). Aplicaciones del factor de transcripción Rosea1 de la ruta de las antocianinas como marcador visual en virología molecular y biotecnología de plantas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171455
TESIS
Compendio

An interaction was recently detected between the potyviral protein, genome-linked (VPg) and the Arabidopsis thaliana translation initiation factor eIF(iso)4E (Wittmann et al., 1997).
Here, experiments were undertaken to address biological aspects of the VPg-eIF4E interaction. First, coimmunoprecipitation experiments performed with purified recombinant proteins have shown that VPg not only associates with eIF4E, as was previously published, but also with the larger eIF4F complex, of which eIF4E is a subunit. These results were confirmed by ELISA-type binding assays. It was also shown that there is no direct interaction between VPg and the other subunit of eIF4F, namely eIF4G. Finally, with the same experimental system, it was shown that the presence of eIF4G does not influence the binding affinity of VPg and eIF4E.
The interaction of VPg with the plant translation apparatus suggests that potyviral infection may alter the host protein expression profile. This hypothesis was investigated with the use of a protoplast system. We have shown that the global rates of protein synthesis in protoplasts transfected with an infectious TuMV cDNA clone dropped shortly after transfection, by as much as an estimated 70%. Recovery to normal levels occurred within 48 hours.
Evidence was obtained that the interaction between VPg and eIF4E is instrumental in this transient down-regulation of protein expression: protoplasts transfected with a mutant TuMV cDNA clone, the VPg of which has no affinity for eIF4E, failed to exhibit the drop in protein synthesis observed with the wild-type clone.

A importância da cultura do meloeiro é crescente no Brasil, sobretudo na região Nordeste, tanto pelo volume comercializado como por ser estabelecida geralmente em pequenas propriedades. Diversas enfermidades acometem esta cultura, destacando-se as viroses. Dentre estas, o mosaico, causado pelo Papaya ringspot virus - estirpe melancia (PRSV-W) é das mais importantes. Dentre as estratégias de controle desta doença, o emprego de cultivares resistentes apresenta-se como um método prático e eficiente. O objetivo deste trabalho foi identificar marcadores moleculares do tipo AFLP ligados ao gene Prv1 de resistência a PRSV-W em melão que futuramente pudessem ser utilizados em seleção assistida por marcadores. Para isto, foram analisadas duas linhagens quase-isogênicas (LQI-R e LQI-S) do tipo Amarelo CAC contrastantes para resistência ao vírus e uma linhagem do tipo Charentais doadora do gene de resistência. A LQI resistente foi obtida através de cruzamento entre a linhagem doadora do gene (LRD) e a linhagem recorrente (LQI-S), seguido de cinco retrocruzamentos de plantas resistentes com a linhagem recorrente. A porcentagem do genoma do parental recorrente recuperado na LQI-R foi de aproximadamente 98,44%. Polimorfismos entre as linhagens resistentes e a suscetível foram considerados marcadores candidatos ligados ao gene de reistência Prv1. Para análise de co-segregação entre gene e marcadores candidatos, foi utilizada uma população RC1F1, fenotipada para resistência a PRSV-W, obtida a partir do cruzamento entre as LQIs. Para cálculo da distância entre o gene e os marcadores foi utilizada fórmula de Kosambi para porcentagens de indivíduos recombinantes maiores que 1% e para porcentagens menores admitiu-se ser a distância em centiMorgans equivalente a esta porcentagem. A técnica AFLP em conjunto com a utilização de linhagens quase-isogênicas mostrou-se eficiente na detecção de marcadores moleculares em melão. Para digestão do DNA, foram utilizadas três combinações diferentes de enzimas de restrição (EcoRI/MseI, HindIII/MseI e PstI/MseI), sendo avaliados perfis eletroforéticos gerados a partir da amplificação com 474 combinações diferentes de iniciadores. Aproximadamente 28.700 fragmentos foram analisados, sendo verificada diversidade genética de 8,6% (2462 fragmentos polimórficos) entre as linhagens quase-isogênicas e a linhagem doadora Charentais. Apenas três fragmentos mostraram-se polimórficos na análise da população RC1F1, estando ligados ao gene de resistência a PRSV-W, de acordo com o teste de co-segregação. Os fragmentos EA270 e HF155 mostramse ligados entre si e estão localizados a uma distância aproximada de 40,9 cM do gene Prv1, enquanto o fragmento EK190 mostrou-se ligado ao gene a uma distância de 0,526 cM. Pela sua proximidade ao gene Prv1, o marcador EK190 pode ser utilizado em programas de seleção assistida por marcadores moleculares visando o melhoramento de linhagens com resistência ao vírus PRSV-W.
The growing importance of melon in Brazil is due to the increased production, especially in the Northern region, where crops are established in small properties. Several diseases affect melons. Among the viruses, the mosaic, caused by Papaya ringspot virus – type watermelon (PRSV-W) is the most important. The use of resistant cultivars is a practical and effective method of disease control. The objective of this work was to identify AFLP markers linked to the Prv1 gene that confers resistance to PRSV-W, that in the future could be used in marker assisted selection. Two near isogenic lines (LQI-R and LQI-S) of the Amarelo CAC type that differ with respect to the presence of Prv1 and one Charentais type line donor of the resistance gene were analyzed. The resistant LQI was obtained through the crossing between the donor line (LRD) and the recurrent line (LQI-S), followed by five backcrosses between resistant plants and the recurrent line. The percentage of recurrent parental genome recovered in the LQI-R was approximately 98.44%. Polymorphisms between resistant and susceptible lines were considered as candidate markers linked to the Prv1 resistance gene. An RC1F1 population obtained from a cross between the LQIs lines and screened for resistance to PRSV-W was used in co-segregation analyses. The distance between markers and resistance gene was calculated using the Kosambi equation for recombination fractions higher than 1%. For lower values, the percentage of recombinants was considered equal to the distance in centiMorgans. The AFLP technique combined with the use of nearisogenic lines seemed to be efficient in detecting molecular markers in melon. DNA digestion was performed with three combinations of different enzymes (EcoRI/MseI, HindIII/MseI and PstI/MseI), and electrophoretic profiles of fragments obtained from 474 combinations of different primers were evaluated. Approximately 28,700 fragments were analyzed. Genetic diversity was estimated as 8.6% (2,462 polymorphic fragments) between near-isogenic lines and the donor Charentais line. Only three fragments were found to be polymorphic and linked to the resistance gene. The markers EA270 and HF155 are linked to each other and located 40.9 cM of the Prv1 gene. The fragment EK190 is linked to the same gene with a distance of 0.526 cM. Because EK190 fragment is very close to the resistance gene, it is a suitable marker to be used in marker-assisted selection aiming to develop melon cultivars resistant to PRSV-W.

The aphid transmitted Polerovirus Turnip yellows virus (TuYV) was found to be widespread with high incidences in oilseed rape (OSR) across Europe. UK, France, Germany and Poland all having >90% TuYV incidence in some OSR crops. From the 179 whole TuYV genomes sequenced in this study the phylogenetic analyses indicated three distinct genetic groups in the UK, two of which were also detected in Europe. These three genotypes were also distinct from the original sequenced TuYV-FL. These groups are proposed to be distinct species due to their genetic distance based on the most variable gene ORF5 and phylogenetic analyses of ORF1, ORF3, ORF4 and ORF5. Mixed TuYV infection was uncommon and only two plant samples had genetically distinct isolates. Whole genome analysis also provided valuable information on two recombination hotspots located within TuYV genes ORF3 and ORF5. Investigation into the epidemiology of TuYV revealed many weed and crop species as hosts, including sugar beet, which it was previously thought not to infect. TuYV isolates detected infecting weed plants in the UK were successfully transmitted to OSR. Previously undescribed hosts, verbascum, geranium, teasel, spear thistle, dock and previously described hosts in the Brassicaceae, Compositae and Lepidium families were found in the UK. A full-length infectious clone of a UK isolate of TuYV has been produced, this will allow further assessment of TuYV in the future. The infectious clone was able to cause systemic infection of TuYV and was aphid transmissible. The Arabidopsis thaliana gene knock-out study did not reveal a single eIF gene or gene linked to virus movement or silencing that could provide extreme broad-spectrum resistance. The gene eIF(iso)4G.1 was able to give a broad-spectrum quantitative resistance, and the potential of eIF3D.2 as well as sucrose symporters SUC1 and SUC2 as candidates for extreme TuYV resistance were discovered. This understanding of the epidemiology and diversity of TuYV is being used to develop strategies for control.

Turnip mosaic virus (TuMV) infects a variety of crops, worldwide, including the economically relevant Brassicacea family. It was previously demonstrated that TuMV infection in tobacco protoplasts leads to an overall decrease of host protein. However, it remains unclear whether this phenomenon is due to the repression of plant gene transcription during the infection period or due to viral inhibition of host translation. In this study, quantification of various transcripts and protein products from infected tobacco was performed via real-time RT-PCR and ELISA. In comparison to the gamma-tubulin endogenous control, gene expression for the tobacco H3, HSP70 and granule-bound starch synthase was affected by TuMV infection with time.
Tobacco protein accumulation in whole leaf tissues was also significantly affected by increase of virus particles.

Responses from pedigree selection for resistance to gibberella ear rot were assessed in four maize (Zea mays L.) populations, two selected after inoculation of Fusarium graminearum (Schwabe) macroconidia into the silk channel and two selected after inoculation into developing kernels. Responses were significant in both populations selected for silk resistance and in one of the populations selected for kernel resistance. Selection was more effective in later generations and genetic gains were associated with among-family selection but not with within-family selection. Results obtained here indicate that responses to selection could be more efficiently obtained by applying high selection intensities in advanced generations, by managing earlier generations as bulks and by reducing the number of plants per family. In another experiment, a wide sample of Argentine maize germplasm was evaluated for silk and kernel resistance to gibberella ear rot and to fusarium ear rot (caused by F. verticillioides (Saccardo) Nirenberg [=F. moniliforme (Sheldon)]. Several entries exhibited disease resistance in comparison with local check hybrids, particularly for fusarium ear rot, the most prevalent ear rot in Argentina. Results obtained in this study suggested the presence of general mechanisms controlling silk and kernel resistance to both diseases. In a supplementary study, viral diseases were surveyed in maize fields from the provinces of Ontario and Quebec in 1999 and 2000. Barley yellow dwarf was found in 1999. Sugarcane mosaic, maize dwarf mosaic and wheat streak mosaic were found in 2000. These diseases were not important for grain-maize planted in May, the most prevalent kind of maize crop in these provinces. Some of these diseases, such as sugarcane maize mosaic and maize dwarf mosaic were found important only in maize fields planted during or after the month of June, and this is of commercial relevance only for sweet corn.