Student Number : 9610284H - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Cassava Mosaic Disease is the most devastating disease affecting cassava (Manihot esculenta Crantz) crops worldwide. This disease is associated with eight species of geminiviruses, all belonging to the genus Begomovirus of the family Geminiviridae. In South Africa, in particular, CMD is caused by South African cassava mosaic virus (SACMV). Currently, there are no adequate methods for control of this disease as mechanisms within virus-host interactions are poorly understood. This brings about the need for development of virus-disease control strategies. This study was therefore conducted to identify the host’s response to an invading virus. The model plant, Arabidopsis was chosen as it is a well-characterized plant system, with expression databases readily available as its entire genome has been sequenced. This study was conducted, firstly, to phenotypically determine if Arabidopsis was resistant or susceptible to SACMV infection, and secondly, to identify the host’s response to pathogen infection on a molecular level through gene expression studies utilizing microarrays. Results from the symptomatology study revealed that Arabidopsis plants were fully symptomatic 28 days post-inoculation, displaying characteristic disease symptoms such as stunting, yellowing, and leaf deformation. This indicated that Arabidopsis was susceptible to SACMV infection. Microarray analyses revealed 86 differentially expressed genes, of which 48 showed up-regulation and 38 down-regulation. Relative quantification real-time PCR was performed on selected genes to confirm these results. Many up-regulated genes were shown to be primarily involved in a general stress response induced by the host, whereas those genes that were downregulated seemed to be involved in more specific responses to viral invasion, probably a consequence of suppression of host genes by SACMV to enhance its own replication. The majority of genes identified fell under the predominant functional categories involved in metabolism, transcription, and transport. To our knowledge, this is the first study in which a DNA geminivirus has been used in a host-pathogen interaction utilizing microarrays.

Cassava (Manihot esculenta Crantz) is a basic staple food crop in Tanzania. Cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses (CMGs) constitutes a major limiting factor to cassava production in the country. This study was undertaken to characterize the CMGs occurring in Tanzania using molecular techniques and to map their geographical distribution to generate information on which the formulation of control measures can be based. Using Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) for analysis of CMGs DNA-A genomes, different CMGs were found to be associated with CMD. Higher molecular diversity was observed among East African cassava mosaic viruses (EACMVs) than African cassava mosaic viruses (ACMVs), which was confirmed later by complete nucleotide sequence analysis. In addition to EACMV and ACMV isolates, two isolates of EACMV Cameroon virus (EACMCV) were found in Tanzania. These were confirmed to be strains of EACMCV Cameroon, originally described in Cameroon, West Africa and here named EACMCV- [TZ1] and EACMCV-[TZ7]. They had high (92%) overall DNA-A nucleotide sequence identity and EACMCV-[TZ1] was widespread in the southern part of the country. A subgenomic DNA form of CMG that appeared to be truncated was identified in a CMD-infected cassava plant. It was confirmed upon sequence analysis to be a defect of EACMV DNA-A and had a capacity of attenuating symptoms when coinoculated with wild-type EACMV. In addition, this study revealed for the first time the presence of two novel non-geminivirus single-stranded DNA (ssDNA) sub-genomic molecules associated with CMG infection. They were shown to be dependent on CMG for replication and movement within the plants, confirming their status as satellite molecules named here as satDNA-II and satDNA-III. When present in coinfection with CMGs, they enhance symptoms and can break high levels of resistance in a cassava landrace. Finally a simple, inexpensive technique is described of archiving, transporting and recovering plant DNA for downstream geminivirus characterisation. / Thesis (PhD)--University of Pretoria, 2007. / Microbiology and Plant Pathology / Unrestricted

Submitted by Bruno Rossitto de Marchi (bruno_dmarchi@hotmail.com) on 2018-08-08T18:14:08Z No. of bitstreams: 1 Bruno De Marchi Tese Definitivo.pdf: 5408910 bytes, checksum: 6c7b13538e592fdcfc85ae1e3eb12212 (MD5) / Approved for entry into archive by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2018-08-08T18:50:48Z (GMT) No. of bitstreams: 1 de marchi_ br_dr_botfca.pdf: 5386545 bytes, checksum: 8a0580cd031695c504bc6270ee8317c9 (MD5) / Made available in DSpace on 2018-08-08T18:50:48Z (GMT). No. of bitstreams: 1 de marchi_ br_dr_botfca.pdf: 5386545 bytes, checksum: 8a0580cd031695c504bc6270ee8317c9 (MD5) Previous issue date: 2018-07-31 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A mosca-branca, Bemisia tabaci (Gennadius) é uma praga de distribuição global que afeta centenas de diferentes plantas hospedeiras incluindo grandes culturas, olerícolas e ornamentais. B. tabaci causa danos principalmente através da transmissão de vírus de plantas como os begomovirus, crinivirus, ipomovirus, torradovirus e carlavirus. Atualmente, B. tabaci é considerada um complexo de pelo menos 40 espécies crípticas que apresentam diversidade genética, biológica e diferentes composições de bactérias endossimbiontes facultativas. No Brasil, tanto espécies nativas quanto exóticas de mosca-branca são encontradas e ainda há uma escassez de dados genômicos destas populações e das bactérias endossimbiontes. Na África Oriental, altas populações de moscas-brancas estão disseminando diferentes vírus de plantas que causam epidemias na cultura da mandioca (Manihot esculenta) e com perdas na produtividade. A principal forma de manejo desses vírus na África é através da utilização de variedades tolerantes. Portanto, é essencial a identificação de novos genes de resistência para o desenvolvimento de variedades e um manejo eficiente das doenças. O sequenciamento de transcriptomas é uma ferramenta que possibilita uma análise genômica da mosca-branca, dos vírus transmitidos por ela, das bactérias endossimbiontes e das plantas hospedeiras dessa praga. Portanto, os dados genômicos obtidos dão suporte para o desenvolvimento de novas técnicas que podem se tornar futuras alternativas de controle de mosca-branca e dos vírus associados. No Capítulo 1, dados de transcriptomas foram obtidos das diferentes espécies de B. tabaci encontradas no Brasil, tendo sido possível a obtenção de genomas mitocondriais completos de espécies exóticas e nativas de mosca-branca, além de genomas parciais do endossimbionte facultativo Hamiltonella. A análise filogenética revelou que as diferenças genéticas presentes no gene mtCOI entre as espécies nativas e as espécies exóticas se estendem ao genoma mitocondrial e ao endossimbionte facultativo Hamiltonella. Além disso, foi possível verificar uma deleção de aminoácidos somente no gene GroEL de Hamiltonella que está presente em populações de moscas-brancas nativas. Esse gene é conhecido por estar associado a transmissão de vírus de plantas. No Capítulo 2, foram sequenciados transcriptomas de plantas de mandioca naturalmente infectadas com vírus coletadas no campo em diferentes países da África. A partir desses dados, foi avaliada a diversidade de genes do cloroplasto e a relação com diferentes espécies de vírus. Há uma baixa diversidade dentre as cultivares de mandioca atualmente plantadas na África e não foi possível verificar nenhuma relação entre os genes de cloroplastos avaliados e as espécies de vírus detectadas ocorrendo naturalmente no campo. Os dados obtidos reforçam a necessidade da introdução de novos materiais genéticos para aumentar a diversidade genética das cultivares de mandioca plantadas nesses países, a fim de melhorar as alternativas de manejo das epidemias de vírus. / The whitefly, Bemisia tabaci (Gennadius) is a global pest that affects hundreds of different plant hosts including vegetable, fiber and ornamental crops. B. tabaci causes damage mainly by the transmission of plant viruses such as begomoviruses, criniviruses, ipomoviruses, torradoviruses, and carlaviruses. Currently, B. tabaci is known as a complex of at least 40 putative cryptic species that shows genetic and biological diversity and a different composition of bacterial facultative endosymbionts. In Brazil, both exotic and indigenous species of whiteflies are found and there is still a lack of genomic data available among these populations and also their bacterial endosymbionts. In East Africa, high populations of whiteflies are transmitting different plant viruses that are causing epidemics in cassava crops (Manihot esculenta) and leading to great yield losses. Currently, the management of these viral diseases in Africa is carried out mainly by growing cassava tolerant varieties. Therefore, is essential to identify new target genes for the development of new varieties for an efficient management of viral diseases. Transcriptome sequencing is a tool that allows a genomic analysis of the whitefly, whitefly-transmitted viruses, bacterial endosymbionts and plant hosts. Therefore, the genomic data obtained gives support for the development of new technics that might aid for future management alternatives of whiteflies and their associated viruses. In Chapter 1, transcriptome data were obtained from different B. tabaci species found in Brazil which allowed to obtain complete mitochondrial genomes from different whitefly species and draft genomes of the facultative endosymbiont Hamiltonella. The phylogenetic analysis revealed that the genetic differences among exotic and native populations present in the mtCOI gene extends to the mitochondrial genome and to the facultative endosymbiont Hamiltonella. In addition, it was verified an amino acid deletion in the GroEL gene from Hamiltonella present only in native populations of whiteflies. This gene is known to be associated with the transmission of plant viruses. In Chapter 2, transcriptome data were obtained from virus-infected cassava plants collected in the field in different African countries. The data allowed to evaluate the diversity of chloroplast genes and their relationship with different virus species. The data revealed a low genetic diversity among cassava currently grow in East Africa. In addition, there was no direct relationship between the evaluated chloroplast genes and the virus species detected. The obtained data reinforce the need of introduction of new genetic accession to increase the genetic diversity of the currently grown cassava in Africa in order to improve the alternatives of management of viral diseases. / CNPq 131324/2015-5 / CNPq 200826/2015-8

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 4 uppsatser.