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Fusarium graminearum is a major fungal pathogen of wheat and other small grain cereal crops globally, causing Fusarium ear blight (FEB) disease. Like many other plant pathogens, F. graminearum is predicted to produce in planta secreted effector proteins that modulate plant metabolism to suppress or re-programme plant defences. Understanding the molecular functions of Fg effectors will help to elucidate the processes underlying wheat spike colonisation and fungal pathogenicity. With the aim of identifying Fg effector proteins that can suppress host plant defences, I selected using next generation sequencing and bioinformatic analysis, a set of small secreted proteins (SSP) to express in planta using the Barley stripe mosaic virus over-expression system (BSMV-VOX). I then tested whether expression of any of these SSPs enhanced Fg fungal infection of susceptible wheat spikes. Amongst the set of Fg SSP tested, FgSSP8, which encodes a ribonuclease protein, induced strong symptoms of necrosis in N. benthamiana leaves when infiltrated via the BSMV:FgSSP8. Three other genes tested (FgSSP7, FgSSP6 and FgSSP5) enhance FEB disease formation in the majority of the experiments when overexpressed in wheat ears prior to infecting with F. graminearum. FgSSP6 and FgSSP7 belong to the cerato-platanin protein (CPP) family. In several other plant pathogenic fungi, CPPs have been implicated in a number of virulence and plant protection mechanisms, including induction of host plant cell death, binding specific polymers and/or expansin-like activity. FgSSP5 encodes a protein that possesses the pfam domain RALF (Rapid alkalinization factor; PF05498.6). RALF domain-containing proteins are predominately found in plants and play a role in plant development regulating tissue expansion and/or negatively regulating pollen tube elongation. BLAST analyses identified RALF domain containing proteins in a restricted range of different pathogen species. Based on the VOX results and biochemical tests, our hypothesis is that pre-elevated cerato-platanins (FgSSP6 and FgSSP7) levels in the apoplast/surrounding the hyphae could initially shield the hyphae from detection by the plant, but late induce an intense defence response culminating in cell death to benefit the necrotrophic phase of Fg by increasing nutrient availability. FgSSP5 may be a specific virulence factor that manipulates a key plant process, by alkalinising the plant environment during infection, and using the same plant receptor repertoire used to recognise plant proteins. Once the mechanisms are further understood, these genes/proteins could potentially be novel intervention targets either for conventional chemistries and/or for methods such as host-induced gene silencing to achieve FEB disease and/or mycotoxin control. The characterisation of single and double gene deletion F. graminearum mutants is in progress.

Made available in DSpace on 2015-08-20T17:09:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-13. Added 1 bitstream(s) on 2015-08-20T17:26:38Z : No. of bitstreams: 1 000844728.pdf: 1148176 bytes, checksum: d142a0d79c9df0edb442320f2bc91ef8 (MD5) / A concentração atmosférica de CO2, principal gás responsável pela intensificação do efeito estufa, vem aumentando significativamente nas últimas décadas. A alfafa é uma importante forrageira que pode ter suas características alteradas pelo aumento da concentração de CO2 atmosférico, favorecendo a ocorrência de doenças e intensificando a emissão de gases de efeito estufa após sua ingestão por animais. Diante desse cenário, o objetivo desse trabalho foi verificar o impacto do aumento da concentração de CO2 do ar em doenças da alfafa, crescimento das plantas e composição bromatológica. Os ensaios foram realizados em campo, com semeadura direta de sementes de alfafa da cultivar crioula em estufas de topo aberto (OTC), adotando o delineamento experimental em blocos casualizados, com três repetições. Os tratamentos foram: testemunha sem OTC, com a concentração de 390 μmol mol-1 de CO2; OTC sem injeção de CO2, com 400 μmol mol-1 de CO2; e OTC com injeção de CO2 até atingir 500 μmol mol-1 de CO2. Na parte aérea das plantas, foram realizadas análises para determinar a matéria seca, matéria mineral, matéria orgânica, nitrogênio em fibra detergente neutro (n-FDN), nitrogênio em fibra detergente ácido (n-FDA), proteína bruta e lignina e análise química foliar. Além dessas, foram realizadas medidas da altura das plantas, relação entre o comprimento da haste e da raiz, peso da biomassa da raiz e da parte aérea e a contagem das inflorescências. Uma doença foi avaliada nos dois primeiros ensaios: a ferrugem, causada por Uromyces striatus, e a segunda foi avaliada nos dois últimos ensaios: murcha de fusário, causada por Fusarium oxysporum f. sp. medicaginis. A severidade das doenças foi avaliada por meio de notas, semanalmente, durante 28 dias ... / The atmospheric CO2 concentration, the main gas responsible for intensifying the greenhouse effect, has increased significantly in recent decades. Alfalfa is important forage that can have its characteristics altered by increased atmospheric CO2 concentration, facilitating the occurrence of diseases and enhancing the emission of greenhouse gases after its ingestion by animals. Given this scenario, the objective of this study was to assess the impact of increased CO2 concentration in the air on alfalfa diseases, plant growth and chemical composition. Assays were performed in the field with direct seeding of alfalfa seed cultivar Creole in open top chambers (OTC), adopting the trial randomized block design with three replications. The treatments were: control without OTC, with the concentration of 390 μmol mol-1 of CO2; OTC without CO2 injection with 400 μmol mol-1 of CO2; and OTC injected CO2 with target concentration of 500 μmol mol-1. In the shoots, analyzes were performed to determine dry matter, ash, organic matter, nitrogen in neutral detergent fiber (NDF-N), nitrogen in acid detergent fiber (ADF-N), crude protein and lignin and foliar analysis. In addition to these measures of plant height, stem length and root weight of biomass of the root and shoot and inflorescence. One disease was evaluated during the second trials: rust, caused by Uromyces striatus and one were evaluated during the two last trials: the wilt of fusarium caused by Fusarium oxysporum f. sp. medicaginis. The diseases severity was assessed weekly by notes during 28 days. The defoliation caused by rust was evaluated by the percentage of leaves. Height of stems, the relationship between the length of stem and root, biomass of roots and shoots were higher in the treatment with CO2 injection. Dry matter, ash, organic matter, n- NDF, ADF-n, crude protein and lignin showed no statistical difference between the treatments. The rust ...

Doctor of Philosophy / Department of Plant Pathology / Bikram S. Gill / Wheat is the most widely grown and consumed grain crop in the world. In order to meet future agricultural production requirements of a growing population, it is essential that we achieve an increased understanding of the basic components and mechanisms shaping growth and productivity of the polyploid wheat plant. Fusarium head blight (FHB) (syn. "scab") poses a serious threat to the quantity and safety of the world's food supply. The resistance locus Fhb1 has provided partial resistance to FHB of wheat for nearly four decades. Map-based cloning of Fhb1 is justified by its significant and consistent effects on reducing disease levels, the importance of FHB in global wheat production and food safety, and because this gene confers partial resistance to this disease and does not appear to behave in a gene-for-gene manner. A bacterial artificial chromosome (BAC) contig spanning the Fhb1 region was developed from the cultivar 'Chinese Spring', sequenced and seven candidate genes were identified in an ~250 kb region. Cosmid clones for each of the seven candidate genes were isolated from a line containing Fhb1 and used for genetic transformation by biolistic bombardment. Transgenic lines were recovered for five candidate genes and evaluated for FHB resistance. All failed to complement the Fhb1 phenotype. Fhb1 is possibly one of the two remaining candidate genes, an unknown regulatory element in this region, or is not present in Chinese Spring. Traditional views on the effects of polyploidy in allohexaploid wheat have primarily emphasized aspects of coding sequence variation and the enhanced potential to acquire new gene functions through mutation of redundant loci. At the same time, the extent and significance of regulatory variation has been relatively unexplored. Recent investigations have suggested that differential expression of homoeologous transcripts, or subfunctionalization, is common in natural bread wheat. In order to establish a timeline for such regulatory changes and estimate the frequency of non-additive expression of homoeologous transcripts in newly formed T. aestivum, gene expression was characterized in a synthetic T. aestivum line and its T. turgidum and Aegilops tauschii parents by cDNA-SSCP and microarray expression experiments. The cDNA-SSCP analysis of 30 arbitrarily selected homoeologous transcripts revealed that four (~13%) showed differential expression of homoeoalleles in seedling leaf tissue of synthetic T. aestivum. In microarray expression experiments, synthetic T. aestivum gene expression was compared to mid-parent expression level estimates calculated from parental expression levels. Approximately 16% of genes were inferred to display non-additive expression in synthetic T. aestivum. Six homoeologous transcripts classified as non-additively expressed in microarray experiments were characterized by cDNA-SSCP. Expression patterns of these six transcripts suggest that cis-acting regulatory variation is often responsible for non-additive gene expression levels. These results demonstrate that allopolyploidization, per se, results in rapid initiation of differential expression of homoeologous loci and non-additive gene expression in synthetic T. aestivum.

Made available in DSpace on 2014-06-12T15:49:40Z (GMT). No. of bitstreams: 2 arquivo1529_1.pdf: 841640 bytes, checksum: a1668107b89c5355d88180dbb4cb329e (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Objetivando estudar o efeito de isolados de bactérias promotoras do crescimento de plantas (BPCP) e fungos micorrízicos arbusculares (FMA) foram realizados três experimentos. Primeiramente, bactérias epifíticas e endofíticas foram isoladas de raízes de bananeiras sadias de seis cultivares e caracterizadas quanto à produção de pectinase, celulase, ácido indolacético, β-1,3-glucanase e quitinase. No 1º. experimento, as rizobactérias foram avaliadas quanto à promoção do crescimento de bananeiras micropropagadas cv. Grande Naine em casa de vegetação, sendo 74 tratamentos de bacterização + um tratamento controle, com 8 repetições. Mudas com aproximadamente 10 cm foram transplantadas e bacterizadas em potes contendo 1 kg de solo desinfestado. No 2º. experimento, foi observada a ação antagônica in vitro das RPCP selecionadas, contra Fusarium oxysporum f. sp. cubense. No 3º experimento, em casa de vegetação, foi estudado o efeito da associação RPCP x FMA no desenvolvimento de mudas de bananeiras cv. Grand Naine. Foi aplicado DIC com 7 tratamentos de bacterização com RPCPs (BAN29, BAN36, BAN81, BAN82, S1, S2 e controle) × 3 tratamentos de inoculação com FMAs (Glomus etunicatum, Glomus clarum e controle) × 7 repetições. A inoculação (200 glomerosporos/planta) ocorreu no transplantio das mudas para sacos contendo 1,5 Kg de solo desinfestado. Em seguida, as mudas foram bacterizadas com seis isolados pré-selecionados no Experimento 1. As suspensões bacterianas (50 mL/planta) utilizadas nos experimentos em casa de vegetação foram preparadas e ajustadas em fotocolorímetro (A580) em água destilada esterilizada (ADE) visando obter para 108 UFC/mL. O tratamento controle sem FMA e sem bactérias recebeu ADE. Para o isolamento de bactérias endofíticas foram testados diferentes substâncias desinfestantes e tempos de exposição, com melhor resultado obtido com hipoclorito de cálcio a 5%, por 20 minutos. Foram obtidos 80 isolados bacterianos, com predominância de bactérias epifíticas (53,7%) e de Gram-positivas (67,1%). Nove isolados produziram pectinase, 37 ácido indolacético e dois β-1,3-glucanase. Nenhum dos isolados foi capaz de produzir celulase, ácido cianídrico, quitinase ou solubilizar fosfato. Incrementos no desenvolvimento de mudas micropropagadas de bananeiras cv. Grand Naine foram evidenciados a partir dos 30 dias após bacterização com 40 isolados. Houve incremento de até 82% para a biomassa fresca (BF) da parte aérea e de até 263% para a BF de raízes das mudas associadas com BAN29, BAN36, BAN81, BAN82, S1 e S2 (Experimento 1). A BAN36 foi capaz de inibir o crescimento micelial de Fusarium oxysporum f. sp. cubense em placas de Petri (até 67%) (Experimento 2). Em geral, a inoculação conjunta RPCP × FMA não proporcionou incremento no crescimento, sendo observada interação positiva apenas entre FMAs e os isolados S1 e S2. Apenas na ausência dos FMAs, as mudas tratadas com BAN29, BAN36, BAN81 e BAN82 diferiram em relação ao controle. Houve estímulo na colonização das raízes de bananeiras por G. clarum quando associadas com BAN82. Isolados bacterianos epifíticos e endofíticos de raízes de bananeiras são capazes de produzir substâncias, in vitro, com potencial uso biotecnológico para promoção do crescimento de mudas micropropagadas de bananeira da cultivar Grand Naine visando o controle de F. oxysporum f. sp. cubense. A interação RPCPs e FMAs demonstrou resultados conflitantes, concluindo-se que o efeito associado depende do isolado específico de cada microrganismo

M.Sc. / An increasing problem in subtropical regions, such as South Africa, is the susceptibility of various banana varieties to Fusarium wilt by the soil borne pathogen Fusarium oxysporum tsp. cubense (FOC). In this study the problem of fungal susceptibility of banana was addressed by investigating the biochemical aspects of cell wall strengthening in banana roots. Defence responses were induced in both adult and tissue culture tolerant Goldfinger and susceptible Williams banana cultivars by treatment of the plants with a heat-released elicitor preparation from the mycelial cell walls of FOC race 4 and the crude filtrate. Banana plants were maintained in a hydroponic system, before being inoculated with the elicitor and crude filtrate preparation. Differences in lignin content, callose deposition, phenolics and the enzymes involved in cell wall strengthening; (PAL, CAD, POD and PPO) between the tolerant and susceptible banana cultivars were investigated. Differences in defence responses after treatment with elicitor and with crude filtrate were observed, but it was shown that the former is a more efficient experimental system for the characterisation of susceptible and tolerant responses in banana cultivars. An elicitor concentration of 45 4g/m1 greatly induced cellular POD, PPO, PAL and CAD activity in Goldfinger, whereas no significant increase was observed for Williams. Lignin content increased significantly in Goldfinger compared to Williams. The quantitative determination of induced total phenolics, phenolic glycosides, phenolic esters and cell wall-bound phenolic acids were higher in Goldfinger than in Williams. These increases in the four phenolic subfractions were clearly confirmed by reverse phase HPLC. No significant increase in callose accumulation was observed for both cultivars. The obtained results indicate an important role for cell wall strengthening as an inducible defence mechanism of banana roots against FOC race 4.