Global ETD Search

1	Cloning and characterization of an asparagus wound-induced gene Warner, Simon A. J. January 1992 (has links) Following previous studies, Asparagus officinalis single cell suspensions were hypothesized to be a rich source of wound-inducible mRNAs. A previously isolated clone, DDl-34, was shown to hybridize to wound-inducible transcript. This sequence was used to isolate the AoPR1 (Asparagus officinalis Pathogenesis Related cDNA clone 1). Data from the isolation and analysis of genomic clones hybridizing to DDl-34 probe suggested that these clones were unlikely to contain the upstream regulatory sequences of the AoPR1 gene and that the genomic arrangement of these sequences is complex. Inverse polymerase chain reactions (IPCR) were used to amplify AoPR1 genic sequences directly from the asparagus genome. Two products were cloned and sequenced, demonstrating that the correct sequences, upstream and downstream of the primers, had been amplified. The downstream IPCR product's sequence overlaps with AoPR1 coding sequence and contains an intron sequence. The upstream IPCR product partially overlaps with the start of AoPR1 coding sequence and was successfully used in transcript mapping experiments. Translational fusions were constructed between this fragment and the -glucuronidase (gus) reporter gene. GUS analysis demonstrated that this fragment, containing the AoPR1 promoter, was sufficient to drive wound-inducible transcription in transgenic tobacco. A smaller upstream fragment was insufficient to drive wound-inducible transcription. GUS expression was also detectable in tissues such as the xylem parenchyma, mature pollen and coloured regions of the petal. AoPR1-gus transgene expression correlates with the spatial expression patterns of phenylpropanoid biosynthesis pathway genes. The nature of the fusion suggested that the AoPR1 protein is intracellular. This is the first example of the cloning and analysis of a monocotyledon gene belonging to the 'intracellular pathogenesis related protein' class. The analysis and application of AoPR1 sequences are discussed. 572.8 Plant defence genes
2	DEFENCE GENE EXPRESSION IN THE TOMATO-VERTICILLIUM PATHOSYSTEM Castroverde, Christian Danve 22 April 2010 (has links) In tomato (Solanum lycopersicum), race-specific resistance against the fungal wilt pathogen Verticillium dahliae race 1 (Vd1) is established in the stem. However, the molecular factors and mechanisms leading to this resistance response are still unknown. In this study, Craigella resistant (CR) and susceptible (CS) tomato plants were successfully infected with Vd1 and this was verified by fungal quantification and symptom score assays. Previous microarray results showed interesting patterns of defence gene expression that correlated with biological phenomena. Plant defence genes code for proteins that are responsible for or associated with the plant resistance response. Through RT-PCR, this thesis set out to confirm these microarray observations and also to generate expression data for genes in which sensitivity was an issue in the microarray. The standard RT-PCR data confirmed a number of the microarray results, but some conflicts remained. From the defence genes investigated, there was agreement between the microarray data and the RT-PCR data for pre-mRNA processing factor 8, class IV chitinase, cyclin-dependent kinase inhibitor and IMP dehydrogenase/GMP reductase. Partial agreement was observed for genes coding for ethylene response factor 2, phenylalanine ammonia lyase and P6 protein. However, there was total disagreement for 14-3-3, beta-glucanase, P1a, RNA-binding protein, calcium-binding protein and S-Adenosyl-L-methionine: hydroxide adenosyltransferase. Real-time RT-PCR was attempted to clarify the remaining issues but further discrepancies arose, particularly in the Ve resistance genes. To resolve these discrepancies, two approaches were designed: (1) one based on the use of a universal internal control and (2) another based on restriction enzyme digestion. In general, the results were more consistent with standard RT-PCR. Overall, this study showed that standardization of a system involving vascular pathogens, leading to reproducible analysis, was possible but only with proper controls and additional validation. Standard RT-PCR appeared to offer a more accurate picture of the expression of defence genes in the tomato-Verticillium pathosystem. The defence gene expression results confirmed in this study remain as potential insights into the molecular mechanisms for Verticillium resistance in tomato plants. Real-time PCR Microarray RFLP Internal control Plant resistance RT-PCR Ve genes Gene expression Defence genes Plant defence Solanum lycopersicum Verticillium
3	PLANT-ENDOPHYTE INTERPLAY PROTECTS TOMATO AGAINST A VIRULENT VERTICILLIUM DAHLIAE Shittu, Hakeem Olalekan 05 October 2010 (has links) When tomato Craigella is infected with Verticillium dahliae Dvd-E6 (Dvd-E6), a tolerant state is induced with substantial pathogen load, but few symptoms. Unexpectedly, these plants are more robust and taller with Dvd-E6 behaving as an endophyte. Some endophytes can protect plants from virulent pathogens. This research was undertaken to improve understanding of the cellular and molecular nature of Verticillium tolerance in tomato, especially whether infection by Dvd-E6 can protect Craigella from virulent V. dahliae, race 1 (Vd1). To permit mixed infection experiments a restriction fragment length polymorphism (RFLP)-based assay was developed and used for differentiating Dvd-E6 from Vd1, when present in mixed infections. The results suggested that protection involves molecular interplay between Dvd-E6 and Vd1 in susceptible Craigella (CS) tomatoes, resulting in restricted Vd1 colonization. Further studies showed a dramatic reduction of Vd1 spores and mycelia. To examine genetic changes that account for these biological changes, a customized DNA chip (TVR) was used to analyze defense gene mRNA levels. The defense gene response was categorized into four groups. Group 1 was characterized by strong induction of defense genes followed by suppression. However, Vd1-induced gene suppression was blocked by Dvd-E6 in mixed infections. These genes included some transcription factors and PR proteins such as class IV chitinases and beta glucanases which are known to target fungal spores and mycelia. Experiments also were repeated with a Craigella resistant (CR) isoline containing a fully active Ve locus (Ve1+ and Ve2+). The biological results showed that the presence of the Ve1+ allele resulted in restricted Vd1 colonization and, in a mixed infection with Dvd-E6, Vd1 was completely eliminated from the plant stem. Surprisingly, there was no significant increase in defense gene mRNAs. Rather, elevated basal levels of defense gene products appeared sufficient to combat pathogen attack. To investigate functional effects of the genetic changes observed, an inducible RNAi knockdown vector for a defense gene (TUS15G8) with unknown function (pMW4-TUS15G8) as well as the Ve2 resistance gene (pMW-Ve2) was prepared as a initial step for future transformation analyses. Taken together the results reveal intriguing but complex biological and molecular changes in mixed infections, which remain a basis for future experiments and potential agricultural benefits. / Canadian Commonwealth Scholarship and Fellowship Plan Tomato-Verticillium response Pathogenesis related (PR) protein Resistant relationship Susceptible relationship Endophyte-induced protection Tolerant relationship Compatible interaction Incompatible interaction Defence protein Mycelia Fungal spores Mixed infection Agrobacterium Functional analysis RNAi construct pMW-Ve2 pMW4-TUS15G8 Gene expression Silencing Ve gene Susceptibility Resistance Defence genes plant-pathogen interaction RFLP Verticillium dahliae race 1 Dvd-E6 Craigella RT-PCR Tolerance Endophyte Plant transformation RNAi Microarray Tomato Verticillium

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