Tesi sul tema "Cotton leaf curl virus"

A serious virus disease of cotton in Pakistan is distantly related to cotton leaf crumple in Arizona. It is much more destructive on cotton than leaf crumple, and has never been found in the western hemisphere. Cotton leaf crumple in Arizona causes only modestly damaging midseason infections, while leaf curl, has had a major impact on the crop in Pakistan. Modern transportation and the increasing movement of living plants in global trade has resulted in them recent introduction of a similar disease of another crop to the western hemisphere.

Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the beta C1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, V b, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T-1 plants harbored a single copy of the V beta transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/ CLCuMB complex. Among the test plants, plant V beta-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic V beta-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.

Cultivars and germplasm lines of cotton, Gossvpium hirsutum L., differed in response to infection by the cotton leaf crumple virus (CLCV). The most widely grown cultivars in Arizona and southern California, 'Deltapine 90' and 'Deltapine 61', are susceptible, while ' Cedix', developed in El Salvador, and 'Coral', developed in Nicaragua, are highly resistant or immune. Nineteen other lines from a resistance breeding project in Nicaragua showed highly variable responses.

Pathogen-derived resistance (PDR) strategies, particularly those based on post-transcriptional gene silencing, have been used with great success for the generation of transgenic plants with resistance to RNA viruses. In contrast, a suitable strategy for transgenic resistance to ssDNA plant viruses, including those viruses belonging to the Geminiviridae, has remained elusive. Further, there is no convincing evidence that either post-transcriptional gene silencing, or pathogen-derived resistance in general, would be broadly applicable to ssDNA plant viruses. Researchers at QUT have been developing a novel resistance strategy against ssDNA viruses based on virus-activated expression of a stably integrated suicide gene. The strategy, based on InPAct (In Plant Activation) technology, relies on a "split" suicide gene cassette being arranged in such a way that expression of a lethal ribonuclease (barnase) is dependent on the virus-encoded replication-associated protein (Rep). Upon infection, Rep mediates the release of the construct resulting in the reconstitution of a transcribable and translatable episomal suicide gene expression cassette. The research for this PhD describes the development of an InPAct vector designed to confer resistance to Tomato leaf curl begomovirus (ToLCV), a major cause of disease in Solanaceous crops in the tropics and subtropics. ToLCV-based InPAct vectors were constructed based upon two ToLCV isolates from Australia and North Vietnam. Prior to the generation of InPAct cassettes, the entire ToLCV-[Au] and ToLCV-Vie intergenic regions (IRs) were embedded within the castorbean catalase intron of a β-glucuronidase expression vector to determine the effect of the IR upon transcript processing. Using transient reporter gene assays in tobacco NT-1 cells, it was demonstrated that the ToLCV IRs both contained cryptic intron splice sites which interfered with efficient transcript processing and GUS expression. A series of truncations to the IRs were subsequently made to identify the potential cryptic intron splice sites and/or interfering sequences in both the ToLCV-[Au] and ToLCV-Vie IRs. The final truncated IRs, which were used in the construction the InPAct cassettes, comprised approximately 100 bp and appeared to contain all the necessary cis-acting elements required for efficient rolling circle replication (RCR). Using histochemical GUS assays and Southern analyses, the InPAct cassettes were shown to be activated and replicated only in the presence of the cognate viral Rep. GUS expression levels were shown to be further enhanced in the presence of the ToLCV replication-enhancer protein (REn) and by the addition of the Tobacco yellow dwarf mastrevirus origin of second strand synthesis into the cassette. Under these conditions, Rep-activated GUS expression from the InPAct vectors was found to reach levels similar to that of the benchmark CaMV 35S promoter. Fifteen independent transgenic lines containing the ToLCV-[Au] and -Vie InPAct-GUS cassettes were generated by Agrobacterium-mediated transformation of tobacco leaf discs. Using agroinfiltration and histochemical assays, Rep-mediated activation of the InPAct cassettes and subsequent GUS expression was demonstrated in 11 out of the 15 lines tested; six of which showed expression levels equivalent to, or higher than, that obtained using a CaMV 35S promoter control. Evidence for activation of the integrated InPAct cassettes at the molecular level was provided by Southern analyses, with showed both linear and open circular forms of the replicating InPAct episome in genomic DNA extracted from infiltrated leaf tissue. Following the demonstration of Rep-activatable reporter gene expression and episomal replication of the ToLCV-based InPAct-GUS vectors using transient and stable tobacco transformation assays, new ToLCV-based InPAct vectors were designed to express the lethal RNase, barnase, in an attempt to generate virus resistant plants. Although transient assays in NT-1 cells demonstrated some "leaky" expression of barnase from the InPAct vectors, the level of barnase-mediated cell death from the InPAct vectors was found to be significantly increased in the presence of the cognate Rep and REn. Thirteen independently transformed tobacco lines containing the ToLCV-[Au] InPAct-barnase cassette were generated by Agrobacterium-mediated transformation of tobacco leaf discs. However, agroinfiltration of these plants with ToLCV Rep and REn failed to activate a barnase response. Subsequent molecular analyses on two transgenic lines revealed that both contained mutations in the barnase-coding gene in a region known to encode the active site. These mutations were presumed to result from the leaky barnase expression during initial stages of the Agrobacterium transformation which would favour the selection of barnase mutant InPAct plants. To overcome the problems associated with leaky expression of barnase, a barstar-expression cassette was included in the ToLCV-[Au] InPAct-barnase cassette. Transient assays in non-transgenic tobacco leaves demonstrated that the basal levels of barstar expressed from the modified InPAct vector were sufficient to negate the effects of leaky barnase expression. Importantly, however, the level of barnase expression in the presence of Rep and REn was shown to be sufficient to overcome the basal levels of barstar. Seventeen independently transformed lines were generated with the ToLCV-[Au] InPAct-barnase/barstar cassette, and analysis of one line revealed the presence of an uncorrupted barnase-coding region. Using transient agroinfiltration assays, seven of the transgenic lines showed varying levels of cognate Rep and REn-activated, barnase-induced cell death. Fifteen transgenic lines were challenged with ToLCV-[Au] by injection of recombinant Agrobacteria containing an infectious ToLCV clone. Unfortunately, all lines displayed typical ToLCV symptoms and tested positive for virus by PCR at 28 days post-inoculation. The inability of the InPAct cassette to confer resistance to ToLCV may have been due to one or a combination of factors, including (i) a delay in barnase-induced cell death, (ii) homology-dependent silencing of the integrated cassette, (iii) generally low-level, Rep-activated barnase expression or (iv) excessive virus load due to the artifical method of inoculation. This study details the first report of a ToLCV-based InPAct system for Rep-induced transgene expression in planta. Despite failing to generate ToLCV-resistant plants, the research findings will provide a solid foundation to develop a more effective InPAct vector and ultimately assist in the generation of transgenic plants with resistance to ToLCV and potentially other ssDNA plant viruses, particularly the begomoviruses.

Pathogen-derived resistance (PDR) strategies, particularly those based on post-transcriptional gene silencing, have been used with great success for the generation of transgenic plants with resistance to RNA viruses. In contrast, a suitable strategy for transgenic resistance to ssDNA plant viruses, including those viruses belonging to the Geminiviridae, has remained elusive. Further, there is no convincing evidence that either post-transcriptional gene silencing, or pathogen-derived resistance in general, would be broadly applicable to ssDNA plant viruses. Researchers at QUT have been developing a novel resistance strategy against ssDNA viruses based on virus-activated expression of a stably integrated suicide gene. The strategy, based on InPAct (In Plant Activation) technology, relies on a "split" suicide gene cassette being arranged in such a way that expression of a lethal ribonuclease (barnase) is dependent on the virus-encoded replication-associated protein (Rep). Upon infection, Rep mediates the release of the construct resulting in the reconstitution of a transcribable and translatable episomal suicide gene expression cassette. The research for this PhD describes the development of an InPAct vector designed to confer resistance to Tomato leaf curl begomovirus (ToLCV), a major cause of disease in Solanaceous crops in the tropics and subtropics. ToLCV-based InPAct vectors were constructed based upon two ToLCV isolates from Australia and North Vietnam. Prior to the generation of InPAct cassettes, the entire ToLCV-[Au] and ToLCV-Vie intergenic regions (IRs) were embedded within the castorbean catalase intron of a β-glucuronidase expression vector to determine the effect of the IR upon transcript processing. Using transient reporter gene assays in tobacco NT-1 cells, it was demonstrated that the ToLCV IRs both contained cryptic intron splice sites which interfered with efficient transcript processing and GUS expression. A series of truncations to the IRs were subsequently made to identify the potential cryptic intron splice sites and/or interfering sequences in both the ToLCV-[Au] and ToLCV-Vie IRs. The final truncated IRs, which were used in the construction the InPAct cassettes, comprised approximately 100 bp and appeared to contain all the necessary cis-acting elements required for efficient rolling circle replication (RCR). Using histochemical GUS assays and Southern analyses, the InPAct cassettes were shown to be activated and replicated only in the presence of the cognate viral Rep. GUS expression levels were shown to be further enhanced in the presence of the ToLCV replication-enhancer protein (REn) and by the addition of the Tobacco yellow dwarf mastrevirus origin of second strand synthesis into the cassette. Under these conditions, Rep-activated GUS expression from the InPAct vectors was found to reach levels similar to that of the benchmark CaMV 35S promoter. Fifteen independent transgenic lines containing the ToLCV-[Au] and -Vie InPAct-GUS cassettes were generated by Agrobacterium-mediated transformation of tobacco leaf discs. Using agroinfiltration and histochemical assays, Rep-mediated activation of the InPAct cassettes and subsequent GUS expression was demonstrated in 11 out of the 15 lines tested; six of which showed expression levels equivalent to, or higher than, that obtained using a CaMV 35S promoter control. Evidence for activation of the integrated InPAct cassettes at the molecular level was provided by Southern analyses, with showed both linear and open circular forms of the replicating InPAct episome in genomic DNA extracted from infiltrated leaf tissue. Following the demonstration of Rep-activatable reporter gene expression and episomal replication of the ToLCV-based InPAct-GUS vectors using transient and stable tobacco transformation assays, new ToLCV-based InPAct vectors were designed to express the lethal RNase, barnase, in an attempt to generate virus resistant plants. Although transient assays in NT-1 cells demonstrated some "leaky" expression of barnase from the InPAct vectors, the level of barnase-mediated cell death from the InPAct vectors was found to be significantly increased in the presence of the cognate Rep and REn. Thirteen independently transformed tobacco lines containing the ToLCV-[Au] InPAct-barnase cassette were generated by Agrobacterium-mediated transformation of tobacco leaf discs. However, agroinfiltration of these plants with ToLCV Rep and REn failed to activate a barnase response. Subsequent molecular analyses on two transgenic lines revealed that both contained mutations in the barnase-coding gene in a region known to encode the active site. These mutations were presumed to result from the leaky barnase expression during initial stages of the Agrobacterium transformation which would favour the selection of barnase mutant InPAct plants. To overcome the problems associated with leaky expression of barnase, a barstar-expression cassette was included in the ToLCV-[Au] InPAct-barnase cassette. Transient assays in non-transgenic tobacco leaves demonstrated that the basal levels of barstar expressed from the modified InPAct vector were sufficient to negate the effects of leaky barnase expression. Importantly, however, the level of barnase expression in the presence of Rep and REn was shown to be sufficient to overcome the basal levels of barstar. Seventeen independently transformed lines were generated with the ToLCV-[Au] InPAct-barnase/barstar cassette, and analysis of one line revealed the presence of an uncorrupted barnase-coding region. Using transient agroinfiltration assays, seven of the transgenic lines showed varying levels of cognate Rep and REn-activated, barnase-induced cell death. Fifteen transgenic lines were challenged with ToLCV-[Au] by injection of recombinant Agrobacteria containing an infectious ToLCV clone. Unfortunately, all lines displayed typical ToLCV symptoms and tested positive for virus by PCR at 28 days post-inoculation. The inability of the InPAct cassette to confer resistance to ToLCV may have been due to one or a combination of factors, including (i) a delay in barnase-induced cell death, (ii) homology-dependent silencing of the integrated cassette, (iii) generally low-level, Rep-activated barnase expression or (iv) excessive virus load due to the artifical method of inoculation. This study details the first report of a ToLCV-based InPAct system for Rep-induced transgene expression in planta. Despite failing to generate ToLCV-resistant plants, the research findings will provide a solid foundation to develop a more effective InPAct vector and ultimately assist in the generation of transgenic plants with resistance to ToLCV and potentially other ssDNA plant viruses, particularly the begomoviruses.

The 1985 and 1986 Cotton Reports have the same publication and P-Series numbers.
Cotton leaf crumple (CLC) is incited by a whitefly- transmitted plant virus that infects primarily species within the genus Gossvpium. An extensive host range study was undertaken to identify other hosts which could serve as virus reservoirs in cotton growing areas. More than 20 plant species within the Leguminosae and Malvaceae were identified as CLCV hosts, some of which may be important in the epidemiology of the disease both in cotton and in newly recognized host crop species.

Tesis por compendio
[ES] En las últimas décadas, se ha observado un incremento en la emergencia de nuevas virosis en los cultivos del sudeste español, principal región abastecedora de frutas y hortalizas al resto de Europa. Entre las virosis emergentes más recientes en esta área de cultivo, se encuentra el tomato leaf curl New Delhi virus (ToLCNDV), un begomovirus de genoma bipartito transmitido por moscas blancas (Bemisia tabaci). En 2012 se detectó por primera vez en las provincias de Murcia y Almería, y desde entonces se está propagando por otros países de la cuenca del Mediterráneo, generando graves daños en los cultivos de cucurbitáceas, y limitando la producción, principalmente, de calabacín (Cucurbita pepo) y melón (Cucumis melo). En esta tesis doctoral se ha desarrollado un programa de mejora genética enfocado a la obtención de variedades de cucurbitáceas resistentes al aislado español del ToLCNDV(-ES). Por primera vez, hemos identificado fuentes de resistencia al ToLCNDV en el género Cucurbita y en pepino (Cucumis sativus). Todas las accesiones resistentes que describimos pertenecen a tipos silvestres o variedades locales, la mayoría originarias de la India, donde se describió por primera vez el ToLCNDV. Es posible que en esta área se haya producido un fenómeno de co-evolución entre plantas silvestres de cucurbitáceas y este begomovirus. Dos accesiones de Cucurbita moschata han mostrado un elevado nivel de resistencia, y a pesar de su cruzabilidad intermedia con C. pepo, ha sido posible la obtención de descendencia interespecífica y la transferencia parcial de la resistencia. Las accesiones resistentes identificadas se han caracterizado para determinar el tipo de herencia que regula la resistencia al ToLCNDV. Mediante el desarrollo de poblaciones segregantes de mejora y el aprovechamiento de herramientas genéticas de mapeo y cartografía hemos identificado tres QTLs que controlan la resistencia al ToLCNDV en melón, uno de efecto mayor y herencia parcialmente dominante en el cromosoma 11 y dos que modifican su efecto mediante interacciones epistáticas en los cromosomas 2 y 12. Siguiendo esta estrategia, también hemos identificado un locus de resistencia recesiva a ToLCNDV en el cromosoma 8 de C. moschata, aunque la penetración incompleta cuando se intenta transferir a C. pepo pone de manifiesto la influencia del fondo genético en el carácter. Las regiones genómicas involucradas en la resistencia de ambas especies son sinténicas y agrupan un conjunto de genes comunes no descritos previamente en la resistencia a otros virus en cucurbitáceas. Los genotipados mediante colecciones de SNPs han permitido identificar marcadores moleculares ligados a la resistencia al ToLCNDV en melón, calabaza y calabacín. Estos marcadores suponen un valioso recurso en programas de mejora, ya que permiten transferir de manera asistida las resistencias identificadas a fondos genéticos comerciales. Para profundizar en el conocimiento de los mecanismos moleculares que dan lugar a la resistencia, hemos realizado un ensayo de RNA-seq, comparando los transcriptomas de un genotipo resistente y otro susceptible de melón durante la infección con ToLCNDV. Los resultados obtenidos al analizar la expresión diferencial son compatibles con el tipo de herencia cuantitativa de la resistencia y reflejan un complejo sistema de regulación transcripcional. La infección sistémica del ToLCNDV en melón se ve influenciado por la desregulación de genes implicados en la ruta de señalización hormonal del ácido jasmónico, transportadores transmembrana, fotosíntesis y factores de transcripción. Además, hemos observado cambios en la expresión de genes implicados en la metilación del DNA, tanto en el genotipo resistente como susceptible, lo que sugiere que el silenciamiento génico mediado por RNA puede estar involucrado en la inhibición de la transcripción del genoma viral, favoreciendo la resistencia.
[EN] Different factors promote the emergence of new viruses in crops of the Southeastern Spain, the main supplier region of vegetables and fruits to the rest of European countries. A new strain of tomato leaf curl New Delhi virus (ToLCNDV) is among the most recent emergent viral diseases in this farming area, a bipartite begomovirus naturally transmitted by whiteflies (Bemisia tabaci). This strain was first detected in 2012 in the provinces of Murcia and Almeria, from where it propagated to other Mediterranean countries. ToLCNDV generates severe damages in cucurbits, decreasing yields mainly in squash (Cucurbita pepo) and melon (Cucumis melo) crops. In this doctoral thesis, a breeding program has been developed to obtain cucurbit varieties with resistance to the Spanish strain of ToLCNDV(-ES). Although cucurbit germplasm with resistance genes to this virus is poor and localized in few accessions, in this work we have identified for the first-time resistance sources to ToLCNDV in Cucurbita spp. and cucumber (Cucumis sativus). All resistant accessions here described are landraces or wild types not domesticated or with low levels of genetic manipulation. Moreover, most of these genotypes are originals from India, where ToLCNDV was first described. Likely, the occurrence of co-evolution events took place between wild cucurbits plants and this begomovirus. Despite not identifying resistance sources in squash, two Cucurbita moschata accessions have shown high resistance levels. Both species present intermediate crossability, even so the obtention of interspecific offspring and the partial transference of the resistance was possible. The resistant accessions identified in the works of this thesis and those identified by our group in previous assays, have been characterized to determine the heritage controlling ToLCNDV resistance. The development of breeding segregating populations and exploiting getenic tools for mapping and cartography, we identifed three QTLs controlling resistance to ToLCNDV in melon, one partially dominant with mayor effect in chromosome 11, and two minor modifiers in chromosomes 2 and 12. Epistatic interactions between them have been detected. Following the same strategy, we have also identified a locus with recessive heritage in chromosome 8 of C. moschata. However, when we tried to transfer it to C. pepo incomplete penetrance occurrence was observed, reflecting the influence of the genetic background on the trait. The genomic regions identified in both species are synthenic and share a common cluster of genes not described previously in cucurbits conferring resistance to other viruses. The advances in next generation sequencing technologies have offered us a huge quantity of genomic and transcriptomic information. Genotyping by SNPs collections allowed the identification of molecular markers linked to ToLCNDV resistance in melon, pumpkin and squash. These markers suppose a valuable resource in breeding programs, since can be used to assist the transference of the identified resistances to commercial genetic backgrounds. To further understand the molecular mechanism regulating the resistance, we have performed an RNA sequencing assay (RNAseq), comparing transcriptomes between a resistant and a susceptible accession of C. melo in the time course of ToLCNDV infection. The results obtained from the analysis of differential expression levels are compatible whit a complex transcriptional regulation. Deregulation of genes involved in hormonal jasmonic signaling, transmembrane transport, photosynthesis and transcription factors are involved in the ToLCNDV systemic infection through melon plants. Furthermore, in both resistant and susceptible genotypes, we observed expression changes of genes described in DNA methylation pathway. This suggests that RNA silencing mechanisms might be responsible of inhibit viral genome transcription, enhancing the resistance response.
[CA] Durant les últimes dècades, s'ha observat un increment en l'emergència de noves virosis als cultius del sud-est espanyol, principal regió proveïdora de fruites i hortalisses a la resta d'Europa. Entre les virosis emergents més recents en aquesta àrea de cultiu, es troba un nou aïllat del tomato leaf curl New Delhi virus (ToLCNDV), un begomovirus de genoma bipartit transmés per mosca blanca (Bemisia tabaci). En 2012 es va detectar per primera vegada a les províncies de Múrcia i Almeria, i des d'aleshores s'està propagant per altres països de la conca del Mediterrani, ací genera greus danys en els cultius de cucurbitàcies, i hi limita la producció, principalment de carabasseta (Cucurbita pepo) i meló (Cucumis melo). En aquesta tesi doctoral s'ha desenvolupat un programa de millora genètica enfocat a l'obtenció de varietats de cucurbitàcies resistents a l'aïllat espanyol del ToLCNDV(-ES). Hem pogut identificar per primera vegada fonts de resistència al ToLCNDV en el gènere Cucurbita i en cogombre (Cucumis sativus). Totes les accesions resistents que descrivim pertanyen a tipus silvestres o varietats locals, la major part originaris de l'Índia, on es va descriure per primera vegada el ToLCNDV. És possible que en aquesta àrea s'haja produït un fenomen de coevolució entre plantes silvestres de cucurbitàcies i aquest begomovirus. Dos accesions de Cucurbita moschata han mostrat un elevat nivell de resistència, i tot i la compatibilitat intermèdia que presenten ambdues espècies, ha sigut possible l'obtenció de descendència interespecífica i la transferència parcial de la resistència. Les accessions resistents identificades s'han caracteritzat per tal de determinar el tipus d'herència que regula la resistència al ToLCNDV. Mitjançant el desenvolupament de poblacions segregants de millora i l'aprofitament de ferramentes genètiques de mapeig i cartografia, hem identificat tres QTLs que controlen la resistència al ToLCNDV en meló, un d'efecte major i herència parcialment dominant en el cromosoma 11 i dos que modifiquen el seu efecte mitjançant interaccions epistàtiques als cromosomes 2 i 12. Seguint aquesta estratègia, també hem identificat un locus de resistència recessiva a ToLCNDV en el cromosoma 8 de C. moschata, encara que la penetració incompleta quan s'intenta transferir a C. pepo evidència la influència del fons genètic en el caràcter. Les regions genòmiques involucrades en la resistència de les dos espècies són sintèniques i agrupen un conjunt de gens comuns no descrits prèviament en la resistència a altres virus en cucurbitàcies. Els genotipats utilitzant col·leccions de SNPs han permés identificar marcadors moleculars lligats a la resistència al ToLCNDV en meló, carabassa i carabasseta. Aquests marcadors suposen un valuós recurs en programes de millora, ja que permeten transferir de forma assistida les resistències identificades a fons genètics comercials. Per tal d'aprofundir en el coneixement dels mecanismes moleculars que confereixen la resistència, hem realitzat un assaig de seqüenciació del RNA (RNA-seq), i hem comparat els transcriptomes d'un genotip resistent i altre susceptible de meló durant la infecció amb ToLCNDV. Els resultats obtinguts en analitzar l'expressió diferencial són compatibles amb el tipus d'herència quantitativa de la resistència i reflecteixen un complex sistema de regulació transcripcional. La infecció sistèmica del ToLCNDV en meló es veu influenciada per la desregulació de gens implicats en la ruta de senyalització hormonal de l'àcid jasmònic, transportadors transmembrana, fotosíntesi i factors de transcripció. A més, hem observat canvis en l'expressió de gens implicats en la metilació del DNA, tant en el genotip resistent com susceptible, el que suggereix que el silenciament gènic mediat per RNA pot estar involucrat en la inhibició de la transcripció del genoma viral, afavorint la resistència.
La Conselleria d’Educació, Investigació, Cultura i Esports (Generalitat Valenciana) y el Fondo Social Europeo (FSECV 2014-2020) han cofinanciado la contratación de la doctoranda como personal investigador de carácter predoctoral (ACIF/2016/188) y dos estancias predoctorales fuera de la Comunitat Valenciana (BEPFI/2017/011 y BEFPI/2018/028). La realización de esta tesis doctoral también se ha realizado en el marco de dos proyectos de investigación del Ministerio de Ciencia, Innovación y Universidades, con cofinanciación de fondos FEDER [Proyectos AGL2017-85563-C2-1-R y RTA2017-00061-C03-03 (INIA)] y del proyecto PROMETEO para grupos de excelencia (2017/078) de la Conselleria d’Educació, Investigació, Cultura i Esports (Generalitat Valenciana).
Sáez Sánchez, C. (2020). Mejora Genética de la Resistencia al tomato leaf curl New Delhi virus en Cucurbitaceas [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153801
TESIS
Compendio

Les virus du genre Begomovirus (famille Geminiviridae) sont fréquemment détectés en association avec des ADN satellites appelées alphasatellite et betasatellite qui font la moitié de la taille du génome viral. L’alphasatellite est autonome pour sa réplication et dépend du virus pour son mouvement et son encapsidation tandis que le betasatellite est dépendant de ces fonctions virales. L’alphasatellite a rarement été montré comme ayant un impact sur le virus assistant, contrairement au betasatellite qui augmente la virulence de son virus assistant. En dehors des bégomovirus tels que le Cotton leaf curl virus (CLCuV) qui ont besoin d’un betasatellite pour initier une infection symptomatique dans leur hôte naturel, la plupart des bégomovirus peuvent causer des symptômes, même sans les satellites avec lesquels ils sont parfois détectés. Le Tomato yellow leaf curl virus (TYLCV), un des virus les plus dommageables dans le monde a rarement été détecté associé à des ADN satellites. Les souches méditerranéennes qui sont aussi les plus invasives, n’ont jamais été détectées avec des ADN satellites, bien qu’elles soient capables en conditions artificielles de les assister avec pour conséquence une considérable augmentation de la virulence en cas de co-inoculation avec un betasatellite. Le risque potentiel d’association de satellites avec le TYLCV-Mld a été évalué en testant divers facteurs potentiellement impliqués dans le maintien de l’association TYLCV-satellite: (i) l'accumulation relative intra-plante du TYLCV et des satellites, (ii) la fréquence de co-infection au niveau cellulaire du TYLCV et des satellites, et (iii) l'efficacité de transmission des satellites par le vecteur Bemisia tabaci. Trois satellites précédemment isolés sur coton au Burkina Faso ont été montrés comme pouvant être assistés par le TYLCV dans des plantes de tomate: Cotton leaf curl Gezira betasatellite (CLCuGB), Cotton leaf curl Gezira alphasatellite (CLCuGA) et Okra leaf curl Burkina Faso alphasatellite (OLCBFA). La quantification par PCR quantitative des ADN du TYLCV et des trois satellites entre 11 et 150 jours après inoculation (dpi) révèle qu’en général, les satellites ont une accumulation supérieure à celle du virus, et que, contrairement aux alphasatellites qui n’ont aucun impact, le betasatellite affecte l’accumulation du TYLCV-Mld. Bien que le rapport des quantités de virus/satellites varie au cours du temps, les satellites sont maintenus avec le TYLCV-Mld au temps tardif de 150 dpi et sont transmis par B. tabaci à 32 et 150 dpi. Le TYLCV-IL interagit différemment avec le CLCuGB car son accumulation n’est pas affectée dans les plantes coinfectées.L’estimation par la technique FISH à 18 et 32 dpi de la fréquence d’association des molécules au niveau cellulaire montre que plus de la moitié des cellules infectées sont coinfectées par le TYLCV et un satellite. Ce résultat est cohérent avec la fréquence observée d’ADN satellite dans les plantes. Cependant, on observe de manière inattendue un nombre important de cellules ne semblant contenir que le betasatellite, ce qui pose des questions sur le fonctionnement des associations virus/satellites. Comme la multiplicité d'infection (MOI) des bégomovirus et des satellites est attendue pour être un facteur déterminant de l’efficacité de la co-infection cellulaire, deux variants équi-competitifs de TYLCV ont été préparés afin de déterminer ce paramètre. Enfin, des amorces PCR permettant la détection générique de betasatellites ont été dessinées pour être utilisées dans le diagnostic par l'Agence française pour l'alimentation, l'environnement et la santé et sécurité au travail (ANSES). Outre les conséquences agronomiques d’un maintien possible des satellites avec le TYLCV, les résultats de cette étude donnent un aperçu novateur sur les interactions entre les bégomovirus et les satellites, au niveau de la plante, au niveau cellulaire et moléculaire
Begomoviruses (family Geminiviridae) are frequently detected with half genome sized defective virus DNAs, and for some of them with satellite DNAs of similar size, i.e. alphasatellite and betasatellite. Both molecules rely on the virus for maintenance in plant. The alphasatellite was rarely proved to have an impact on the helper virus but the betasatellite was often shown to increase its virulence. Except some begomoviruses, like Cotton leaf curl virus (CLCuV) which rely on a betasatellite for a full symptomatic infection in its natural host plant, most of the begomoviruses which were frequently detected with satellites do not rely on them for infectivity. Tomato yellow leaf curl virus (TYLCV) is one of the most damaging begomovirus worldwide. The Mediterranean IL and Mld strains, the most invasive ones, were never detected in association with satellites, although they were experimentally proved to readily assist them for replication and movement in plant. This was particularly true for betasatellites and resulted in a dramatic increase in the virulence of TYLCV.The potential of a TYLCV-satellite association was assessed by testing various factors involved in the maintenance of both molecules in tomato plants: (i) the relative intra-plant accumulation of TYLCV and satellites, (ii) the frequency of host cells co-infected with TYLCV and satellites, and (iii) the transmission efficiency of satellites by the natural whitefly vector of TYLCV, Bemisia tabaci. Three satellites previously isolated from okra in Burkina Faso, were shown here to be assisted by TYLCV in tomato plants: Cotton leaf curl Gezira betasatellite (CLCuGB), Cotton leaf curl Gezira alphasatellite (CLCuGA) and Okra leaf curl Burkina Faso alphasatellite (OLCBFA). The dynamic of TYLCV and satellite DNAs monitored between 11 and 150 days post-inoculation (dpi) by quantitative PCR revealed that satellites accumulated at a higher level than the virus, and that, in contrast with alphasatellites which have no impact, betasatellites affected TYLCV-Mld accumulation. Although the ratio of virus/satellite amounts varies over time, satellites were maintained in all test plants up to 150 dpi and were readily transmitted at 32 and 150 dpi. TYLCV-IL interacts differentially with CLCuGB as its accumulation was not affected in the coinfected plants.At 32 dpi, the TYLCV/satellite infection status of plant cells was determined by FISH and more than 50% of the monitored infected cells were co-infected with TYLCV and a satellite. The infection status was consistent with the frequency of satellite DNA in plants. Unexpectedly a substantial number of cells were positive only for betasatellite, suggesting that the coinfection with the virus could be dispensable for replication. This observation raises question on the functioning of virus/satellite association or multipartite viruses. As the multiplicity of infection (MOI) of begomoviruses and satellites is expected to be a determinant of the efficiency of virus/satellite cell coinfection, two equi-competitive TYLCV variants were prepared to determine this parameter for TYLCV. Finally, PCR primers designed for the generic detection of betasatellites were designed to be used as a diagnostic tool by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES).Besides the agronomic concern of the possible maintenance of DNA satellites with TYLCV, the results of our study are expected to provide a new insight on the interactions between begomovirus and satellites, at the plant, cellular and molecular levels

BACKGROUND:Tomato leaf curl Sudan virus (ToLCSDV) is a single-stranded DNA begomovirus of tomato that causes downward leaf curl, yellowing, and stunting. Leaf curl disease results in significant yield reduction in tomato crops in the Nile Basin. ToLCSDV symptoms resemble those caused by Tomato yellow leaf curl virus, a distinct and widespread begomovirus originating in the Middle East. In this study, tomato samples exhibiting leaf curl symptoms were collected from Gezira, Sudan. The associated viral genome was molecularly characterized, analyzed phylogenetically, and an infectious clone for one isolate was constructed.FINDINGS:The complete genomes for five newly discovered variants of ToLCSDV, ranging in size from 2765 to 2767-bp, were cloned and sequenced, and subjected to pairwise and phylogenetic analyses. Pairwise analysis indicated that the five Gezira isolates shared 97-100% nucleotide identity with each other. Further, these variants of ToLCSDV shared their highest nucleotide identity at 96-98%, 91-95%, 91-92%, and 91-92% with the Shambat, Gezira, Oman and Yemen strains of ToLCSDV, respectively. Based on the high maximum nucleotide identities shared between these ToLCSDV variants from Gezira and other previously recognized members of this taxonomic group, they are considered isolates of the Shambat strain of ToLCSDV. Analysis of the complete genome sequence for these new variants revealed that they were naturally occurring recombinants between two previously reported strains of ToLCSDV. Finally, a dimeric clone constructed from one representative ToLCSV genome from Gezira was shown to be infectious following inoculation to tomato and N. benthamiana plants.CONCLUSION:Five new, naturally occurring recombinant begomovirus variants (>96% shared nt identity) were identified in tomato plants from Gezira in Sudan, and shown to be isolates of the Shambat strain of ToLCSDV. The cloned viral genome was infectious in N. benthamiana and tomato plants, and symptoms in tomato closely resembled those observed in field infected tomato plants, indicating the virus is the causal agent of the leaf curl disease. The symptoms that developed in tomato seedlings closely resembled those observed in field infected tomato plants, indicating that ToLCSDV is the causal agent of the leaf curl disease in Gezira.

Sabe-se que a grande maioria dos fitopatógenos depende quase que exclusivamente de vetores para disseminarem-se para novos hospedeiros, porém pouco foi estudado no que se refere aos efeitos dos micro-organismos sobre seus insetos vetores. Sendo Bemisia tabaci uma praga de elevada importância e vetora de inúmeros vírus para plantas cultiváveis, é de extrema relevância estudar os efeitos provocados pelos vírus sobre seu desempenho biológico. Assim, esta pesquisa objetivou: a) avaliar os efeitos direto e indireto dos begomovírus Tomato severe rugose vírus (ToSRV) e Tomato yellow leaf curl virus (TYLCV) sobre parâmetros biológicos de B. tabaci biótipo B e Q respectivamente, dos quais: duração e viabilidade do período ninfal; razão sexual; fecundidade; fertilidade e longevidade. Os resultados encontrados variam de positivos, neutros a negativos, dependendo do parâmetro, efeito e espécie do vírus estudado. Sendo assim, observou-se que há efeito direto de ambos os vírus na duração do período ninfal, sendo menor em insetos virulíferos que em sadios. Ademais, há um incremento no número médio de ovos depositados por insetos virulíferos com ToSRV (225,2 ovos/fêmea) quando comparado com insetos sadios (180,1 ovos/fêmea). Já TYLCV afetou diretamente a longevidade de machos os quais tiveram a duração da fase adulta incrementada quando virulíferos (30 dias) e 24 dias quando sadios. Há um efeito indireto negativo de ToSRV sobre a viabilidade ninfal de seu vetor, sendo de 52% quando estes são mantidos em plantas infectadas e 86% em plantas sadias de tomate. A razão sexual também foi afetada por este vírus, favorecendo as fêmeas, sendo de 2:1 a proporção entre fêmeas e machos em plantas infectadas. Ademais, a longevidade de machos foi reduzida drasticamente quando em plantas infectadas com ToSRV. Também foi detectado um efeito indireto positivo do TYLCV sobre a fecundidade de fêmeas de B. tabaci biótipo Q, as quais depositaram em média maior quantidade de ovos em plantas infectadas que em plantas sadias de tomate, sendo 52,8 e 33,2 ovos respectivamente. Tais resultados permitem concluir que, nas condições em que os ensaios foram realizados, ToSRV afeta diretamente de forma positiva seu vetor, enquanto possui efeitos indiretos principalmente negativos sobre parâmetros biológicos de B. tabaci biótipo B. Igualmente TYLCV possui efeitos diretos positivos sobre o biótipo Q da espécie de mosca-branca. Já indiretamente este vírus, diferentemente de ToSRV, afeta positivamente a biologia de seu vetor B. tabaci biótipo Q, favorecendo a fecundidade dos indivíduos que se desenvolveram em plantas infectadas de tomate.
It is known that the vast majority of pathogens relies almost exclusively vector for spreading to new hosts, but little has been studied regarding the effects of micro-organisms on its insect vectors. Bemisia tabaci is a pest of high importance vector of numerous virus to cultivated plants, it is extremely important to study the effects caused by viruses on its biological performance. Thus, this study aimed to: a) evaluate the direct and indirect effects of the begomovirus Tomato severe rugose virus (ToSRV) and Tomato yellow leaf curl virus (TYLCV) on biological parameters of B. tabaci biotype B and Q respectively, of which duration and viability of nymphal development; sex ratio; fecundity; fertility and longevity. The results range from positive, neutral to negative, depending on the parameter, effect and species of the virus studied. Thus, it was observed that there is a direct effect of the both virus in the duration of nymphal development, being less than viruliferous insects in healthy individuals. Moreover, there is an increase in the average number of eggs laid by viruliferous insects with ToSRV (225,2 eggs / female) when compared with healthy insects (180,1 eggs / female). Already TYLCV directly affected the longevity of males which lasted adulthood increased when viruliferous (30 days and 24 when healthy). There is an indirect negative effect on the viability of ToSRV nymphal of its vector, and 52% when they are kept in infected plants and 86% in healthy tomato plants. The sex ratio was also affected by this virus, favoring females, with a ratio of 2:1 between females and males in infected plants. Furthermore, the longevity of males was reduced dramatically when plants infected with ToSRV. We also detected a positive indirect effect of TYLCV on fertility of female B. tabaci biotype Q, which placed greater average number of eggs in infected plants than on healthy plants of tomato, 52,8 and 33,2 eggs respectively. These results indicate that, under conditions in which the tests were performed, ToSRV directly affects positively its vector, while indirect effects has mostly negative on biological parameters of B. tabaci biotype B. TYLCV also has positive direct effects on the Q biotype of the whitefly species. Already indirectly this virus, unlike ToSRV, positively affects the biology of its vector B. tabaci biotype Q, favoring the fecundity of individuals that developed in infected tomato plants.

This project investigated new strategies to improve viral resistance in crops against the increasing global threat of DNA viruses. The research showed that a combination of protective strategies is more likely to be effective and durable than a single approach. A gene from a wild relative was identified as a new potential source of DNA virus resistance.

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Kyoto University (京都大学)
0048
新制・論文博士
博士(農学)
乙第13023号
論農博第2833号
新制||農||1043(附属図書館)
学位論文||H28||N4968(農学部図書室)
32951
(主査)教授 佐久間 正幸, 教授 松浦 健二, 教授 奥野 哲郎
学位規則第4条第2項該当

1) Geminiviruses are a group of plant viruses which contain circular single stranded DNA molecules as their genomes and the capsid consists of two icosahedra fused together to form twinned or geminate particles. The largest genus in the family Geminiviridae is that of begomoviruses which are of two kinds; the monopartite begomoviruses which contain only one circular single stranded DNA molecule as their genome and the bipartite begomoviruses which contain two circular single stranded DNA molecules (designated DNA-A and DNA-B) as their genomes. In bipartite viruses, the two DNA molecules are enclosed in separate geminate capsids. 2) In bipartite begomoviruses, the DNA-A encodes the proteins essential for replication and encapsidation of the viral genome while the DNA-B encodes the proteins involved in movement. The DNA-B encodes two proteins: the BV1 or the nuclear shuttle protein (NSP) and BC1 or the cell-to-cell movement protein. Geminiviruses have DNA genomes which replicate inside the host cell nucleus. The NSP, which contains nuclear localization signal, brings the viral DNA from nucleus to the cytoplasm while the BC1 serves to take the viral genome to the cell periphery for movement to the neighbouring cell through the plasmodesmata. 3) The monopartite begomoviruses do not contain DNA-B (which, in bipartite begomoviruses, encodes the proteins involved in movement) and it has been suggested that some of the proteins encoded by DNA-A take up the movement function. Based on studies on TYLCV and CLCuV, a model has been proposed for the movement of monopartite begomoviruses according to which the coat protein (CP) of monopartite begomoviruses serves as the functional equivalent of the NSP of bipartite begomoviruses 4) The present thesis deals with the biochemical characterization of the C4 protein of the monopartite begomovirus CLCuKV-Dab. As stated in statement (3) above, the V2 and C4 proteins of monopartite begomoviruses have been implicated to be involved in cell-to¬cell movement of the viral genome. In TYLCV, both the proteins were shown to be localized to the cell periphery and could move from one cell to another through the plasmodesmata. Further, the V2 protein of CLCuKV-Dab was shown to interact with the coat protein and bind to single stranded DNA. The biochemical properties of the C4 protein needed to be elucidated in order to strengthen the proposal of its probable involvement in movement. 5) The objectives of the present study were: i) Bioinformatic analysis of the C4 protein of CLCuKV-Dab ii) Biochemical characterization of ATPase and pyrophosphatase activities of the C4 protein. iii) Studies on the effect of V2-C4 interaction on the enzymatic properties of C4. iv) Functional characterization of C4 in planta. 6) The FoldIndex© and PONDR analyses predicted the C4 protein of CLCuKV-Dab to be natively unfolded. Similarly, in PSIpred analysis, most of the C4 protein was predicted to be a random coil without any well-defined secondary structure. Further, the protein sequence was analyzed using the motifscan server. However, no motif for any specific function was predicted in the C4 Protein. 7) The C4 gene was initially cloned into pRSET-C vector and overexpressed as histidine tagged protein and the solubility of the protein was tested in various conditions including low temperature (18° C) after inducing the expression of the protein, buffers of various pH and different salt concentrations but the protein remained insoluble. Subsequently, the protein was purified under denaturing conditions and attempts were made to refold the protein but the protein precipitated during refolding. In order to get the C4 protein in soluble form, the C4 gene was subcloned into pGEX-5X2 vector and overexpressed as a GST-tagged fusion protein (GST-C4). Some of the GST-C4 protein was soluble which was purified by using GST-bind resin. The purified fusion protein was observed as a 37 kDa band on SDS-PAGE gel. The purified protein was accompanied by a degraded product of approximately 30 kDa size. Both the intact GST-C4 protein and the degraded product were detected in western blot analysis using anti-GST antibody. 8) Because C4 has been implicated to be involved in movement of monopartite begomoviruses and movement is an energy requiring process, it was of interest to determine if GST-C4 possesses ATPase activity. The purified GST-C4 protein was incubated with γ-[32P]-ATP, the product of the reaction was separated by thin layer chromatography and the chromatography plate was analyzed by phosphorimager. The hydrolysis of ATP by GST-C4 and the release of inorganic phosphate was clearly observed, suggesting that GST-C4 might possess ATPase activity. 9) The reaction conditions for the ATPase activity of GST-C4 were standardized. The activity increased linearly upto 2.60μM of the protein. The optimum temperature and pH for the ATPase activity were found to be 30 C and 6.0 respectively. The activity was inhibited by EDTA, suggesting that it is dependent on divalent metal ions. The activity was stimulated by Mg+2, Mn+2 and Zn+2 but inhibited by Ca+2ions. Further, in the time course experiment, it was observed that the ATPase activity increased linearly upto one hour. 10) The Km, Vmax and kcat for the ATPase activity of GST-C4 were found to be 51.72 ± 2.5 µM, 7.2 ± 0.54 nmoles/min/mg of the protein and 0.27 min-1 respectively. Some of the other virally encoded ATPases have been found to exhibit kcat similar to that found for GST-C4 but it is much lower than those of most of the prokaryotic and eukaryotic ATPases (as mentioned in Table 3.3, page 100, chapter 3). Further, the presence of the degraded product did not affect the kinetic constants as described in chapter 3, pages 95¬-98. It is possible that the enzymatic activity might increase upon interaction with some ligand. 11) In the absence of any putative ATP binding motifs, systematic deletions from N-and C-termini were made to delineate the regions of C4 important for the ATPase activity. GST-N∆15-C4 and GST-N∆30-C4 exhibited approximately 70 % reduction in the ATPase activity while all the C-terminal deletion mutants (GST-C∆10-C4, GST-C∆20¬C4 and GST-C∆30-C4) retained the activity similar to the full length GST-C4 protein. This suggested that the N-terminal region of C4 may contain the residues important for the ATPase activity of GST-C4. 12) In the N-terminal region of C4, there is a sequence CSSSSR which closely resembles the sequence present at the active site of phosphotyrosine phosphatases (CXXXXXR). However, GST-C4 did not catalyze the hydrolysis of p-Nitrophenyl phosphate, a substrate analogue commonly used to assay phosphotyrosine phosphatase activity. It was of interest to determine if the cysteine and arginine in this sequence are important for the ATPase activity of GST-C4. GST-R13A-C4 exhibited an approximately two fold reduction in Vmax suggesting that R13 in C4 may be catalytically important for the ATPase activity of GST-C4. On the other hand, the C8A mutation did not affect the ATPase activity of GST-C4. 13) The GST-C4 protein was tested for its ability to hydrolyze several other phosphate containing compounds as mentioned chapter 2, pages 53-55. Among these compounds, GST-C4 catalyzed the hydrolysis of sodium pyrophosphate, that is, GST-C4 exhibited an inorganic pyrophosphatase activity. 14) The reaction conditions for the inorganic pyrophosphatase activity of GST-C4 were initially standardized. The pyrophosphatase activity of GST-C4 increased linearly upto 3.38 µM of the protein. The optimum temperature and pH for the pyrophosphatase activity were found to be 37° C and 7.0 respectively. The pyrophosphatase activity was inhibited by EDTA, suggesting that it is dependent on divalent metal ions. The activity was most efficiently stimulated by Mg+2, although it was also stimulated by Mn+2and Zn+2but inhibited by Ca+2ions. Thus, the pyrophosphatase activity of GST-C4 resembles the family I inorganic pyrophosphatases in metal ion requirements. Further, the pyrophosphatase activity increased linearly upto 1 hour 30 minutes. 15) The Km, Vmax and Kcat for the pyrophosphatase activity of GST-C4 were found to be 0.76 ± 0.04 mM, 141.16 ± 20 nmoles/min/mg of the protein and 5.2 minrespectively. The kcat for the pyrophosphatase activity was approximately 20 fold higher than that for the ATPase activity (0.27 min-1). 16) GST-N∆15-C4 and GST-N∆30-C4 exhibited >70 % reduction in the pyrophosphatase activity, a finding similar to that for the ATPase activity. On the other hand, while GST-C∆10-C4 retained the activity similar to the full length GST-C4 protein, GST-C∆20-C4 and GST-C∆30-C4 exhibited 20 % and 60 % reduction in the pyrophosphatase activity, respectively, as compared to the full length GST-C4 protein. This suggested that the C-terminal region of C4 may also contain the residues important for the pyrophosphatase activity of GST-C4. However, the C-terminal deletion mutants retained the ATPase activity similar to the full length protein. 17) The pyrophosphatase activity of GST-C4 was stimulated more than three fold by several reducing agents. The C4 protein contains only one cysteine (at position 8 in the C4 sequence). This was the first clue that the cysteine may be important for the pyrophosphatase activity of the GST-C4 protein. Further, the pyrophosphatase activity of GST-C4 did not exhibit preference for a particular kind of reducing agent like that of the pyrophosphatase activity in Streptococcus faecalis. 18) GST-C8A-C4 exhibited more than two fold reduction in Vmax, suggesting that the C8 may be catalytically important for the pyrophosphatase activity of GST-C4. On the other hand, the R13A mutation did not affect the pyrophosphatase activity of the GST-C4 protein. Thus, it is possible that during catalysis, the cysteine thiolate of C4 makes a 19) The pyrophosphatase activity of GST-C4 was inhibited by vanadate and fluoride. Vanadate was found to be a competitive inhibitor with Ki 0.33 mM while fluoride was a non-competitive inhibitor with Ki 2.82 mM. A comparative account of the two enzymatic activities of GST-C4 is presented in table 6.1

1) Geminiviruses are a group of plant viruses which contain circular single stranded DNA molecules as their genomes and the capsid consists of two icosahedra fused together to form twinned or geminate particles. The largest genus in the family Geminiviridae is that of begomoviruses which are of two kinds; the monopartite begomoviruses which contain only one circular single stranded DNA molecule as their genome and the bipartite begomoviruses which contain two circular single stranded DNA molecules (designated DNA-A and DNA-B) as their genomes. In bipartite viruses, the two DNA molecules are enclosed in separate geminate capsids. 2) In bipartite begomoviruses, the DNA-A encodes the proteins essential for replication and encapsidation of the viral genome while the DNA-B encodes the proteins involved in movement. The DNA-B encodes two proteins: the BV1 or the nuclear shuttle protein (NSP) and BC1 or the cell-to-cell movement protein. Geminiviruses have DNA genomes which replicate inside the host cell nucleus. The NSP, which contains nuclear localization signal, brings the viral DNA from nucleus to the cytoplasm while the BC1 serves to take the viral genome to the cell periphery for movement to the neighbouring cell through the plasmodesmata. 3) The monopartite begomoviruses do not contain DNA-B (which, in bipartite begomoviruses, encodes the proteins involved in movement) and it has been suggested that some of the proteins encoded by DNA-A take up the movement function. Based on studies on TYLCV and CLCuV, a model has been proposed for the movement of monopartite begomoviruses according to which the coat protein (CP) of monopartite begomoviruses serves as the functional equivalent of the NSP of bipartite begomoviruses 4) The present thesis deals with the biochemical characterization of the C4 protein of the monopartite begomovirus CLCuKV-Dab. As stated in statement (3) above, the V2 and C4 proteins of monopartite begomoviruses have been implicated to be involved in cell-to¬cell movement of the viral genome. In TYLCV, both the proteins were shown to be localized to the cell periphery and could move from one cell to another through the plasmodesmata. Further, the V2 protein of CLCuKV-Dab was shown to interact with the coat protein and bind to single stranded DNA. The biochemical properties of the C4 protein needed to be elucidated in order to strengthen the proposal of its probable involvement in movement. 5) The objectives of the present study were: i) Bioinformatic analysis of the C4 protein of CLCuKV-Dab ii) Biochemical characterization of ATPase and pyrophosphatase activities of the C4 protein. iii) Studies on the effect of V2-C4 interaction on the enzymatic properties of C4. iv) Functional characterization of C4 in planta. 6) The FoldIndex© and PONDR analyses predicted the C4 protein of CLCuKV-Dab to be natively unfolded. Similarly, in PSIpred analysis, most of the C4 protein was predicted to be a random coil without any well-defined secondary structure. Further, the protein sequence was analyzed using the motifscan server. However, no motif for any specific function was predicted in the C4 Protein. 7) The C4 gene was initially cloned into pRSET-C vector and overexpressed as histidine tagged protein and the solubility of the protein was tested in various conditions including low temperature (18° C) after inducing the expression of the protein, buffers of various pH and different salt concentrations but the protein remained insoluble. Subsequently, the protein was purified under denaturing conditions and attempts were made to refold the protein but the protein precipitated during refolding. In order to get the C4 protein in soluble form, the C4 gene was subcloned into pGEX-5X2 vector and overexpressed as a GST-tagged fusion protein (GST-C4). Some of the GST-C4 protein was soluble which was purified by using GST-bind resin. The purified fusion protein was observed as a 37 kDa band on SDS-PAGE gel. The purified protein was accompanied by a degraded product of approximately 30 kDa size. Both the intact GST-C4 protein and the degraded product were detected in western blot analysis using anti-GST antibody. 8) Because C4 has been implicated to be involved in movement of monopartite begomoviruses and movement is an energy requiring process, it was of interest to determine if GST-C4 possesses ATPase activity. The purified GST-C4 protein was incubated with γ-[32P]-ATP, the product of the reaction was separated by thin layer chromatography and the chromatography plate was analyzed by phosphorimager. The hydrolysis of ATP by GST-C4 and the release of inorganic phosphate was clearly observed, suggesting that GST-C4 might possess ATPase activity. 9) The reaction conditions for the ATPase activity of GST-C4 were standardized. The activity increased linearly upto 2.60μM of the protein. The optimum temperature and pH for the ATPase activity were found to be 30 C and 6.0 respectively. The activity was inhibited by EDTA, suggesting that it is dependent on divalent metal ions. The activity was stimulated by Mg+2, Mn+2 and Zn+2 but inhibited by Ca+2ions. Further, in the time course experiment, it was observed that the ATPase activity increased linearly upto one hour. 10) The Km, Vmax and kcat for the ATPase activity of GST-C4 were found to be 51.72 ± 2.5 µM, 7.2 ± 0.54 nmoles/min/mg of the protein and 0.27 min-1 respectively. Some of the other virally encoded ATPases have been found to exhibit kcat similar to that found for GST-C4 but it is much lower than those of most of the prokaryotic and eukaryotic ATPases (as mentioned in Table 3.3, page 100, chapter 3). Further, the presence of the degraded product did not affect the kinetic constants as described in chapter 3, pages 95¬-98. It is possible that the enzymatic activity might increase upon interaction with some ligand. 11) In the absence of any putative ATP binding motifs, systematic deletions from N-and C-termini were made to delineate the regions of C4 important for the ATPase activity. GST-N∆15-C4 and GST-N∆30-C4 exhibited approximately 70 % reduction in the ATPase activity while all the C-terminal deletion mutants (GST-C∆10-C4, GST-C∆20¬C4 and GST-C∆30-C4) retained the activity similar to the full length GST-C4 protein. This suggested that the N-terminal region of C4 may contain the residues important for the ATPase activity of GST-C4. 12) In the N-terminal region of C4, there is a sequence CSSSSR which closely resembles the sequence present at the active site of phosphotyrosine phosphatases (CXXXXXR). However, GST-C4 did not catalyze the hydrolysis of p-Nitrophenyl phosphate, a substrate analogue commonly used to assay phosphotyrosine phosphatase activity. It was of interest to determine if the cysteine and arginine in this sequence are important for the ATPase activity of GST-C4. GST-R13A-C4 exhibited an approximately two fold reduction in Vmax suggesting that R13 in C4 may be catalytically important for the ATPase activity of GST-C4. On the other hand, the C8A mutation did not affect the ATPase activity of GST-C4. 13) The GST-C4 protein was tested for its ability to hydrolyze several other phosphate containing compounds as mentioned chapter 2, pages 53-55. Among these compounds, GST-C4 catalyzed the hydrolysis of sodium pyrophosphate, that is, GST-C4 exhibited an inorganic pyrophosphatase activity. 14) The reaction conditions for the inorganic pyrophosphatase activity of GST-C4 were initially standardized. The pyrophosphatase activity of GST-C4 increased linearly upto 3.38 µM of the protein. The optimum temperature and pH for the pyrophosphatase activity were found to be 37° C and 7.0 respectively. The pyrophosphatase activity was inhibited by EDTA, suggesting that it is dependent on divalent metal ions. The activity was most efficiently stimulated by Mg+2, although it was also stimulated by Mn+2and Zn+2but inhibited by Ca+2ions. Thus, the pyrophosphatase activity of GST-C4 resembles the family I inorganic pyrophosphatases in metal ion requirements. Further, the pyrophosphatase activity increased linearly upto 1 hour 30 minutes. 15) The Km, Vmax and Kcat for the pyrophosphatase activity of GST-C4 were found to be 0.76 ± 0.04 mM, 141.16 ± 20 nmoles/min/mg of the protein and 5.2 minrespectively. The kcat for the pyrophosphatase activity was approximately 20 fold higher than that for the ATPase activity (0.27 min-1). 16) GST-N∆15-C4 and GST-N∆30-C4 exhibited >70 % reduction in the pyrophosphatase activity, a finding similar to that for the ATPase activity. On the other hand, while GST-C∆10-C4 retained the activity similar to the full length GST-C4 protein, GST-C∆20-C4 and GST-C∆30-C4 exhibited 20 % and 60 % reduction in the pyrophosphatase activity, respectively, as compared to the full length GST-C4 protein. This suggested that the C-terminal region of C4 may also contain the residues important for the pyrophosphatase activity of GST-C4. However, the C-terminal deletion mutants retained the ATPase activity similar to the full length protein. 17) The pyrophosphatase activity of GST-C4 was stimulated more than three fold by several reducing agents. The C4 protein contains only one cysteine (at position 8 in the C4 sequence). This was the first clue that the cysteine may be important for the pyrophosphatase activity of the GST-C4 protein. Further, the pyrophosphatase activity of GST-C4 did not exhibit preference for a particular kind of reducing agent like that of the pyrophosphatase activity in Streptococcus faecalis. 18) GST-C8A-C4 exhibited more than two fold reduction in Vmax, suggesting that the C8 may be catalytically important for the pyrophosphatase activity of GST-C4. On the other hand, the R13A mutation did not affect the pyrophosphatase activity of the GST-C4 protein. Thus, it is possible that during catalysis, the cysteine thiolate of C4 makes a 19) The pyrophosphatase activity of GST-C4 was inhibited by vanadate and fluoride. Vanadate was found to be a competitive inhibitor with Ki 0.33 mM while fluoride was a non-competitive inhibitor with Ki 2.82 mM. A comparative account of the two enzymatic activities of GST-C4 is presented in table 6.1

Viruses are submicroscopic obligate parasites and depend on the host cell for their growth and reproduction. Plants are infected by diverse group of viruses that mostly possess RNA as their genome. As exception, viruses belonging to the family Geminiviridae are DNA viruses and infect both mono and dicotyledonous plants causing a large economic loss. These viruses are smaller in size encoding fewer proteins and employ the host cell machinery for successful infection and spread. Geminiviruses undergo frequent recombinations due to mixed infection resulting in vast diversity across the family and account for driving evolution in these viruses. Movement in these viruses is complex since they have to cross two important barriers, nuclear and cell wall barrier to establish systemic spread. All these factors play very important role while designing control measures against these viruses. Thus a detailed understanding of these processes at molecular level is essential. Cotton is the major cash crop in Indian subcontinent with huge export values. India has become the second largest producer of cotton in the world. However, the major constraint in cotton cultivation has been crop loss due to diseases caused by viruses, particularly the cotton leaf curl disease (CLCuD) caused by begomoviruses. Present thesis deals with the analysis of genetic variability of CLCuD in India and functional characterization of proteins involved in the movement of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab). CLCuKV-Dab belongs to family Geminiviridae and subgroup Begomovirus. A review of the literature on Geminiviridae classification, genome organization, virus entry, replication, transcription, translation, assembly and movement is presented in Chapter 1. This chapter also includes the review of host factors involved in replication, geminiviral proteins involved in gene silencing and a detailed report on CLCuD complexes and sub viral DNAs that are associated with CLCuD. The materials used in this study and the experimental protocols followed such as construction of recombinant clones, their overexpression in both bacterial and baculovirus expression systems, Protein purification techniques, site directed mutagenesis and all other biochemical, molecular biology and cell biology methods are described in detail in Chapter 2. Previous study has reported the complete genomic sequences of CLCuKV-Dab and Tomato leaf curl Bangalore virus-cotton [Fatehabad] (ToLCBV-Cotton [Fat]) and partial sequence of CLCuKV-Gang and the Cotton leaf curl Rajasthan virus (CLCuRV-Ban). Phylogenetic analysis of DNA-A sequences of these viruses with other CLCuD causing viruses is discussed in detail in Chapter 3. Chapter 4 deals with overexpression, purification and functional characterization of CLCuKV-Dab CP in terms of its interaction with DNA, the kinetics and its role in cell to cell movement. The proposed partners to CP in the cell to cell movement of monopartite begomoviruses are AV2 and AC4. Thus the Chapter 5 describes the functional characterization of recombinant AV2 of CLCuKV-Dab. Chapter 6 deals with expression of CP and AV2 as GFP fusion proteins in insect cells using baculovirus expression system to study the localization patterns of these proteins. Chapter 7 describes functional characterization of CLCuKV-Dab AC4. Bioinformatic analysis of AC4 showed that it belongs to the rare group of natively unfolded proteins that are functionally active In conclusion, there is a large genetic variability that exists among the begomoviruses and in particular, among the CLCuD causing begomoviruses in India. Functional characterization of the proteins involved in the cell to cell movement in CLCuKV-Dab led to a possible model for its movement; the CP translated in the cytoplasm is targeted the nucleus via its NLS and there binds to progeny ssDNA and exports the ssDNA out of nucleus through its export signals. AC4 or some other host proteins yet to be identified transports the ssDNA-CP complex from the nuclear periphery to AV2 present at the cell periphery. The complex is then transported from one cell to the neighboring cell via plasmodesmata. AC4 being an ATPase/NTPase could provide energy for the process.

Viruses are submicroscopic obligate parasites and depend on the host cell for their growth and reproduction. Plants are infected by diverse group of viruses that mostly possess RNA as their genome. As exception, viruses belonging to the family Geminiviridae are DNA viruses and infect both mono and dicotyledonous plants causing a large economic loss. These viruses are smaller in size encoding fewer proteins and employ the host cell machinery for successful infection and spread. Geminiviruses undergo frequent recombinations due to mixed infection resulting in vast diversity across the family and account for driving evolution in these viruses. Movement in these viruses is complex since they have to cross two important barriers, nuclear and cell wall barrier to establish systemic spread. All these factors play very important role while designing control measures against these viruses. Thus a detailed understanding of these processes at molecular level is essential. Cotton is the major cash crop in Indian subcontinent with huge export values. India has become the second largest producer of cotton in the world. However, the major constraint in cotton cultivation has been crop loss due to diseases caused by viruses, particularly the cotton leaf curl disease (CLCuD) caused by begomoviruses. Present thesis deals with the analysis of genetic variability of CLCuD in India and functional characterization of proteins involved in the movement of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab). CLCuKV-Dab belongs to family Geminiviridae and subgroup Begomovirus. A review of the literature on Geminiviridae classification, genome organization, virus entry, replication, transcription, translation, assembly and movement is presented in Chapter 1. This chapter also includes the review of host factors involved in replication, geminiviral proteins involved in gene silencing and a detailed report on CLCuD complexes and sub viral DNAs that are associated with CLCuD. The materials used in this study and the experimental protocols followed such as construction of recombinant clones, their overexpression in both bacterial and baculovirus expression systems, Protein purification techniques, site directed mutagenesis and all other biochemical, molecular biology and cell biology methods are described in detail in Chapter 2. Previous study has reported the complete genomic sequences of CLCuKV-Dab and Tomato leaf curl Bangalore virus-cotton [Fatehabad] (ToLCBV-Cotton [Fat]) and partial sequence of CLCuKV-Gang and the Cotton leaf curl Rajasthan virus (CLCuRV-Ban). Phylogenetic analysis of DNA-A sequences of these viruses with other CLCuD causing viruses is discussed in detail in Chapter 3. Chapter 4 deals with overexpression, purification and functional characterization of CLCuKV-Dab CP in terms of its interaction with DNA, the kinetics and its role in cell to cell movement. The proposed partners to CP in the cell to cell movement of monopartite begomoviruses are AV2 and AC4. Thus the Chapter 5 describes the functional characterization of recombinant AV2 of CLCuKV-Dab. Chapter 6 deals with expression of CP and AV2 as GFP fusion proteins in insect cells using baculovirus expression system to study the localization patterns of these proteins. Chapter 7 describes functional characterization of CLCuKV-Dab AC4. Bioinformatic analysis of AC4 showed that it belongs to the rare group of natively unfolded proteins that are functionally active In conclusion, there is a large genetic variability that exists among the begomoviruses and in particular, among the CLCuD causing begomoviruses in India. Functional characterization of the proteins involved in the cell to cell movement in CLCuKV-Dab led to a possible model for its movement; the CP translated in the cytoplasm is targeted the nucleus via its NLS and there binds to progeny ssDNA and exports the ssDNA out of nucleus through its export signals. AC4 or some other host proteins yet to be identified transports the ssDNA-CP complex from the nuclear periphery to AV2 present at the cell periphery. The complex is then transported from one cell to the neighboring cell via plasmodesmata. AC4 being an ATPase/NTPase could provide energy for the process.

The tissue and intracellular distribution of the monopartite Tomato leaf curl virus (TLCV) was investigated by in situ hybridization. contrary to the previous understanding of geminiviral localization, single stranded (SS) DNA of TCLV accumulated in the cytoplasm. TCLV ssDNA was also found in the nucleus, as were levels of replicative form doubl-stranded (ds)DNA.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2007

[EN] Tomato yellow leaf curl disease (TYLCD) causes great damage in tomato (Solanum lycopersicum L.) crops in south-eastern Spain and in many tropical and subtropical areas in the world. The disease is caused by a complex of viruses, all belonging to the genus Begomovirus, family Geminiviridae. Nine species have been reported causing TYLCD and six more have been proposed as tentative species. Four viral species associated with TYLCD are present in Spain. Preventive measures to fight the disease, as well as measures based on controlling the insect vector (Bemisia tabaci Gen.) are not effective, so the development of resistant varieties seems the best long term strategy. Given that resistance has not been reported in the cultivated species, screening for resistance has been focused on wild tomato relatives. Resistance has been found in different wild species and some resistant breeding lines and commercial varieties have been developed. Ty-1, derived from S. chilense LA1969, is the most frequently used gene. Advances in genetic engineering techniques have also been exploited in developing plant material with pathogen derived resistance. However, resistant varieties currently available are not a solution, as with high inoculum pressure conditions and early infections, plants still develop symptoms and yield losses are caused. For that reason, many research groups continue working worldwide to obtain plants with high levels of resistance to TYLCD. Current breeding objectives are the development of broad spectrum resistance to several begomovirus, the accumulation of resistance genes from different sources to increase the levels of resistance and the identification of molecular markers tightly linked to resistance genes, which allows shortening breeding programmes and accumulating different resistance genes in the same plant material. This work has been developed in the research group ‘Breeding for resistance to Tomato yellow leaf curl disease’ of the Institute for Conservation and Improvement of Agrodiversity (COMAV). When this project was initiated, several resistant plant materials had been developed from previous works of the group. Twelve breeding lines derived from S. chilense LA1932 and LA1938 were available. These lines were resistant to Tomato yellow leaf curl Sardinia virus (TYLCSV), the first viral species described in Spain causing TYLCD. It was of interest the evaluation of resistance in these lines to Tomato yellow leaf curl virus (TYLCV), introduced later in Spain and spread worldwide. Six breeding lines showed high levels of partial resistance to TYLCSV and TYLCV. The resistance consisted on attenuation and delay in symptom development, as well as reduction in viral accumulation. Significant yield losses due to viral infection were not observed in these lines. These lines also show good horticultural traits which make them appropriate to be base material for developing commercial hybrids resistant to both, TYLCSV and TYLCV. High levels of resistance have also been identified in S. pimpinellifolium UPV16991. The resistance has already been fixed in the genetic background of S. lycopersicum. It was convenient to determine the genetic control of the resistance and the expression in tomato background, before using it in breeding programmes. For these purposes L102 was selected. L102 belongs to the F6 generation, after the initial cross S. lycopersicum NE-1 x S. pimpinellifolium UPV16991. Resistance to TYLCV in L102 is controlled by one gene, with partial recessiveness and incomplete penetrance. Moreover, the expression of resistance strongly depends on S. lycopersicum background in which it is introgressed. The highest levels of resistance are obtained when crossing L102 with vigorous lines. So, we recommend to use UPV16991-derived resistance in the development of vigorous hybrids in homozygosis or combined with resistance from other sources. To exploit resistance derived from UPV16991, L102 and some other lines with the same origin were crossed with a breeding line homozygous for Ty-1. Resistance was then evaluated in several plant material heterozygous for both, Ty-1 and the resistance gene from UPV16991. These plant materials showed higher levels of resistance than heterozygotes for each of the genes. Resistance in one of the hybrids was even higher than in homozygotes for each of the genes. These results show that combining resistance from UPV16991 with Ty-1 is the most practical approach to exploiting this resistance, since it allows the development of hybrids without the need of fixing the resistance gene in both parents. Finally, availability of molecular markers tightly linked to the resistance genes is essential to accumulate resistance from different sources. Some molecular markers tightly linked to Ty-1 have been reported. However, in all cases, S. peruvianum-derived Mi gene interferes with these markers, causing false positive results. In this work, a molecular marker, JB-1, tightly linked to Ty-1 has been identified. This is a CAPS (Cleaved Amplified Polymorphic DNA) marker. The presence of Mi, as well as introgressions from other wild tomato relatives such as S. lycopersicum (formerly Lycopersicon esculentum var. cerasiforme), S. habrochaites and S. pimpinellifolium do not interfere with the results for this marker. In addition, the analysis of several plant material with introgressions from different wild tomato relatives has allowed the location of CT21, the RFLP (Restriction Fragment Length Polymorphism) marker from which JB-1 was designed.
[ES] La enfermedad del rizado amarillo del tomate (Tomato yellow leaf curl disease, TYLCD) causa graves daños en los cultivos de tomate (Solanum lycopersicum L.) del sudeste español y de la mayor parte de las zonas tropicales y subtropicales de todo el mundo. La enfermedad está causada por un complejo de virus pertenecientes al género Begomovirus, familia Geminiviridae. Se han descrito nueve especies causantes de TYLCD y otras seis han sido propuestas. En España están presentes cuatro de las especies virales asociadas a TYLCD. Las medidas preventivas de lucha contra la enfermedad, así como las basadas en el control del insecto vector transmisor (Bemisia tabaci Gen.) no resultan efectivas por si mismas, de forma que el desarrollo de materiales resistentes supone la mejor estrategia de lucha a largo plazo. Dado que en la especie cultivada no se han descrito entradas resistentes, la búsqueda de fuentes de resistencia se ha centrado en las especies silvestres del género relacionadas con el tomate. Se ha encontrado resistencia en distintas entradas de algunas de estas especies y se han desarrollado líneas de mejora y materiales comerciales con resistencia procedente de algunas de ellas. El gen Ty-1, derivado de la entrada LA1969 de S. chilense, ha sido el más empleado para la obtención de líneas e híbridos comerciales resistentes. Por otra parte, haciendo uso de los avances en las técnicas de ingeniería genética, también se han desarrollado materiales con resistencia derivada del patógeno. Sin embargo, los materiales resistentes disponibles hasta el momento no suponen una solución definitiva al problema, ya que, con presiones fuertes de inóculo o infecciones tempranas, las plantas desarrollan síntomas de la enfermedad, produciéndose pérdidas de producción. Por este motivo, numerosos grupos de investigación continúan trabajando a nivel mundial con la finalidad de obtener materiales con niveles elevados de resistencia a TYLCD. Entre los objetivos actuales de mejora se incluyen el desarrollo de resistencia de amplio espectro a varios begomovirus, la combinación de genes de distinta procedencia para conseguir mayores niveles de resistencia y la identificación de marcadores moleculares ligados a los genes de resistencia que permitan acortar los programas de mejora y acumular en un mismo material genes de resistencia de distintas fuentes. El grupo de “Mejora para la resistencia a la enfermedad del rizado amarillo del tomate”, del Instituto de Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), en el que se ha realizado la presente tesis doctoral, disponía al inicio de la misma de distintos materiales con resistencia a TYLCD, desarrollados en trabajos previos. Entre estos materiales se encontraban 12 líneas de mejora derivadas de la entradas LA1932 y LA1938 de S. chilense, seleccionadas por su resistencia a la especie Tomato yellow leaf curl Sardinia virus (TYLCSV), la primera especie viral causante de TYLCD detectada en España. Resultaba de interés evaluar la respuesta de estos materiales a la especie Tomato yellow leaf curl virus (TYLCV), introducida posteriormente en España y más extendida a nivel mundial. Seis de las líneas evaluadas han mostrado niveles elevados de resistencia parcial a TYLCSV y TYLCV, consistente en la atenuación en la manifestación de síntomas y retraso en el momento de aparición de los mismos, además de en la reducción de la acumulación viral. Además, no se han observado en estas líneas pérdidas significativas en el rendimiento como consecuencia de la infección por TYLCV. Las características agronómicas las hacen apropiadas como parentales para el desarrollo de híbridos con elevada resistencia a TYLCSV y TYLCV. Por otra parte, se habían identificado niveles elevados de resistencia en la entrada UPV16991 de S. pimpinellifolium. Una vez fijada la resistencia en el fondo genético de S. lycopersicum, y como paso previo al empleo en programas de mejora, era conveniente determinar el control genético de la misma, así como conocer su expresión en el fondo genético de la especie cultivada. Para ello se ha empleado la línea L102, que corresponde a la quinta generación de autofecundación a partir del cruce inicial S. lycopersicum NE-1 x S. pimpinellifolium UPV16991. Se ha comprobado que la resistencia parcial a TYLCV de la línea L102 está controlada por un gen con recesividad parcial y penetración incompleta. Además, la expresión de la misma depende considerablemente del fondo genético de S. lycopersicum en el que se introgresa, obteniéndose los mayores niveles de resistencia en cruces con líneas vigorosas. Por todo esto, se recomienda el uso de esta resistencia bien en homocigosis en el desarrollo de híbridos vigorosos, bien en combinación con resistencia procedente de otras fuentes. En este sentido, se decidió evaluar la resistencia en materiales que combinaban el gen Ty-1 y el gen derivado de UPV16991, ambos en heterocigosis. El nivel de resistencia en estos materiales ha sido superior al mostrado por los heterocigotos para cada uno de los genes, e incluso en algún caso se ha superado la resistencia de los homocigotos para cada uno de los genes. Esto indica que la combinación de la resistencia derivada de UPV16991 con el gen Ty-1 es la aproximación más práctica para la utilización de esta resistencia, ya que evita la necesidad de fijar el gen de resistencia en ambos parentales. Por último, para la acumulación de resistencia de distinta procedencia en un mismo material, resulta imprescindible disponer de marcadores moleculares ligados a los genes de resistencia. Se han descrito algunos marcadores ligados al gen Ty-1, sin embargo, la presencia del gen Mi, derivado de S. peruvianum, interfiere con los resultados para estos marcadores, obteniéndose falsos positivos. En este trabajo se ha identificado un marcador molecular, JB-1, tipo CAPS (Cleaved Amplified Polymorphic DNA) ligado al gen de resistencia Ty-1. La presencia del gen Mi, así como introgresiones de otras especies como S. lycopersicum (antes Lycopersicon esculentum var. cerasiforme), S. habrochaites y S. pimpinellifolium, no interfieren con los resultados para este marcador. Además, el análisis de materiales con introgresiones de distintas especies silvestres relacionadas con el tomate para varios marcadores de la región del Ty-1 ha permitido localizar el marcador CT21, el RFLP (Restriction Fragment Length Polymorphism) a partir del cual se desarrolló JB-1.
[CAT] La malaltia de l’arrissat groc de la tomaca (Tomato yellow leaf curl disease, TYLCD) causa greus danys als cultius de tomaca (Solanum lycopersicum L.) del sud-est espanyol i de la major part de les zones tropicals i subtropicals de tot el món. La malaltia està causada per un complex de virus pertanyents al gènere Begomovirus, família Geminiviridae. S’han descrit nou espècies causants de TYLCD i altres sis han sigut proposades. A Espanya estan presents quatre de les espècies virals associades a TYLCD. Les mesures preventives de lluita contra la malaltia, així com les basades en el control de l’insecte vector transmissor (Bemisia tabaci Gen.) no resulten efectives per si mateixes, de manera que el desenvolupament de materials resistents suposa la millor estratègia de lluita a llarg termini. Atés que en l’espècie cultivada no s’han descrit entrades resistents, la recerca de fonts de resistència s’ha centrat en les espècies silvestres del gènere relacionades amb la tomaca. S’ha trobat resistència en distintes entrades d’algunes d’estes espècies i s’han desenvolupat línies de millora i materials comercials amb resistència procedent d’algunes d’elles. El gen Ty-1, derivat de l’entrada LA1969 de S. chilense, ha sigut el més utilitzat per a l’obtenció de línies i híbrids comercials resistents. D’altra banda, fent ús dels avanços en les tècniques d’enginyeria genètica, també s’han desenvolupat materials amb resistència derivada del patogen. No obstant, els materials resistents disponibles fins al moment no suposen una solució definitiva al problema, ja que, amb pressions fortes d’inòcul o infeccions primerenques, les plantes desenvolupen símptomes de la malaltia, produint-se pèrdues de producció. Per este motiu, nombrosos grups d’investigació continuen treballant a nivell mundial amb la finalitat d’obtindre materials amb nivells elevats de resistència a TYLCD. Entre els objectius actuals de millora s’inclouen el desenvolupament de resistència d’ampli espectre a diversos begomovirus, la combinació de gens de distinta procedència per a aconseguir majors nivells de resistència i la identificació de marcadors moleculars lligats als gens de resistència que permeten acurtar els programes de millora i acumular en un mateix material gens de resistència de distintes fonts. El grup de “Millora per a la resistència a la malaltia de l’arrissat groc de la tomaca”, de l’Institut de Conservació i Millora de l’Agrodiversitat Valenciana (COMAV), en el que s’ha realitzat la present tesi doctoral, disposava a l’inici de la mateixa, de distints materials amb resistència a TYLCD, desenvolupats en treballs previs. Entre estos materials es trobaven 12 línies de millora derivades de l’entrades LA1932 i LA1938 de S. chilense, seleccionades per la seua resistència a l’espècie Tomato yellow leaf curl Sardinia virus (TYLCSV), la primera espècie viral causant de TYLCD detectada a Espanya. Resultava d’interés avaluar la resposta d’estos materials a l’espècie Tomato yellow leaf curl virus (TYLCV), introduïda posteriorment a Espanya i més estesa a nivell mundial. Sis de les línies avaluades han mostrat nivells elevats de resistència parcial a TYLCSV i TYLCV, consistent en l’atenuació en la manifestació de símptomes, retard en el moment d’aparició dels mateixos i reducció de l’acumulació viral. A més, no s’han observat en estes línies pèrdues significatives en el rendiment com a conseqüència de la infecció per TYLCV. Les característiques agronòmiques les fan apropiades com a parentals pel desenvolupament d’híbrids amb elevada resistència a TYLCSV i TYLCV. D’altra banda, s’havien identificat nivells elevats de resistència en l’entrada UPV16991 de S. pimpinellifolium. Una vegada fixada la resistència en el fons genètic de S. lycopersicum, i com a pas previ a l’ús en programes de millora, era convenient determinar el control genètic de la mateixa, així com conéixer la seua expressió en el fons genètic de l’espècie cultivada. Per a això s’ha emprat la línia L102, que correspon a la quinta generació d’autofecundació a partir del creuament inicial S. lycopersicum NE-1 x S. pimpinellifolium UPV16991. S’ha comprovat que la resistència parcial a TYLCV de la línia L102 està controlada per un gen amb recessivitat parcial i penetració incompleta. A més, l’expressió de la mateixa depén considerablement del fons genètic de S. lycopersicum en el que s’introgresa, obtenint-se els majors nivells de resistència en creuaments amb línies vigoroses. Per tot açò, es recomana l’ús d’esta resistència bé en homozigosis en el desenvolupament d’híbrids vigorosos, bé en combinació amb resistència procedent d’altres fonts. En este sentit, es va decidir avaluar la resistència en materials que combinaven el gen Ty-1 i el gen derivat d’UPV16991, ambdós en heterozigosis. El nivell de resistència en estos materials ha sigut superior al mostrat pels heterozigots per a cada un dels gens, i fins i tot en algun cas s’ha superat la resistència dels homozigots per a cada un dels gens. Açò indica que la combinació de la resistència derivada d’UPV16991 amb el gen Ty-1 és l’aproximació més pràctica per a la utilització d’esta resistència, ja que evita la necessitat de fixar el gen de resistència en ambdós parentals. Finalment, per a l’acumulació de resistència de distinta procedència en un mateix material, resulta imprescindible disposar de marcadors moleculars lligats als gens de resistència. S’han descrit alguns marcadors lligats al gen Ty-1, no obstant, la presència del gen Mi, derivat de S. peruvianum, interferix amb els resultats per a estos marcadors, obtenint-se falsos positius. En este treball s’ha identificat un marcador molecular, JB-1, tipus CAPS (Cleaved Amplified Polymorphic DNA) lligat al gen de resistència Ty-1. La presència del gen Mi, així com introgresions d’altres espècies com S. lycopersicum (abans Lycopersicon esculentum var. cerasiforme), S. habrochaites i S. pimpinellifolium, no interferixen amb els resultats per a este marcador. A més, l’anàlisi de materials amb introgresions de distintes espècies silvestres relacionades amb la tomaca per a diversos marcadors de la regió del Ty-1 ha permés localitzar el marcador CT21, el RFLP (Restriction Fragment Length Polymorphism) a partir del qual es va desenvolupar JB-1.
This research was supported by the “Ministerio de Ciencia y Educación”, project number AGL2001-1857-C04-03.
Pérez De Castro, AM. (2007). Mejora genética para la resistencia a los geminivirus tomato yellow leaf curl virus (TYLCV) y tomato yellow leaf curl sardinia virus (TYLCSV) en tomate [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/135825
TESIS

碩士
國立中興大學
生物科技學研究所
101
Squash leaf curl virus (SqLCV) is a species of the which harbors single-stranded, circular DNA (sscDNA) genomes. The genetic information of SqLCV is divided into two sscDNAs, designated DNA A and DNA B. Cucurbit plants infected by SqLCV exhibit various degrees of leaf curling symptoms which impair the growth and fruiting of the crops and cause severe losses to agricultural economy. In this study, leaf samples of the bottle gourd (Lagenaria siceraria) showing very severe curling symptoms were collected from Peitou township in Changhua County, which provided convenient source for the study of factors involved in leaf curling symptom in cucurbits. Therefore, the specific aims of this study are the isolation and sequence analyses of the SqLCV from the bottle gourd, the construction of infectious clones for the study of symptom expression, and the production of specific antibodies against SqLCV. Genomic DNAs of SqLCV were amplificed by Rolling Circle Amplification (RCA), followed by cloning and sequence analyses. To construct the infectious clones of SqLCV, tandem dimmers of DNA A and B were generated by partial restriction enzyme digestion of the RCA products and subcloned into the binary vector pBin19. Two independent SqLCV infectious clones of each DNA A and DNA B, designated BGSQ1A, BGSQ5A, BGSQ1B and BGSQ2B, have been constructed. Infectivity assays showed that the combination of BGSQ5A and BGSQ1B or BGSQ2B could elicit leaf curl symptoms on L. siceraria following the vacuum inoculation method developed in this study. Furthermore, SqLCV coat protein gene has been cloned in the pCyCP vector, over-expressed and purified as a fusion protein, and subjected to the production of specific antibodies. The results in this study provided useful tools for further study of the mechanism underlying the control of leaf curl symptoms.

"Jamuary, 2005"
Bibliography: leaves 124-144.
xviii, 160 leaves : ill., plates, photos ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, Discipline of Plant and Pest Science, 2005?

碩士
國立臺灣大學
昆蟲學研究所
103
Whiteflies not only damage plants through feeding but also serve as vectors transmitting plant viruses. Whitefly-transmitted begomoviruses threaten a wide range of crops worldwide. Tomato yellow leaf curl Thailand virus (TYLCTHV) and Tomato leaf curl Taiwan virus (ToLCTWV) are two most prevalent viruses in tomato fields in Taiwan. Coat protein (CP), the only structural protein of begomoviruses, is hypothesized to interact with a putative receptor in the midgut of Bemisia tabaci. In vivo binding assay demonstrated recombinant TYLCTHV CP specifically bound to the midgut cells of B. tabaci. The objective of this study was to determine if preacquisition of TYLCTHV CP competes the putative receptor in the midgut of B. tabaci hence suppresses the acquisition and vector transmission of TYLCTHV. Preacquisition of recombinant TYLCTHV CP decreased the accumulation level of TYLCTHV in the midgut cells of B. tabaci but did not reduce the acquisition of TYLCTHV in B. tabaci. Further, preacquisition of recombinant TYLCTHV CP suppressed the whitefly transmission of TYLCTHV. Similar experiments were conducted to investigate if preacquisition of TYLCTHV CP (homologous to ToLCTWV CP) would reduce virus accumulation and acquisition in B. tabaci, and the whitefly transmission of ToLCTWV. The results demonstrated that preacquisition of recombinant TYLCTHV CP did not influence the accumulation and acquisition of ToLCTWV in B. tabaci and did neither reduce the whitefly transmission of ToLCTWV. In conclusion, TYLCTHV CP functioned as an attachment protein and preacquisition of TYLCTHV CP reduced the whitefly transmission of TYLCTHV. The failure that using TYLCTHV CP to inhibit the whitefly transmission of ToLCTWV suggested that the interaction between TYLCTHV CP and the putative receptor in the midgut of B. tabaci is specific. Understanding the CP-midgut interaction would unveil the mechanism of whitefly transmission of begomoviruses and warrant further study to develop strategies for disease control.

碩士
國立中興大學
生物科技學研究所
106
Geminiviruses usually inflict typical leaf curling symptoms on infected plants, with severe growth retardation and yield reduction, resulting in serious losses in agricultural economy. Previous studies in our lab have revealed that the C4 proteins of geminiviruses are involved in the determination of the direction of leaf curling symptoms. However, the underlying mechanisms remained to be explored. Therefore the objectives of this study are to identify the key amino acid residues involved in the determination of leaf curling directions. Previously, it has been shown by our group that two amino acids in the C4 protein of Ageratum yellow vein virus (AYVV) played key roles in the determination of leaf curling direction and severity. In this study, the C4 protein of Tomato leaf curl virus Tai-Nan Strain (TLCV-TN), which caused severe downward leaf curl symptom on Nicotiana benthamiana, was used as the primary material for mutational analysis. Since the C4 gene open reading frame overlaps that of the Rep protein and at least two copies of origin of replication (Ori) are required for the infectivity of the clones, different constructs were designed and tested in this study. Accordingly, infectious clones of two mutants, designated A-, and B-mut, were constructed and assayed in N. benthamiana. The results revealed that two amino acids in A-mut and B-mut could attenuate the downward leaf curling symptoms. Thus, the results elucidated key amino acids in C4 proteins that are involved in the modulation of leaf curling symptoms. It is expected that the results from this study may contribute to the knowledge on the modulation of leaf curling symptom, and may be applied in the development of effective management strategy for viral diseases.

碩士
國立臺灣大學
昆蟲學研究所
105
Tomato yellow leaf curl Thailand virus (TYLCTHV) is a member of the genus Begomovirus in the family Geminiviridae, and it is associated with tomato yellow leaf curl disease (TYLCD) in China, Myanmar, Thailand, and Taiwan. TYLCD is one of the major constraint on tomato production worldwide and often leads to 20-100% reduction in the yield of tomato particularly in tropical and subtropical areas. The genome of TYLCTHV is composed of bipartite DNA that are designated DNA-A and DNA-B. DNA-A carries six genes that encode the CP, the AV2 protein, the Rep protein, the TrAP, the REn protein, and the AC4 protein. DNA-B encodes two viral proteins, the NSP and the MP. To better understand the functions of the TYLCTHV proteins, green fluorescence protein (GFP) fused with each viral protein was expressed in planta by agroinfiltration. Further, the protein-protein interaction was examined by bimolecular fluorescence complementation (BiFC) assay which provides simultaneous protein interaction and localization data in living cells. The results showed that the GFP-CP fusion and the GFP-NSP fusion both localized exclusively to the nucleus with an accumulation in the nucleolus. The GFP-Rep fusion distributed throughout the nucleus excluding the nucleolus. The GFP-AV2, GFP-TrAP, the GFP-AV2, GFP-TrAP, GFP-REn, GFP-AC4, and GFP-MP fusions localized to both the cytoplasm and the nucleus. My results demonstrated that the subcellular localizations of the TYLCTHV proteins in the virus-infected plant leaves were identical to those in the healthy plant leaves. Besides, none of the protein-protein interactions of TYLCTHV were detected by BiFC assay. The results of the unchanged subcellular localizations of the TYLCTHV proteins in the healthy and in the TYLCTHV-infected N. benthamiana leaves are in agreement with the results of no protein-protein interactions examined by BiFC assay. Viral protein localizations in host cells provides valuable information about the biological roles of viral proteins. More studies are needed to further understand the relationship between the TYLCTHV proteins and their interactions with host proteins. The information will help us to further understand the molecular mechanisms that underlie the processes of virus replication, movement, and infection.

碩士
國立中興大學
農藝學系所
99
Geminiviruses caused severe disease on important economic crops in recent years. Tomato leaf curl New Delhi virus (ToLCNDV) belonging to geminivirus was identified from many plants in tropical and subtropical countries. ToLCNDV contains bipartite genome, designated as DNA-A and DNA-B and each is approximately 2.7 kb nucleotides in size. DNA-A encodes coat protein, replication-associated proteins, and proteins involved in gene expression. DNA-B encodes proteins responsible for viral movement. In April 2007, a new ToLCNDV isolate designated as ToLCNDV-OM was identified from oriental melon. This isolate could infect some important cucurbit crops like cucumber, sponge gourd, and zucchini. Most geminiviruses cannot be mechanically transmitted to their original hosts. However, ToLCNDV-OM could be transmitted to its original host by mechanical inoculation. In order to investigate the factors affecting the mechanical transmissibility of ToLCNDV-OM, the additional 13 amino acids of N terminus of nuclear shuttle protein (NSP) from non-mechanical transmissible isolate ToLCNDV-Severe compared to mechanical transmissible isolate ToLCDNV-OM and ToLCNDV-Potato was targeted. We established ToLCNDV-OM and ToLCNDV-OMa39NSP which contains additional 13 amino acids from ToLCNDV-Severe to N terminus of NSP of ToLCNDV-OM of agro-infectious clones. The virus inocula were prepared from infected Nicotiana benthamiana by agroinfection and then were mechanically introduced into N. benthamiana, oriental melon, and tomato to evaluate the mechanical transmissibility of the virus. Results showed that ToLCNDV-OMa39NSP did not change its mechanical transmissibility. Consequently, the N terminus of NSP may be not the critical factor affecting mechanical transmissibility of ToLCNDV. After that the DNA-A and DNA-B of ToLCNDV-OM were reassorted with those of a non-mechanical transmissible ToLCNDV cucumber isolate (ToLCNDV-CB). Results indicated that the combination of DNA-A of non-mechanical transmissible ToLCNDV-CB with DNA-B of ToLCNDV-OM became mechanically transmissible whereas that of DNA-A of ToLCNDV-OM with DNA-B of ToLCNDV-CB did not. It clearly suggested that DNA-B of ToLCNDV-OM contains the critical factors for mechanical transmissibility. DNA-B encodes two proteins, including NSP and movement protein (MP). In order to find the determinant of mechanical transmissibility, the NSP or MP of ToLCNDV-CB and ToLCNDV-OM were exchanged. Results showed that the exchange of NSP between these two isolates did not change the mechanical transmissibility, whereas that the exchange of MP affected the mechanical transmissibility of each isolate; e.g., ToLCNDV-CB became transmissible whereas ToLCNDV-OM lost its transmissibility. Consequently, MP of ToLCNDV-OM plays the critical role for mechanical transmissibility. Understanding the key element for mechanical transmission provides useful information for managing geminiviruses.

碩士
國立中興大學
生物科技學研究所
99
Squash leaf curl virus (SqLCV) is a member of the family Geminiviriade, genus Begomovirus. In recent years, SqLCV causes severe economic loses of muskmelon in central and southern area of Taiwan. Previous studies of our laboratory revealed that SqLCV-infected Nicotiana benthamiana plants display various degrees of severity of downward leaf curling symptoms. Further studies showed that C4 protein might be the determinant of the directions of leaf curling in N. benthamiana. However, there is no previous evidence indicating that C4 protein is the pathogenecity determinant in muskmelon. Therefore, the objective of this study is to analyze the factors involved in SqLCV pathogenicity through sequence comparisons and infection assays using infectious clones of different isolates from different hosts and localities. The genomes of various SqLCV isolates from central and southern area of Taiwan were amplified by Rolling Circle Amplification (RCA), and cloned into pUC119 vector for full-length sequence analyses. Infectious clones were constructed using dimers of SqLCV DNA A and DNA B from partial restriction digestion of RCA products and subsequent cloning into pBin19 vector. Four different SqLCV infectious clones, designated SQ2, SQ3, SQ8, and SQ11, have been constructed. When N. benthamiana infected individually by SQ2, SQ8 or SQ3, the leaves display downward curling phenomenon. SqLCV DNAs were also detected in the upper uninoculated leaves, indicating successful systemic infections by these constructs. No obvious symptoms were observed when N. benthaimiana were inoculated with SQ11, but SqLCV DNAs were detected by PCR and RCA in systemic leaves, indicating that SQ11 is a mild strain of SqLCV. When N. benthamiana infected by DNA A of SQ11 plus DNA B of SQ8, the leaves displayed severe downward curling symptom in the pseudo-recombination experiment. SqLCV DNAs were also detected in the upper uninoculated leaves. On the other hand, no symptoms were observed when N. benthamiana were inoculated DNA A of SQ8 plus DNA B of SQ11. Only DNA A, but not DNA B, could be detected in the upper uninoculated leaves. It is speculated that SQ11 DNA B may cause mild symptom in N. benthamiana. In order to investigate whether C4 protein is the pathogenecity determinant in muskmelon, a new infectious clone, designated SQ8mAC4, was constructed using inverse PCR to mutate the C4 protein coding region of SqLCV. No symptoms were observed when muskmelons were inoculated with SQ8mAC4 and SqLCV DNA were not detect in the inoculated and systemic leaves, suggesting that C4 protein might be required for the accumulation of SqLCV DNAs. The feasibility of cross protection was tested using SQ11 as the protecting mild strain. When N. benthamiana was challenged by SQ8 after inoculation with SQ11 for 10 days, the upper uninoculated leaves displayed downward curling symptom in the cross protection assay. The lack of cross-protection may result from the recombination between the genomes of SQ8 and SQ11, or synergistic effects of the two isolates. In the future work, the detailed mechanisms controlling the severity of symptoms by SqLCV will be further analyzed using the infectious clones constructed in this study. It is expected that through the understanding of this mechanism, effective disease management measures for geminivirus infections will be established.

碩士
國立中興大學
植物病理學系所
102
Members in the Geminiviridae have wide host range including economically important crops and are cosmopolitan in distribution. Tomato leaf curl New Delhi virus (ToLCNDV), a member of genus Begomovirus, infects a wide range of plants in tropical and subtropical regions, especially cucurbitaceous and solanaceous crops. It was isolated from tomato in India and Pakistan and from cucurbitaceous plants in Thailand. ToLCNDV contains bipartite genome, designated as DNA-A and DNA-B and are approximately 2.7 kb each in size. DNA-A encodes coat protein, replication-associated proteins and proteins involved in gene expression. Similarly, DNA-B encodes proteins responsible for viral movement. In April 2007, a mechanically transmissible isolate of ToLCNDV which was later designated as ToLCNDV oriental melon (ToLCNDV-OM) was isolated from oriental melon (C. melo var. makuwa cv. Sliver Light) in Taiwan. This ToLCNDV-OM isolate can also infect tomato (L. esculentum Mill. var. Farmers 301). However, another isolate designated as ToLCNDV cucumber (ToLCNDV-CB) and was originally isolated from cucumber can not infect tomato. In order to investigate the factors that affect its host adaptability, gene recombination between ToLCNDV-OM and ToLCNDV-CB was done. Tobacco (N. benthamiana) and tomato plants grown in green house were challenged with the recombinant viruses via agroinfiltration. The inoculation results showed that the exchange of the gene coding for movement protein (MP) affected the virus infectivity on tomato. Furthermore, we have also identified that the 19th amino acid residue (glutamate, E19) of ToLCNDV-OM MP gene determines the tomato adaptation via the protein point mutation protocol. It was also shown that not only19th amino acid residue of ToLCNDV-CB MP gene, but the 3rd amino acid residue (isoleucine, I3) also has influence in its adaptability in the tomato. In this study, we used two isolates of one virus to prove that the MP of ToLCNDV is the critical factor for its host adaptability via gene recombination and point mutation.

碩士
國立彰化師範大學
生物學系
106
Tomato (Solanum lycopersicum) is one of the most important crops worldwide, and is affected by whitefly-transmitted tomato leaf curl Taiwan virus (ToLCTWV) in Taiwan. The symptoms such as stunted growth, leaf curling, abnormal development of epidermal tissues and delayed flowering were observed in tomato plants affected by ToLCTWV. The virus then caused reduced fruiting. ToLCTWV belongs to the geminiviruses (Begomovirus). The geminivirus-encoded V2 protein, known as pre-coat protein, was identified as a major factor of pathogenicity. The viral proteins may counteract with each other to against host resistance. However, the molecular functions of viral proteins pathogenicity and counterreactions are still unknow. In this study, we analyzed the (1) pathogenicity and counterreactions of V2 protein, (2) transcription activation and transcriptome analysis of V2 protein, and (3) counter-defence of host transcriptional gene silencing and ubiquitin-proteasome system by viral proteins of ToLCTWV in Solanum lycopersicum cv. Micro-Tom and Nicotiana benthamiana as host plants. The results indicated that viral V2 protein caused the most severe symptoms in plant, which may via activating the coat protein transcription and interfering with plant salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) mediated signaling pathway. Additionally, C2 protein can counteract the necrosis induced by V2 protein in N. benthamiana. Bimolecular fluorescence complementation (BiFC) showed interactions between C2 and V2 proteins. Furthermore, both V2 and C2 protein suppressed transcriptional gene silencing (TGS) and interacted with each other to prevent the degradation of viral proteins from ubiquitin-proteasome system (UPS) in N. benthamiana. These results revealed the viral strategies of counter-defence to host plants.

博士
國立中興大學
植物病理學系所
98
Abstract Whitefly-transmitted geminiviruses (Geminiviridae) and thrips-borne tospoviruses (Bunyaviridae) are two groups of extremely important plant viruses. Current transgenic approach based on genetic engineering technology provides an efficient strategy to breed plants to resist viral infection. However, public concerns about the use of antibiotic- and herbicide-resistance genes for the selection of transgenic plants during the transformation process have increased tremendously. Therefore, the objectives of this study were to develop the reliable transformation system that could remove selectable markers while generating transgenic plants that would resist the infection of geminiviruses and tospoviruses. Firstly, Tomato leaf curl Taiwan virus (ToLCTWV), a predominant tomato-infecting geminivirus in Taiwan, was subjected to investigate which viral gene fragments can confer high resistance to geminiviruses in transgenic plants. Individual transgenic constructs covering the entire ToLCTWV genome was transformed into Nicotiana benthamiana plants. Four constructs including IRC1 (intergenic region flanked with 5′ end of C1), C2 (partial C2 ORF), C2C3 (overlapping region of C2 and C3 ORFs) and Rep2 (3′ end of the C1 ORF) of high resistance for ToLCTWV have been observed. The detection of siRNA in transgenic plants confirmed that the mechanism of resistance was via gene silencing. Moreover, the middle half of the N gene of Tomato spotted wilt virus (TSWV), which is the type member of Tospovirus, was fused with the partial C2 ORF of ToLCTWV as the chimeric transgene and transformed into N. benthamiana and tomato to develop transgenic plants with multiple viral resistance. The transgenic plants remained symptomless post agro infected with ToLCTWV and exhibited high resistance to TSWV. The detectable siRNAs demonstrated that the resistance was mediated by gene silencing mechanism. The results also explained that linking multiple gene fragments of two viruses with different genomic organization was an effective strategy to engineer plants against both DNA and RNA viruses. Meanwhile, we developed three strategies of co-transformation to generate marker-free transgenic plants; they were (1) pGANP-CP1/pBin19, which comprises two individual plasmids carrying T-DNA of the target and marker genes separately; (2) pGA2T-CP1, which consists of one plasmid carrying two T-DNAs for the target and marker genes; and (3) pGA2TNH, which contains two T-DNAs in one plasmid in which one T-DNA carries the bi-selectable marker which can be used for more plant species especially those with low sensitivity to kanamycin. The co-transformation frequencies of the R0 transgenic N. benthamiana plants for both selection marker and target gene were similar. The co-transformation frequencies of three vector systems were about 50%. Segregation of transgene and selectable marker gene was revealed in the progeny of some co-transformed lines. The highest production ratio of marker-free transgenic plants was 24.1% in two plasmids system, followed by 18.6% in one plasmid system and 17.5% in bi-selectable marker system. We demonstrated that these strategies were feasible and efficient to eliminate the marker genes, and can provide a practical and simple tool for generating marker-free transgenic plants. Therefore, previously mentioned gene fragments that confer high resistance to ToLCTWV including IRC1, C2, C2C3 and Rep2 were linked together and fused with the middle half of the N gene from TSWV to make a chimeric transgene to be constructed into the binary vector, pGA2TNH, for the generation of viral resistance marker-free transgenic N. benthamiana. The transgenic R0 plants resistant to ToLCTWV were obtained and the marker-free resistant progeny plants were segregated by self-pollination. Overall, the results showed in this study have important implications for field deployment of transgenic strategies to control geminivirus and tospovirus. Moreover, the plant transformation systems that can generate marker-free plants would certainly boost the public acceptance of transgenic crops.

Bibliography: leaves 133-152.
xi, 152 leaves : ill. (some col.), col. map ; 30 cm.
Studies biological relatedness of strains of tomato leaf curl virus and cross-interaction with the replication-associated protein requireed for DNA replication.
Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1997

碩士
中興大學
植物病理學系所
95
Mosaic, leaf curl, and puckering symptoms were observed in Cucumis melo L. var. Silver Light plants in the field during 2007 spring season. Three samples were collected from symptomatic plants. A virus culture SL-1 was isolated and established in the systemic hosts, Nicotiana benthamiana and C. melo L. plants via mechanical inoculation. Viral DNA was extracted from diseased oriental melon plants with leaf curl from field and polymerase chain reaction (PCR) with reported primers was used to detect the presence of begomoviral DNA-A, DNA-B, and associated satellite DNA. Begomoviral DNA-A and DNA-B were detected in all three diseased samples. The PCR-amplified 1.5 kb viral DNA-A and 2.5 kb DNA-B from one positive sample were cloned and sequenced. Base on the assemblled sequence, specific primers were designed for cloning the full-length DNA-A and DNA-B of the isolate SL-1, which were determined as 2739 and 2673, repectively, nucleotides (nt) in length. Sequence analyses were conducted using DNAMAN Sequence Analysis Software. The DNA-A contained the conserved nanonucleotides-TAATATTAC and eight open reading frames, including three in virus sense (AV1 to AV3) and five in virus complementary sense (AC1 to AC5). The DNA-B contained two open reading frames, including the nuclear shuttle protein in virus sense and the moving protein in the virus complementary sense. BLASTn analysis and sequence comparison with those available in the GenBank showed that isolate SL-1 has highest nucleotide identity of 97% and 93%, respectively, with the DNA-A and DNA-B of Tomato leaf curl New Delhi virus (ToLCNDV) from Thailand. Our results indicate that the virus causing leaf curl disease on oriental melon is an isolate of ToLCNDV and designated as ToLCNDV-OM (ToLCNDV-oriental melon isolate). In addition, 38 plants species from 4 families (Solanaceae, Chenopodiaceae, Cucurbitaceae, Leguminosae) were mechanically inoculated with ToLCNDV-OM for host reaction assay. Systemic symptoms were observed on plants of C. melo L., C. sativus L., Luffa aegyptiaca, Cucurbita pepo, Lagenaria siceraria, C. melo Linn, N. benthamiana, and Lycopersicum esculentum. To our knowledge, this is the first report of ToLCNDV causing a severe disease on oriental melon plants and can be transmitted through mechanical inoculation.

碩士
國立彰化師範大學
生物學系
105
Tomato is one of the most important crops worldwide and has been severely affected by geminiviruses. Tomato leaf curl Taiwan virus (ToLCTV), belonging to the geminiviruses, was isolated in Taiwan and causes tremendous crop loss. The geminivirus-encoded C2 proteins are crucial for a successful interaction between the virus and host plants. However, the exact functions of the viral C2 protein of ToLCTV have not been investigated. We analyzed the molecular function(s) of the C2 protein by transient or stable expression in tomato (Solanum lycopersicum cv. Micro-Tom) and Nicotiana benthamiana as host plants. Shoot growth was severely stunted by infection of Micro-Tom and N. benthamiana with ToLCTV as compared with control plants. Expression of ToLCTV C2-green fluorescent protein (GFP) fusion protein was predominately located in the nucleus, which indicates the role of C2 in interference with host factors that are distributed in the organelle. Notably, the C2-GFP fluorescence was distributed in nuclear aggregates. Micro-Tom and N. benthamiana plants inoculated with potato virus X (PVX)–C2 displayed chlorotic lesions and stunted growth. PVX-C2 elicited hypersensitive responses accompanied by production of reactive oxygen species in N. benthamiana plants, which suggests that the viral C2 was a potential target of the host-defense responses. ToLCTV C2 protein was also characterized for its RNA silencing suppressor activities. Transgenic N. benthamiana plants expressing the C2 protein showed decreased expression of the gene encoding chromomethylase (NbCMT3-2) and pNbCMT3-2::GUS (β-glucuronidase) promoter activity. C2 protein may be an important pathogenicity determinant of ToLCTV and interfere with host components involved in DNA methylation.

碩士
國立屏東科技大學
植物保護系
94
Tomato leaf curl Taiwan virus (ToLCTWV) is a serious infected virus of tomato in Taiwan, which causing symptoms of leaf yellowing, leaf-curling, stunting, shortening of internodes and severe reduction of leaf size on tomato. ToLCTWV is a whitefly (Bemisia argentifolii) transmitted geminivirus (Geminiviridae, Begomovirus). In this study, polymerase chain was used to detect ToLCTWV in infected whitefly. Feeding on infected tomato, ToLCTWV can be detected in female whitefly in 5 minutes and is 20 min in male. Vector feeding 24 hours on ToLCTWV-infected tomato was analyzed, amount of 5 pooled crawlers, 2 pooled nymphs, and one single pupa of whitefly shows ToLCTWV can be detected available. However, no ToLCTWV could be detected in eggs production of infected whitefly on watermelon. Detection of ToLCTWV containable study reveals the infected proportion in female was 85.8% and is higher than 70.3% in male. Samples from field collection also indicated the infectious female (33.8%) was higher than that in male (18.7%). DNA template of ToLCTWV-infected whitefly was diluted to see the detected sensitivity and the results were 2-10 in female and 2-5 in male. Analysis shown that ToLCTWV can be detected in whitefly in 30 days. Two variants each from eastern and western Taiwan were cotransmitted to tomato by whiteflies, and PCR-RFLP was used to detect the ToLCTWV variants in feeding whitefly and its feeding site of tomato. Results shown that ToLCTWV can be detected in feeding whitefly and its feeding site of plants, however, the pattern of eastern ToLCTWV were detected more than that of western pattern. In the mixed infection analysis, 75 detections were contained ToLCTWV, 32 of them revealed the coexisted ToLCTWV pattern between feeding whitefly and its feeding site, whereas 10 revealed the reverse ToLCTWV pattern between whitefly and plant. Sixteen and seventeen can be detected only in whitefly and plant, respectively. Our studies provided a simple and rapid ToLCTWV identified method in whitefly, which will apply to the ToLCTWV influence of field vector and tomato.

碩士
國立中興大學
國際農學碩士學位學程
104
Plant virus is one of the pathogens that can cause severe disease in agricultural crops worldwide. Tomato spotted wilt virus (TSWV) (genus Tospovirus) and Tomato yellow leaf curl Thailand virus (TYLCTHV) (genus Begomovirus) belong to the top ten economically important plant viruses. They can cause severe damage in many plants such as maize, sweet potato, rice, wheat, and most especially tomato, which is the most exported fruit in many countries. TSWV, a wide host range virus, was first reported in sweet pepper in Taiwan in 2009, which showed chlorosis and chlorotic spots on leaves. In 2007, TYLCTHV was first reported in Taiwan, it was found to be more virulence and damage the tomato industry in Taiwan. A partial RNA-dependent-RNA polymerase gene (L gene) of TSWV, as well as 3’ end of the C1 gene (Rep), overlapping region of C2 and C3 gene (C2C3), and the intergenic region (IR) of TYLCTHV were developed from the previous study and being used to generate transgenic Nicotiana benthamiana resistant to TYLCTHV and TSWV. A marker-free pGA2T-C1C2C3-RDRin3-IR-TSWV carrying the IR constructed as inverted repeat sequence and inserted into the intron of a tomato RNA-direct RNA polymerase 1 (RdR1) gene with the flank fused Rep, C2C3 and TSWV-L gene was used for Agrobacterium-mediated transformation of N. benthamiana in this study. Transient expression was carried out in order to examine the hairpin construct. N. benthamiana were agro-infiltrated with individual constructs and RNAs were extracted 3 days after agro-infiltration. RT-PCR were performed using specific primers of Rep and C2C3 genes. Targeting regions flanking the intron sequence revealed a fragment of 0.8 kb similar to the control Rep fused with C2C3 fragment which contain no intron. The hairpin fragment inserting in intron fragment was amplified by PCR which revealed a fragment of 2 kb. Transgenic N. benthamiana were grown in greenhouse with suitable conditions and mechanically infected by TSWV and TYLCTHV to observed virus resistance within 30 days. The presence of inserted genes was detected by PCR using specific primers for the selection marker (nptII) and inserted viral fragments (Table. 1). After TYLCTHV inoculation, four out of thirty (13.33%) transgenic N. benthamiana lines showed no symptom within 30 days, fourteen out of thirty (46.67%) showed delayed symptoms within 15-28 days after inoculation. Three out of thirty (10%) transgenic N. benthamiana lines showed no symptom within 30 days and nine out of thirty (30%) showed delayed symptom within 15-28 days after inoculated with TSWV. All resistant transgenic N. benthamiana contained all viral sequence fragments after PCR detection using different primer pairs. Transgenic resistant N. benthamiana were self-fertilized and seeds were collected. After which, R1 transgenic N. benthamiana were challenged with viruses. More than 40% of R1 transgenic N. benthamiana showed no symptom when challenged with TYLCTHV, while 20-55% of R1 transgenic N. benthamiana showed no symptom when challenged with TSWV. Marker-free transgenic N. benthamiana of more than 12% were also obtained and showed resistant to both TSWV and TYLCTHV. These results indicated that pGA2T-C1C2C3-RDRin3-IR-TSWV construct could provide a promising tool for developing resistance against both plant RNA and DNA viruses. Keywords: DNA methylation, hairpin RNA, RNA interference (RNAi), marker-free, Tomato spotted wilt virus, Tomato yellow leaf curl virus, transgenic plant, virus resistanceKeywords: DNA methylation, hairpin RNA, RNA interference (RNAi), marker-free, Tomato spotted wilt virus, Tomato yellow leaf curl virus, transgenic plant, virus resistance

碩士
國立中興大學
生物科技學研究所
98
Tomato Yellow Leaf Curl Virus (TYLCV), a member of the family Geminiviridae, genus Begomovirus, harbors single-stranded circular DNA genome. The intergenic region of TYLCV genomic DNA contains bidirectional promoter elements interspersed within the origin of replication. The replication-associated protein (Rep) is essential for viral DNA replication, in which the Rep protein initiates rolling circle replication by binding to specific repeated sequences (iterons) within the origin of replication and acting as a topoisomerase. The aim of this study is to explore the underlying mechanism for the specific interaction between TYLCV Rep protein and the cognate viral genomic DNA by identifying the sequence and location of TYLCV iterons and DNA-binding domains of TYLCV Rep protein. The TYLCV genome was amplified from Bemisia tabacci (whiteflies) by Rolling Circle Amplification (RCA), and cloned into the pUC119 vector. The full length Rep open reading frame, N-terminus (Rep1-181) and C-terminus (Rep177-361) of Rep were cloned in pET21d and pET29a vectors and expressed in Escherichia coli. The target proteins were purified by electro-elution from polyacrylamide gels, and used to raise Rep-specific antibodies in rabbits. The interactions between Rep proteins and TYLCV genomic DNA were analyzed by Southwestern blot with Rep-specific antibodies. Preliminary results showed that Rep protein specifically interacted with double-strand circular geminivirus DNAs, instead of the linear form DNAs, suggesting that the binding specificity is partially determined by DNA topology. Both full-length Rep and Rep1-181 preferentially interact with IR and the coding regions of Rep and C4 protein, indicating that the main DNA-interacting domain locates at the N-terminus of Rep protein, while Rep177-361 might play the role of specificity determinant. The Type I topoisomerase function of Rep protein were analyzed by a mimicry RCA in vitro by using Φ29 DNA polymerase. The result confirmed that Rep protein possesses nicking / re-ligation activity, providing evidence that the native activity of the Rep protein expressed and purified from prokaryotic cells was still preserved. Through this study, it is expected that practical methods to disrupt the DNA replication cycles could be developed as the effective therapeutics for viral diseases caused by TYLCV and other geminiviruses.

碩士
國立臺灣大學
昆蟲學研究所
95
Bemisia tabaci (Gennadius) is a globally distributed insect pest in the field. It has a wide range of host plants and is thought to be a species complex with many biotypes. In Taiwan, biotypes B, An, and Nauru have been recorded. The economic impacts of this insect pest consist of its damage to host plant health by sucking plant juices, its pollution of plants by secreting honeydew, and its transmission of plant viruses. The most serious damage caused by B. tabaci is its transmission of Tomato leaf curl Taiwan virus (ToLCTWV) to tomato plants in Taiwan. In this research, we collected B. tabaci from many tomato fields in Taiwan, and detected the ToLCTWV from B. tabaci by polymerase chain reaction (PCR). At the same time, specific primer sets were used to identify the biotypes of B. tabaci. In this research, 98.97% of the examined B. tabaci specimens were biotype B, and all of viruliferous B. tabaci specimens with the ToLCTWV were biotype B. Moreover, we studied the phylogeny and phylogeography using molecular markers. The results showed that the ToLCTWV can be separated into western and eastern Taiwan “variants”. Genetic distance analysis revealed that the variants of western Taiwan are closely related. On the contrary, genetic distances among variants of eastern Taiwan were more variable. Furthermore, we studied the retention possibility of the ToLCTWV among B. tabaci biotypes, and found that both the B and Nauru biotypes can maintain the ToLCTWV. In contrast, retention of the ToLCTWV by biotype An was weak after 10 days of infection. In the study of symbionts of B. tabaci, we found that sequence similarity of the protective gene against virus particles was 73% between biotypes Nauru and B. Furthermore, phylogenetic analysis of 16S rDNA showed that the primary symbionts were consistently grouped with B. tabaci biotypes and the geographic distribution.

碩士
國立臺灣大學
園藝學研究所
99
Tomato yellow leaf curl disease (TYLCD) induced by Tomato yellow leaf curl virus is one of the most severe diseases of tomato. TYLCD defeats tomato production worldwide and often causes 100％ economic loss. There is no efficient method to control TYLCD, except for breeding resistant cultivars. However, the plant physiology concerning TYLCD infection remains totally unknown. This research studied the antioxidation and proteomics of tomato plants after infection of TYLCD to understand the resistance mechanism. The resistant tomato line CLN2777G-1-5 and susceptible cultivar Tomatoll which were selected and bred by Asian Vegetable Research and Development Center, and Tomato yellow leaf curl Thailand virus (TYLCTHV) were used as experimental materials. The results indicated that TYLCTHV is transmitted by whitefly that not mechanical. The leaf number, plant height, relative chlorophyll content (SPAD value) and chlorophyll fluorescence Fv/Fm of the infected ‘Tomatoll’ plant, were significantly decreased. All plants of susceptible cultivar were detected the occurrence of TYLCTHV DNA and first symptom appeared on 3 and 7 days after inoculation. While eighty three percent plants of resistant line were not detected the occurrence of virus DNA until 28 days and did not show symptom. Activity of superoxide dismutase (SOD) in leaves of resistant line was significantly lower than susceptible cultivar on 1 and 7 days after inoculation. However, the activities of catalase (CAT)、ascorbate peroxidase (APX)、glutathione reductase (GR) and peroxidase of resistant line raised successively after inoculation. The raised activities of antioxidant enzymes may induce the resistance of CLN2777G-1-5 plants to TYLCTHV. Proteomics of leaves analyzed by ProteomeLabTM PF-2D and matrix-assisted laser desorption ionization time-of-flight, thirty proteins with differential expression were identified from CLN2777G-1-5 and‘Tomatoll’after inoculation with TYLCTHV. These proteins involve photosynthesis, stress defense, protein transport, translation, protein lysis, and oxidation-reduction reaction. The results may be helpful to plan strategy and select index for breeding TYLCTHV resistant tomato.

碩士
國立彰化師範大學
生物學系
105
Geminiviruses possess a small circular single-stranded DNA (ssDNA) as their genome and cause tremendous loss in many economically important crops and vegetables. Tomato (Solanum lycopersicum L.) is one of the most popular and widely consumed vegetable all over the world because of its high nutritional value. Tomato leaf curl Taiwan virus (ToLCTV) belongs to geminiviruses which occurs in tomato-growing area in Taiwan. However, the exact functions of the viral C1, C4 and V2 proteins of ToLCTV have not been characterized and compared. In this study, we analyzed the molecular function(s) of the C1, C4 and V2 protein by potato virus X (PVX)-mediated transient expression in tomato (Solanum lycopersicum cv. Micro-Tom) and Nicotiana benthamiana as host plants. Distinct host responses to transient expression of ToLCTV C1, C4 and V2 were observed. ToLCTV V2 triggered hypersensitive responses (HR) in tomato and N. benthamiana plants but not the viral C1 and C4 proteins. Thus, V2 was a potential target of the host-defense responses. Transient expression of ToLCTV C4 and V2 also suppressed host transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). The results indicate that the viral protein plays distinct roles to counter host defense mechanisms. Comparison of subcellular localization of the three viral proteins reveald that GFP (green fluorescent protein)-C1 were predominant in the cell periphery, whereas GFP-V2 were accumulated in the cell periphery and nucleus. In contrast, C4 was localized in the cytoplasm in a more granular fashion with various sizes. The aggregate of C4 proteins resulted from self-interactions as demonstrated by bimolecular fluorescence complementation (BiFC). Agrregate of the C4 protein was more resistance to host protein degradation systems. In summary, this study characterized the distinct pathogenesis and host responses to transient expression of individual genes encoded by ToLCTV.

"September 2001"
Bibliography: leaves 198-262.
xii, 262 leaves, [5 p.] : ill. (col.), plates (col.) ; 30 cm.
The aim of this research was to express the isolated c4 gene in transgenic plants to determine its function 'in planta' and examine its potential use in a virus resistance strategy.
Thesis (M.Ag.Sc.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2002

"September 2001"
Bibliography: leaves 198-262.
xii, 262 leaves, [5 p.] : ill. (col.), plates (col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
The aim of this research was to express the isolated c4 gene in transgenic plants to determine its function 'in planta' and examine its potential use in a virus resistance strategy.
Thesis (M.Ag.Sc.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2002

"October, 2003"
Bibliography: leaves 130-141.
xvii, 150, [10] leaves : ill. (some col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
This study describes the silencing of tobacco transgenes carrying TLCV promoters following TLCV infection.
Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine. 2003

碩士
國立中興大學
植物病理學系所
106
Geminiviridae is the largest family of plant viruses. The genus Begomovirus, which is the most important genus of geminiviridae, has one or two circular single stranded- DNA genome. Begomoviruses are usually transmitted by whitefly (Bemisia tabaci). Very few begomoviruses are mechanically transmissible by sap-inoculation on leaves. Previous studies in our lab have shown that tomato leaf curl New Delhi virus oriental melon isolate (ToLCNDV-OM) is an isolate of bipartite begomovirus and can infect cucurbits via mechanical transmission. Genome reassortment assay of ToLCNDV-OM and a non-mechanically transmissible ToLCNDV-CB isolate showed that the movement protein (MP) of ToLCNDV-OM plays the key role in mechanical transmission. This is the first identification of a specific geminiviral movement protein as a determinant of mechanical transmissibility. Tomato leaf curl Taiwan virus (ToLCTV) was first detected in Taiwan in 1981 and was endemic throughout the island by the early 1990’s. tomato yellow leaf curl Thailand virus (TYLCTHV), which was first detected in 2005, is now widespread throughout Taiwan. TYLCTHV is a bipartite and mechanically transmissible begomovirus, whereas ToLCTV is a monopartite and not mechanically transmissible. Through monitoring the begomoviruses in Taiwan during 1998-2009, it becomes apparent that the introduced TYLCTHV is now the predominant strain in the most tomato production areas. Based on the result of ToLCNDV study, we first focused on the MP of TYLCTHV and constructed four mutants, each carrying a point mutation within MP. When the 17th or 23rd amino acid (Glutamate, E) was mutated to Alanine (A), the mutants cannot be mechanically transmitted to Nicotiana benthamiana. This result showed that TYLCTHV MP is also the critical viral factor for mechanical transmissibility. Future studies will focus on the identification and isolation of host factors that may play crucial roles in the viral infection process by interacting with MP and assisting mechanical transmissibility of TYLCTHV.