Orientadores: Gonçalo Amarante Guimarães Pereira, Sandra Martha Gomes Dias / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T22:03:27Z (GMT). No. of bitstreams: 1 Barsottini_MarioRamosdeOliveira_M.pdf: 16356564 bytes, checksum: 9cf8e738f13f95639e35471c9a7da160 (MD5) Previous issue date: 2013 / Resumo: O fungo Moniliophthora perniciosa desperta grande interesse agroeconômico, pois é o agente etiológico da Vassoura-de-Bruxa do cacau. A cultura do cacaueiro é de grande importância no cenário nacional e na América Latina, sendo o entendimento dos mecanismos moleculares desta doença de grande valia. Durante a interação entre patógeno e hospedeiro, o primeiro produz moléculas para evadir ou alterar as respostas normais de defesa do segundo. O sequenciamento do genoma de M. perniciosa levou à identificação de proteínas-chave potencialmente envolvidas no processo patogênico do fungo, dentre as quais, estão: proteínas pertencentes à família das Cerato-plataninas (MpCPs), bem como proteínas pertencentes à família das Necrosis- and Ethylene-inducing Proteins (MpNEPs). As CPs são amplamente associadas à interação fungo-hospedeiro, agindo como toxinas, indutoras de resposta de defesa ou alergenos. As NEPs induzem morte celular e necrose em plantas dicotiledônes através da permeabilização da membrana celular. O objetivo desse projeto foi caracterizar funcionalmente as MpCPs e as MpNEPs, determinando assim sua relevância durante a Vassoura-de-Bruxa. A partir do transcriptoma de M. perniciosa e da análise filogenética das doze MpCPs encontradas, foi revelada uma correlação entre grupos de MpCPs e sua expressão diferencial ao longo da doença. Quatro MpCPs foram clonadas, expressas em sistema heterólogo e tiveram sua estrutura cristalográfica resolvida. Ensaios bioquímicos e biofísicos confirmaram que as MpCPs presentes em diferentes grupos filogenéticos apresentam capacidades distintas no tocante à interação com o açúcar N-acetilglicosamina e de formar agregados ordenados. Estudos funcionais indicaram que estas características estão potencialmente relacionadas ao bloqueio de resposta de defesa da planta e ao crescimento do fungo, respectivamente. Quanto às MpNEPs, somente a isoforma MpNEP2 foi detectada durante a Vassoura-de-Bruxa. A partir da estrutura cristalográfica dessa proteína e ensaios de mutação sítio-dirigida, foi identificado um hairpin hidrofóbico exposto ao solvente, possivelemte associado à ancoragem da MpNEP2 na membrana celular, o qual é tão importante quanto o sítio ativo da proteína para a atividade biológica da mesma / Abstract: Moniliophthora perniciosa is the causal agent of Witches' Broom Disease of cocoa and a major agroeconomic concern in Brazil and Latin America. In order to efficiently control this disease, it is crucial to understand the molecular basis underlying its progression. During the attack to the plant, a pathogen releases molecules to suppress or alter the regular defense response of the host. Results obtained from the genome sequencing of M. perniciosa lead to the identification putative virulence factors belonging to the Cerato-platanin protein family (MpCPs), and to the Necrosis- and Ethylene-inducing Proteins (MpNEPs). CPs are important to fungus-host interaction process, acting as toxins, defense response-inducing molecules or allergens. NEPs are toxin-like pore-forming proteins, which affect only dicot plants. This project aimed at the functional characterization of the MpCPs and MpNEPs, as well as understanding their importance for the Witches' Broom Disease progression. Twelve MpCP-coding genes were identified, and comprehensive transcriptome and phylogenetic analyses showed a correlation between MpCPs evolutionary clusters and their expression patterns throughout the disease. Four representative MpCPs had their crystal structure resolved. Biophysical and biochemical characterizations showed a correlation between the MpCP clusters, regarding sugar (N-acetylglucosamine) binding and protein self-assembling, which are possibly related to plant defense response suppression and hyphal growth, respectively. As for the MpNEPs, only the isoform MpNEP2 was shown to be expressed during the Whitches' Broom Disease. Its crystalloghaphic structure, along with site-directed mutagenesis and functional assays revealed that, besides the protein's active site, an hydrophobic hairpin exposed to te solvent is important to the necrosis-promoting activity, probably mediating the contact of MpNEP2 with the cell membrane / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

D.Phil. / The fungus, Verticillium dahliae, is the causative agent of Verticillium wilt, which results in significant cotton (Gossypium hirsutum) crop losses worldwide. This study contributes to the elucidation of cotton defence responses against V. dahliae. The identification, cloning and characterization of three genes that were differentially expressed in response to elicitation with a cell wall-derived (CWD) V. dahliae elicitor are described. It was hypothesized that the molecular architectures of the three characterized genes are supportive of a role in cotton defence against V. dahliae. As one of these genes was present as two homoeologous copies, this study also reports on the molecular characterization of both homoeologs, thus providing further insight into the processes of genomic evolution between homoeologous loci in allotetraploid cotton. The three genes were initially represented as expressed sequence tags (ESTs), obtained from a previous differential display reverse transcription polymerase chain reaction (DDRT-PCR) study by Zwiegelaar (2003), as part of an MSc project. These ESTs, designated C1B10, C4B5 and C4B4, were differentially induced upon elicitation with a CWD V. dahliae elicitor (Zwiegelaar, 2003). In the present study, the genes represented by the three ESTs were identified and characterized by genome walking and 5‘/3‘ rapid amplification of cDNA ends (RACE). Additionally, PCR and reverse-transcription PCR (RT-PCR) were utilized, where necessary, to obtain internal sequences, not covered by the genome walking and RACE reactions. Through the use of these molecular techniques, the full transcript and genomic sequences of each of the three genes was obtained, including their promoters. The promoter of each gene was analyzed for cis-elements driving gene transcription, through bioinformatic analysis. Furthermore, the copy number of each gene was determined through Southern blot analysis. The genes were translated to reveal their encoded protein sequences. The amino acid sequences were submitted to a basic local alignment (BLAST) search of the NCBI database to identify, and align them with, homologous proteins from other plant species (and those from G. hirsutum, if any). An in silico analysis of the encoded protein of each gene was also performed. This examination included domain architecture, post-translational modification, subcellular location and tertiary structure predictions. This study also involved the isolation of the elicitor from the cell walls of V. dahliae fungal cultures. The potency of the freshly-isolated elicitor was investigated with a triphenyltetrazolium chloride (TTC) viability assay on cotton cell suspensions. Its potential to induce PR-proteins was also explored but these results were inconclusive. In addition, expression studies were performed with real-time PCR (q-PCR), to confirm the up- or down-regulation of each gene upon elicitation of cotton cell suspensions with the CWD V. dahliae elicitor, and to investigate the time frame/kinetics of induction. The gene corresponding to the C1B10 EST was designated GhLIPN as this study revealed that it encodes a lipin protein. Lipins are novel proteins with phosphatidate phosphatase 1 (PAP1) activity, exclusive to eukaryotes. They play a fundamental role in the lipid metabolism of organisms ranging in complexity from yeast to animals and plants. In plants, this role includes lipid membrane remodelling during phosphate (Pi) deficiency. During the study of the GhLIPN gene, it was discovered that it occurred as two distinct homoeologous copies from the A- and D-co-resident genomes of allopolyploid G. hirsutum. The GhLIPN homoeologs were named GhLIPN I and N for Insert present and No insert, respectively, based on the presence or absence of a 13 base pair (bp) insertion/deletion (indel) site in intron 6.

M.Sc. (Biochemistry) / Lipopolysaccharide (LPS) is a complex lipoglycan that is found in the outer membrane of Gram-negative bacteria and is composed of three regions namely the fatty acid Lipid A, a core region of short oligosaccharide chains and an O-antigen region of polysaccharides. When LPS is recognized as a microbe-/pathogen-associated molecular pattern (M/PAMP), it not only induces an innate immune response in plants but also stimulates the development of defence responses such as the immediate release of reactive oxygen species/intermediates (ROS/I), pathogen-related (PR) gene expression and activation of the hypersensitive response (HR), resulting in stronger subsequent pathogen interactions. The identification and characterisation of the elusive LPS receptor/receptor complex in plants is thus of importance, since understanding the mechanism of perception and specific signal transduction pathways will clarify, and lead to the advancement of, basal resistance in plants in order to decrease crop plant losses due to pathogen attack. In mammals, LPS binds to a LPS binding protein (LBP) to form a LPS-LBP complex which is translocated to myeloid differentiation 2 (MD2) with the presence/absence of its co-receptor, a glycosylphosphatidylinositol (GPI)-linked protein, CD14. The interaction occurs on the host membrane and triggers an inflammatory defence response through the signalling cascade activated by the interaction with Toll-like receptor 4 (TLR4). A similar LPS-receptor interaction is, however, unknown in plants. To address the LPS perception mechanism in plants, biological binding studies with regard to concentration, incubation time and temperature, affinity, specificity and saturation were conducted in Arabidopsis thaliana protoplasts using LPS labeled with Alexa 488 hydrazide. Quantum dots (Qdots), which allow non-covalent hydrophobic labeling of LPS, were further also employed in similar Arabidopsis protoplast binding studies. These studies were conducted by fluorescence determination through the use of a BD FACS Aria flow cytometer. Although Alexa-labeling does not affect the biological activity in mammalian studies, the same cannot necessarily be said for plant systems, and hence Qdots were included to address this question. The conjugation of Qdots to LPS was confirmed by transmission electron microscopy (TEM) and results illustrated higher fluorescence values as compared to Alexa-LPS fluorescence analysis. Furthermore, inhibition of the perception process is also reported using Wortmannin and Brefeldin A as suitable endo- and exocytosis inhibitors. Affinity, specificity and saturability as well as the role of endo- and exocytosis inhibition in LPS binding to protoplasts was ultimately demonstrated by both fluorophores, with the use of Qdots as a label proving to be a more sensitive strategy in comparison to the conventional Alexa 488 hydrazide label.

TCV (Turnip crinkle virus) coat protein is required for the resistance response in Arabidopsis thaliana Di-17 plants. An aspartate to asparagine mutation at amino acid four of the coat protein is sufficient to result in resistance-breaking. To determine the essential chemical properties responsible for the induction of resistance, a series of site-directed mutants were produced. Serine as well as asparagine at amino acid four induces systemic disease on both Di-3 and Di-17 plants; however, replacement of aspartate with glutamate retains the ability to induce the HR (hypersensitive response) and resist TCV infection with rapid and strong induction of PR-1 gene. These data suggest that the negative charge at the fourth amino acid of the coat protein is critical for the induction of resistance. Taken together with other mutagenesis research, the N-terminus of the coat protein appears to be the sole viral recognition element. The A. thaliana TIP protein is suggested to be involved in resistance, mainly through its C-terminus. Interestingly, one of the resistance-breaking mutants (D4N) produces a HR on Di-3 plants that are normally susceptible. The Di-3 TIP protein has several differences from the Di-17 TIP. To detect whether the delayed HR is related to interaction between Di-3 TIP and D4N mutation, a yeast two-hybrid assay was attempted. Interactions have not yet been detected. There are a number of possible explanations.

Thesis (Ph.D.). / Written for the Dept. of Plant Science. Title from title page of PDF (viewed 2008/01/12). Includes bibliographical references.

Orientador: Alessandra Alves de Souza / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T07:11:43Z (GMT). No. of bitstreams: 1 Pereira_WillianEduardoLino_M.pdf: 4114093 bytes, checksum: f064932e99a8077c41524b40c01c7dbe (MD5) Previous issue date: 2014 / Resumo: A citricultura brasileira representa um setor comercial muito rico, porém a produtividade brasileira é baixa, sobretudo em razão de pragas e doenças, como a Clorose Variegada do Citros (CVC), causada pela bactéria Xylella fastidiosa que afeta todas as variedades de laranja doce (Citrus sinensis). O melhoramento genético de citros é um desafio em virtude da baixíssima variabilidade genética e do grande avanço de pragas e doenças. Uma alternativa para acelerar a obtenção de plantas resistentes a doenças seria a obtenção de plantas transgênicas. A seleção de genes candidatos em citros seria uma excelente estratégia, não fossem dificuldades para transformação genética como escapes, enraizamento, enxertia e longo ciclo da cultura. A solução para acelerar o conhecimento do potencial do gene em conferir resistência ao patógeno seria então o uso de hospedeiros alternativos como Nicotiana tabacum e Arabidopsis thaliana, pois possuem grande informação genética, protocolos simples de transformação e ciclo de vida curto. Devido à identificação prévia por transcritoma de genes possivelmente associados à resistência de Citrus reticulata a X. fastidiosa, torna-se necessário o estudo da função desses genes para aplicação por transgenia visando resistência em C. sinensis. Assim, o objetivo do trabalho foi utilizar plantas modelo (tabaco e Arabidopsis) para estudo funcional desses genes potenciais. Nossos resultados indicaram que Arabidopsis é moderadamente resistente à infecção por X. fastidiosa, uma vez que a bactéria não consegue colonizá-la eficientemente. Utilizando plantas mutantes de Arabidopsis para os genes candidatos RPS5, RAP2.2, MOA2.2, PseudoNBS, ERF73, RD22 e ATJ2 (homólogos aos diferencialmente expressos em C. reticulata sob infecção) verificou-se que dois deles, RPS5 e RAP2.2, estão envolvidos na resistência à bactéria, pois na ausência deles a bactéria colonizou mais eficientemente o hospedeiro, e portanto, são bons candidatos para superexpressão em C. sinensis visando resistência à CVC. RPS5 codifica uma proteína do tipo CC-NBS-LRR, comumente responsável pelo reconhecimento de moléculas dos patógenos e desencadeando vias de sinalização, enquanto o gene RAP2.2 codifica um fator de transcrição relacionado à uma via de sinalização mediada por etileno, de forma a desencadear respostas contra a infecção. Em relação a tabaco foi estudado a colonização da bactéria visando estabelecer um bioensaio quantitativo. Nesse sentido, foi elaborada e validada uma escala diagramática para correta aferição da severidade da doença nas folhas. Também foi estabelecida uma correlação entre níveis de severidade e população bacteriana, permitindo estimar a população bacteriana na folha a partir da análise de severidade. Um bioensaio mais rápido foi estabelecido para avaliação do gene soyBiPD, candidato à conferir resistência a X. fastidiosa. Entretanto, nossos resultados demonstram que esse gene não confere resistência à esse patógeno na planta modelo, não sendo, portanto, candidato para transformar C. sinensis visando resistência a CVC. Num curto período de tempo, oito genes candidatos foram avaliados e dois apresentaram resultados promissores (RPS5 e RAP2.2), sendo selecionados para transformação em C. sinensis visando tolerância a CVC. Isso comprova a importância de plantas modelo no estudo e seleção de genes promissores e a consequente redução de custos, trabalho e tempo para obter respostas acerca da potencialidade dos genes / Abstract: The Brazilian citrus agribusiness is a very important commodity; nevertheless Brazilian citrus productivity is low, mainly due to pests and diseases that affect the culture. The Citrus Variegated Chlorosis (CVC), caused by the bacterium Xylella fastidiosa is one of the most important diseases, since it affects all varieties of sweet oranges. Citrus breeding programs could be used to solve this problem however the breeding is a challenging due to the very low genetic variability and the breakthrough of pests and diseases. An alternative to accelerate the obtaining of resistant plants to diseases could be through of transgenic plants. The selection of candidate genes in citrus would be an excellent strategy, if there were not difficulties in genetic transformation. The solution to accelerate the knowledge of gene to confer pathogen resistance is the use of alternative hosts such as the Nicotiana tabacum and Arabidopsis thaliana. Both hosts have great genetic information, simple protocol of transformation and short life cycle. Due to the prior identification by transcriptome of genes possibly associated with resistance of C. reticulata to X. fastidiosa become necessary to study the function of these genes. Thus, the goal of this study was to use model plants (Arabidopsis and tobacco) for functional study of potential genes to confer X. fastidiosa resistance and use them in C. sinensis in a transgenic approach. Our results indicated that Arabidopsis is moderately resistant to infection by X. fastidiosa, since bacteria cannot colonize it efficiently. Using mutant plants to the candidate genes RPS5, RAP2.2, MOA2.2, PseudoNBS, ERF73, RD22 and ATJ2 (homologous to the differentially expressed in C. reticulata under infection) it was found that RPS5 and RAP2.2 are probably involved in resistance to this bacterium, since the mutants were more susceptible to X. fastidiosa, therefore they are good candidates for overexpression in C. sinensis aiming CVC resistance. The RPS5 gene encodes a protein of the CC-NBS-LRR type commonly responsible for the recognition of molecules of pathogens and triggering signaling pathways, while RAP2.2 gene encodes a transcription factor related to the signaling pathway mediated by ethylene, triggering responses against infection. Regarding tobacco we study the colonization of bacteria to establish a quantitative bioassay. In this sense, was developed and validated a diagrammatic scale for accurate measurement of disease severity in the leaves. A correlation between levels of severity and bacterial population, allowing estimation of the bacterial population in plant from the analysis of severity, was also established. A faster bioassay was established for evaluation of soyBiPD gene, candidate to confer resistance to X. fastidiosa. However, our results demonstrate that this gene does not confer resistance in the plant model, and therefore would not be used to transform C. sinensis. In a short time, eight candidate genes were evaluated and two of them showed promising results (RPS5 and RAP2.2). These results demonstrate the importance of model plants in accelerate the knowledge of candidate genes and consequent the reduction of costs, labor and time to obtain responses about the potentiality of genes to confer pathogen resistance / Mestrado / Genetica Vegetal e Melhoramento / Mestre em Genética e Biologia Molecular

Orientador: Marcos Antonio Machado / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T15:02:08Z (GMT). No. of bitstreams: 1 Oliveira_TiagoSilva_M.pdf: 9792911 bytes, checksum: ad5dca1b90e42dd95db47317b65cb9be (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The abstract is available with the full electronic document / Mestrado / Genetica Vegetal e Melhoramento / Mestre em Genética e Biologia Molecular

Orientadores: Gonçalo Amarante Guimarães Pereira, Odalys Garcia Cabrera / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T17:42:19Z (GMT). No. of bitstreams: 1 Ambrosio_AlinneBatista_D.pdf: 4013772 bytes, checksum: 30fca05155be94194f624c02d6697d87 (MD5) Previous issue date: 2012 / Resumo: O cacaueiro (Theobroma cacao L., Sterculiaceae), planta nativa da região amazônica, é aprincipal espécie dentre as 22 do gênero que é comercialmente explorada para aprodução de sementes, as quais tem grande importância econômica principalmente porser usado na produção de chocolate.Por conta disso, a produção das sementes sempre foiuma grande preocupação para as regiões produtoras, e o fator que causa o maior declínioda produção de cacau é o aparecimento de doenças. No Brasil, o cacaueiro apresentacomo principais doenças causadoras de perdas na produção a vassoura-de-bruxa e o maldo-facão. A vassoura-de-bruxa é causa pelo fungo Moniliophthora perniciosa e tem comosintomas característicos a hiperplasia e hipertrofia das células que dão lugar a tecidosanômalos chamados de vassoura. O mal-do-facão, causado pelo fungo Ceratocystiscacaofunesta, é uma doença assintomática, sendo detectada somente com a morte daplanta. As duas doenças foram estudadas neste trabalho atravez de ferramentas dagenômica na tentativa de identificação de mecanismos de interação patógenohospedeiro.Em Ceratocystis cacaofunesta foi feita uma análise global da mitocôndriaintegrando dados de genômica, transcritômica e proteômica. Foi gerado o primeiroinventário de proteinas mitocondriais de um fitopatógeno, sendo preditas um total de1.124 proteinas, das quais 304 foram identificadas experimentalmente. Dentre asproteinas listadas no inventário, 117 tem função desconhecida e 7 parecem ser específicasdeste fungo.Análises dos dados do genoma de M. perniciosa sugerem que o fungo poderiaestar produzindo giberelina, o que explica muito dos eventos que acontecem durante ainteração M. perniciosa-cacaueiro durante a fase biotrófica do fungo. Este trabalho demonstrou a importância desse hormônio na progressão da doença, assim como aalteração do metabolismo de giberelina na planta durante a interação. Os dados obtidosneste trabalho geraram conhecimento para desenvolvimento de estrátégias de controledas duas doenças, além de direcionar estudos com outros fitopatógenos / Abstract: Cacao tree (Theobroma cacao L., Sterculeaceae) is a native plant from Amazon region andthe main species commercially exploited for seed production among the 22 of the genus.This plant has a great economic importance because of its use in the production ofchocolate. Due to this, producing regions always has been concern to seed production,and diseases are the most important factors of decline in cocoa production. In Brazil,cacao witches' broom and ceratocystis wilt are major diseases causing production losses.The witches' broom is caused by the fungus Moniliophthora perniciosa and thecharacteristic hyperplasia and hypertrophy symptoms of cells gives rise to anomaloustissue called broom. Meanwhile, the ceratocystis wilt caused by Ceratocystis cacaofunestais an asymptomatic disease; it is detected only with the death of the plant. Both diseaseswere studied using genomic tools in an attempt to identify mechanisms of host-pathogeninteraction. In Ceratocystis cacaofunesta, a mitochondrial global analysis integrating datafrom genomics, transcriptomics and proteomics was made. This work generated the firstinventory of mitochondrial proteins of a plant pathogenic fungus, in which 1,124 proteinswere predicted and 304 were identified experimentally. Among the proteins listed in theinventory, 117 have unknown function and 7 appear to be specific to this fungus. Dataanalysis of the genome of M. perniciosa suggests that the fungus could be producinggibberellin, it may explain much of the events that happen during the interaction M.perniciosa-cacao in biotrophic phase of the fungus. Therefore, this work showed theimportance of this hormone in disease progression, as well as altered metabolism ofgibberellin in the plant during the interaction. The data obtained in this work generatedknowledge to develop strategies to control these two diseases, and can drive studies withother pathogens / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Coniothyrium zuluense is the causal agent of a serious Eucalyptus stem canker disease in South Africa (Wingfield et al., 1997). Eucalypts are the most important hardwood plantations in the world, and in South Africa these hardwoods occupy approximately 1.5 million hectares of plantation area, an area that is soon to be increased by an additional 600 000 hectares. As exotics, Eucalyptus plantations are constantly exposed to infection by fungal pathogens such as C. zuluense, which by secreting cell-¬wall degrading enzymes contribute to the degradation of plant cell walls and subsequent reduction and in the quality of timber produced. This ultimately affects the South African paper, pulp and timber industries. Selection of resistant clones through traditional breeding methods is the most common method currently employed in overcoming the problem of fungal infection. The genetic manipulation of Eucalyptus trees for enhanced resistance to fungal diseases is an alternative to the time-consuming and tedious approach of conventional breeding. The identification of several antifungal proteins, particularly polygalacturonase-inhibiting proteins (PGIPs) from various plant species including Eucalyptus, lead to the hypothesis that over-expression of these proteins could potentially reduce pathogen attack. However, prior to the expression of PGIPs in plants, isolation and molecular characterization of these genes are required. The aims of this study were therefore (l) to clone and characterize the complete Eucalyptus grandis pgip gene, (2) to transform Nicotiana tabacum (tobacco) plants with the E. grandis pgip gene and (3) to test for inhibition of C. zuluense PGs by PGIPs extracted from transgenic tobacco plants. This forms the first step towards the generation of E. grandis clones that are more disease tolerant. A review of the role of fungal endopolygalacturonases and polygalacturonase¬inhibitors in plant-pathogen interactions are presented in chapter I. Strategies employed to isolate and characterize pgip genes from a range of plant species are highlighted and the importance ofPGIPs in disease resistance is discussed. In chapter 2, the molecular cloning and characterization of the E. grandis pgip gene is discussed. The work presented in this chapter is a follow up on work previously conducted by Chimwamurombe (2001). Previously, a partial Eucalyptus pgip gene sequence was obtained with the use of degenerate oligonucleotide primers. In this study, the complete Eucalyptus pgip gene was obtained through the employment of genome walking strategies. Transformation of Nicotiana tabacum cv LA Burley plants with the Eucalyptus pgip gene and the molecular characterization of transgenic tobacco plants is discussed in chapter 3. The transformation and expression of foreign genes in tobacco plants is a well-established protocol, making tobacco the most appropriate candidate plant for assessing the functionality of the plant transformation construct. The production of endopolygalacturonases from virulent C. zuluense isolates and the subsequent PGIP assays conducted to determine levels of PG inhibition are included in this chapter. This thesis consists of three independent chapters representing studies on the molecular characterization of an E. grandis pgip gene and focusing on the potential for inhibition of PGs produced by C. zuluense by Eucalyptus PGIP extracted from transgenic tobacco plants. Repetition of certain aspects in the individual chapters has been unavoidable and the thesis is presented following a uniform style. / Dissertation (MSc)--University of Pretoria, 2003. / Genetics / Unrestricted

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Plants have developed sophisticated means of combating plant diseases. The events that prepare the plant for, and follow plant-pathogenic interactions, are extremely complex and have been the topic of intensive investigation in recent years. These interactions involve a plethora of genes and proteins, and intricate regulation thereof; from the host and pathogen alike. Studying the contribution of single genes and their encoded proteins to the molecular dialogue between plant and pathogen has been a focus of plant molecular biologists. To this end, a gene encoding a polygalacturonase-inhibiting protein (PGIP) was recently cloned from Vitis vinifera. These proteins have the ability to inhibit fungal endopolygalacturonases (ePGs), enzymes which have been shown to be required for the full virulence of several fungi on their respective plant hosts. The activity of PGIP in inhibiting fungal macerating enzymes is particularly attractive for the improvement of disease tolerance of crop species. The VvPGIP-encoding gene was subsequently transferred to Nicotiana tabacum for high-level expression of VvPGIP. These transgenic plants were found to be less susceptible to infection by Botrytis cinerea in an initial detached leaf assay. Also, it was shown that ePG inhibition by protein extracts from these lines correlated to the observed decrease in susceptibility to B. cinerea. This study expands on previous findings by corroborating the antifungal nature of the introduced PGIP by whole-plant, timecourse infection assays. Six transgenic tobacco lines and an untransformed wildtype (WT) were infected and the lesions measured daily from day three to seven, and again at day 15. The transgenic lines exhibited smaller lesions sizes from three to seven days post-inoculation, although these differences only became statistically significant following seven days of incubation. At this point, four of the six lines exhibited significantly smaller lesions than the WT, with reductions in disease susceptibility ranging between 46 and 69% as compared to the WT. Two of the lines exhibited disease susceptibility comparable to the WT. In these resistant plant lines, a correlation could be drawn between Vvpgip1 expression, PGIP activity and ePG inhibition. These lines were therefore considered to be PGIP-specific resistant lines, and provided ideal resources to further study the possible in planta roles of PGIP in plant defense. The current hypothesis regarding the role(s) of PGIP in plant defense is twofold. Firstly, PGIPs have the ability to specifically and effectively inhibit fungal ePGs. This direct inhibition results in reduced fungal pathogenicity. Alternatively, unhindered action of these enzymes results in maceration of plant tissue and ultimately, tissue necrosis. Subsequently, it could be shown that, in vitro, the inhibition of ePGs prolongs the existence of oligogalacturonides, molecules with the ability to activate plant defense responses. Thus, PGIPs limit tissue damage by inhibition of ePG; this inhibition results in activation of plant defense responses aimed at limiting pathogen ingress. Several publications reported reduced susceptibility to Botrytis in transgenic plant lines overexpressing PGIP-encoding genes. However, none of these publications could expand on the current hypotheses regarding the possible in planta roles of PGIP in plant defense. In this study we used transgenic tobacco lines overexpressing Vvpgip1 as resources to study the in planta roles for PGIP. Transcriptomic and hormonal analyses were performed on these lines and a WT line, both before and following inoculation with Botrytis cinerea. Transcriptomic analysis was performed on uninfected as well as infected tobacco leaf material utilizing a Solanum tuberosum microarray. From the analysis with healthy, uninfected plant material, it became clear that genes involved in cell wall metabolism were differentially expressed between the transgenic lines and the WT. Under these conditions, it could be shown and confirmed that the gene encoding tobacco xyloglucan endotransglycosylase (XET/XTH) was downregulated in the transgenic lines. Additionally, genes involved in the lignin biosynthetic pathway were affected in the individual transgenic lines. Biochemical evidence corroborated the indication of increased lignin deposition in their cell walls. Additionally, phytohormone profiling revealed an increased indole-acetic acid content in the transgenic lines. These results show that constitutive levels of PGIP may affect cell wall metabolism in the Vvpgip1-transgenic lines which may have a positive impact on the observed reduced susceptibilities of these plants. An additional role for PGIP in the contribution to plant defenses is therefore proposed. PGIP may directly influence defense responses in the plant leading to the strengthening of cell walls. This might occur by virtue of its structural features or its integration in the cell wall. These reinforced cell walls are thus “primed” before pathogen ingress and contribute to the decrease in disease susceptibility observed in lines accumulating high levels of PGIP. Transcriptional and hormonal analyses, at the localized response, were performed on Botrytis-infected leaf tissue of the transgenic lines and a WT line. Several Botrytis responsive genes were found to be upregulated in both the WT and the transgenic lines. Although limited differential expression was observed between the two genotypes, the analyses identified a gene which was upregulated two-fold in the transgenic lines, as compared to WT. This was confirmed by quantitative Real-Time PCR. This gene is involved in the lipoxygenase pathway, specifically the 9-LOX branch, leading to the synthesis of the divinyl ether oxylipins colneleic and colnelenic acid, which show inhibitory effects on Botrytis spore germination. Phytohormone profiling revealed that the transgenic lines accumulated more of the defense-related hormone pool of jasmonates. These are formed via the 13-LOX pathway and have been shown to be important for the restriction of Botrytis growth at the site of infection. Collectively, the results from the infection analyses indicate that in these transgenic lines, both branches of the lipoxygenase pathway are differentially induced at the level of the localized response to Botrytis infection. Similarly, an increased induction of the synthesis of the defense-related hormone salicylic acid could be observed, although this hormone did not accumulate to significantly higher levels. These results are the first report of differential induction of a defense-related pathway in pgip-overexpressing lines and substantiate the proposal that following ePG inhibition by PGIP, signaling which activates plant defense responses, takes place. Taken together, these results significantly contribute to our understanding of the in planta role of PGIP in plant defense responses. / AFRIKAANSE OPSOMMING: Plante het deur evolusie gesofistikeerde meganismes teen die aanslag van plantsiektes ontwikkel. Die gebeure wat die plant voorberei, asook dié wat op plant-patogeen interaksies volg, is uiters kompleks en vorm die kern van verskeie navorsingstemas die afgelope paar jaar. Etlike plant- én patogeengene en proteïene is by hierdie interaksies betrokke en aan komplekse reguleringsprosesse onderworpe. Die bestudering van die bydrae van enkelgene en hul gekodeerde proteïene tot die molekulêre interaksie tussen ‘n plant en patogeen is ‘n sterk fokus van plant-molekulêre bioloë. Met hierdie doel as fokus, is ‘n geen wat vir ‘n poligalakturonaseinhiberende proteïen (PGIP) kodeer, van Vitis vinifera gekloneer. Hierdie proteïene beskik oor die vermoë om fungiese endopoligalakturonases (ePG's), ensieme wat benodig word vir die virulensie van verskeie fungi op hul gasheerplante, te inhibeer. Die inhibisie van ePG's deur PGIP en die gepaardgaande verminderde weefseldegradasie is ‘n baie belowende strategie vir die verbetering van verboude gewasse se patogeentoleransie. Die VvPGIPenkoderende geen is gevolglik na Nicotiana tabacum oorgedra vir hoëvlakuitdrukking van VvPGIP. Daar is gevind dat hierdie transgeniese plante minder vatbaar vir Botrytis cinerea-infeksies was in ‘n inisiële antifungiese toets wat gebruik gemaak het van blaarweefsel wat van die moederplant verwyder is. Daar is ook ‘n korrelasie gevind tussen B. cinerea-siekteweerstand en ePG-inhibisie deur proteïenekstrakte van die transgeniese populasie. Die huidige studie bou voort op en bevestig vorige bevindinge betreffende die antfungiese aard van die heteroloë PGIP in die heelplant en oor tyd. Ses transgeniese tabaklyne en 'n ongetransformeerde wilde-tipe (WT) is geïnfekteer en die lesies is vanaf dag drie tot sewe, en weer op dag 15, gemeet. Die transgeniese lyne het in die tydperk van drie tot sewe dae ná-inokulasie kleiner lesies as die WT getoon, alhoewel hierdie verskille slegs statisties beduidend geword het na sewe dae van inkubasie. Op daardie tydstip het vier van die ses lyne aansienlik kleiner lesies as die WT getoon, en verlagings in siektevatbaarheid het, in vergelyking met die WT, van 46% tot 69% gewissel. Twee van die lyne het siektevatbaarheid getoon wat vergelykbaar was met dié van die WT. In die siekteweerstandbiedende plantlyne was daar 'n verband tussen Vvpgip1-ekspressie, PGIP-aktiwiteit en ePG-inhibisie. Hierdie plantlyne is dus as PGIP-spesifieke siekteweerstandslyne beskou en dien dus as ideale eksperimentele bronne vir die ontleding van die moontlike in plantafunksies van PGIP in plantsiekteweerstandbiedendheid. Die huidige hipotese betreffende die funksie(s) van PGIP in plantsiekteweerstand is tweeledig. Eerstens het PGIP die vermoë om fungusePG's spesifiek en doeltreffend te inhibeer. Hierdie direkte inhibisie veroorsaak ‘n vermindering in patogenisiteit van die fungus op die gasheer. Indien ePG's egter hulle ensimatiese aksie onverstoord voortsit, sal weefseldegradasie en uiteindelik weefselnekrose die gevolg wees. Daar kon ook bewys word dat die in vitroinhibisie van ePG's deur PGIP die leeftyd van oligogalakturoniede, molekules wat die vermoë het om die plantweerstandsrespons aan te skakel, kan verleng. PGIP het dus nie net die vermoë om ePG's, en dus weefseldegradasie, te inhibeer nie; maar hierdie inhibisie lei ook daartoe dat plantweerstandsresponse aangeskakel word met die oog op die vermindering van patogeenindringing. Verskeie publikasies het reeds gerapporteer oor verminderde Botrytisvatbaarheid in PGIP transgeniese plantlyne. Geeneen van hierdie publikasies kon egter uitbrei op die huidige hipotese aangaande die moontlike in planta-funksie van PGIP in plantsiekteweerstand nie. In hierdie studie is transgeniese tabaklyne wat PGIP ooruitgedruk gebruik om hierdie moontlike in planta-funksies vir PGIP uit te klaar. Transkriptoom- en hormonale analises is op hierdie plantlyne en ‘n WT voor en ná inokulasie met die nekrotroof Botrytis cinerea uitgevoer,. Transkriptoomanalises is uitgevoer op ongeïnfekteerde, sowel as geïnfekteerde tabakblaarmateriaal deur gebruik te maak van ‘n Solanum tuberosum-mikroraster. Die analises met gesonde, ongeïnfekteerde plantmateriaal het daarop gewys dat gene betrokke by selwandmetabolisme tussen die transgeniese lyne en die WT verskillend uitgedruk was. Dit kon bewys word dat, sonder infeksiedruk, die geen wat xiloglukaan-endotransglikosilase (XET) kodeer, in die transgeniese lyne afgereguleer was. Gene wat betrokke is in die lignien-biosintetiese pad was ook in die individuele transgeniese lyne beïnvloed. Biochemiese toetse het ook die aanduiding van verhoogde ligniendeposisie in die transgeniese lyne se selwande bevestig. Addisionele fitohormoonprofiele het getoon dat hierdie lyne ook beskik oor verhoogde vlakke van indoolasynsuur (IAA). Hierdie resultate wys daarop dat konstitutiewe vlakke van PGIP selwandmetabolisme in die Vvpgip1-transgeniese lyne moontlik kan beïnvloed, wat plantsiekteweerstand in dié lyne positief kan beïnvloed. Dit wil dus voorkom asof PGIP 'n bykomende funksie in plantsiekteweerstand het. Plantweerstandsreponse kan direk deur PGIP beïnvloed word, wat tot die versterking van plantselwande kan lei; dit kan geskied by wyse van die strukturele eienskappe van die proteïen of die integrasie daarvan in die selwand. Hierdie selwande is dus “voorberei” alvorens patogeenindringing plaasvind en kon bydra tot die verminderde siektevatbaarheid wat waargeneem is in lyne wat hoë vlakke van PGIP akkumuleer. Transkriptoom- en hormonale analises is ook uitgevoer op Botrytisgeïnfekteerde blaarmateriaal van beide die transgeniese lyne en ‘n WT. Verskeie Botrytis-responsgene is in beide die transgeniese lyne en die WT opgereguleer. Differensïele geenekspressie tussen die twee genotipes was taamlik beperk, maar in die analises kon ‘n geen geïdentifiseer word wat tweevoudig in die transgeniese lyne opgereguleer was in vergelyking met die WT. Hierdie resultaat is ook bevestig met behulp van die “Real-Time” Polimerasekettingreaksie (PKR). Hierdie geen is betrokke in die lipoksigenase (LOX) -pad (spesifiek die 9-LOXarm), wat tot die sintese van die diviniel-eter oksilipiene “colneleic-” en “colnelenic”-suur lei. Daar is al bewys dat hierdie twee verbindings Botrytisspoorontkieming kan inhibeer. Fitohormoonprofiele van die geïnfekteerde plante het gewys dat die transgeniese lyne verhoogde vlakke van die poel van jasmonate wat plantsiekteweerstands-hormone is, ná inokulasie akkumuleer. Hierdie hormone word in die 13-LOX-arm van die lipoksigenase pad gevorm en is belangrik vir die beperking van Botrytis by die infeksiesetel. Die resultate van die analises wat op Botrytis-infeksie volg, dui daarop dat beide arms van die lipoksigenasepad in die transgeniese lyne verskillend by die lokale respons geïnduseer word. ‘n Verhoogde induksie van ‘n ander plantsiekteweerstandshormoon, salisielsuur, kon ook opgemerk word, alhoewel die totaal geakkumuleerde vlakke nie beduidend hoër was as dié van die WT nie. Hierdie resultate is die eerste wat onderskeidende induksie van ‘n siekteweerstandspad in enige van die pgip-ooruitgedrukte plantlyne rapporteer. Daarmee ondersteun dit ook die hipotese dat, seintransduksie wat plantweerstandsresponse aanskakel, ná inhibisie van ePG deur PGIP plaasvind. Die resultate wat met hierdie studie verkry is, dra dus beduidend by tot die huidige kennis van die in planta-funksie van PGIP in plantsiekteweerstandsresponse.