Pseudomonas chlororaphis PA23 inhibits several root pathogens in both the greenhouse and field. A LysR-type transcriptional regulator (LTTR) called PtrA (Pseudomonas transcriptional regulator A) that is essential for Sclerotinia sclerotiorum antifungal activity was discovered through transposon mutagenesis. P. chlororaphis PA23 produces the antibiotics phenazine 1-carboxylic acid, 2-hydroxyphenazine and pyrrolnitrin, and several additional products that contribute to biocontrol. Phenotypic assays and proteomic analysis have revealed that production of these secondary metabolites are markedly reduced in a ptrA mutant. Most LTTRs regulate genes that are upstream of and divergently transcribed from the LTTR locus. A short chain dehydrogenase (scd) gene lies immediately upstream of ptrA in the opposite orientation. Characterization of an scd mutant, however, has revealed no significant changes in antifungal activity compared to wild-type PA23. Gene expression analysis of the ptrA mutant indicates that ptrA may exert its regulatory effects through the Gac-Rsm network, and may be controlling expression of the scd gene. Collectively these findings indicate that PtrA is an essential regulator of PA23 biocontrol and is connected to other regulators involved in fungal antagonism.

Plants produce a wide variety of natural products that can be exploited for medicinal purposes. Paclitaxel is a key anti-cancer drug originally isolated from the bark of Taxus spp. that is currently approved for use in the treatment of breast, lung and non-small cell cancers, AIDS-related Kaposi's sarcoma and coronary artery disease. Worldwide demand for paclitaxel is high and plant cell culture (PCC) is an attractive production route. Cultured cambial meristematic cells (CMCs) provide a good platform from which to increase drug production, as they possess superior growth properties on an industrial scale compared to typical dedifferentiated cell culture. Elicitors, such as methyl-jasmonate (MeJA), can up-regulate paclitaxel production in PCC, however the effect is only transient. Identification and characterisation of the key transcriptional regulators that control MeJA induced metabolic reprogramming can provide potential tools to manipulate Taxus CMC culture to produce more paclitaxel. Roche454 sequencing was employed to establish the basic transcriptomic profile of Taxus cuspidata CMCs, which was then utilized as a reference to observe the transcriptional profile of CMCs at three time points after MeJA elicitation (0.5, 2 and 12 h). Analysis of the transcriptional regulatory network identified 19 transcription factors (TFs) that were significantly up-regulated at an early time point (0.5 h) after elicitation. These TFs came from five families – AP2, MYB, NAC, bHLH and WRKY – that are well known to regulate secondary plant metabolism. An Arabidopsis thaliana transient expression assay (TEA) was employed to investigate the regulatory activity of these 19TFs against 10 paclitaxel biosynthetic promoters. The TEA screen identified 79 significant interactions with every promoter interacting with at least three TFs, which could activate or repress activity. A MYB TF was identified that could up-regulate eight out of the ten promoters tested, indicating it maybe a potential overall regulator of paclitaxel biosynthesis. In vitro electromobility shift assays established the possible binding site for this TF as an AC element, with the consensus sequence of A(A/C)C. Repressors of promoter activity were also identified, for example an AP2 TF which contains the well-established ERF associated amphiphilic repression (EAR) motif. The activity of the EAR domain was explored in vivo using a TEA assay and site directed mutagenesis mutants. Activity was lost when the mutation occurred within the domain suggesting the TF was working as an active repressor. TFs can work individually or in combination to achieve metabolic reprogramming after MeJA elicitation. One of the best characterised examples of plant combinatorial control is between particular sub classes of MYB and bHLH TFs. However investigation into possible interactions between the T. cuspidata MYB and bHLH TFs in vivo using yeast two hybrid and TEAs found few combinations that led to a significant change in regulatory activity. The regulatory activity of WRKY TFs was shown to be post-translationally controlled when the TEAs were treated with MeJA, however the mechanism by which this occurs remains to be elucidated. The interactions identified between the 19 TFs and the paclitaxel biosynthetic promoters can be exploited in the future to produce superior Taxus CMC lines with increased paclitaxel yields.

Deformed epidermal autoregulatory factor -1 (DEAF-1) is a transcription factor mapping to the chromosomal region 11p15.5, a region associated with loss of heterozygosity (LOH) in human cancers. Potential DEAF-1 binding motifs were identified in the PTEN promoter and the ability of DEAF-1 to regulate PTEN gene expression was investigated. DEAF-1 increased transcription 10-14 fold with PTEN sequences between -429 and -221, while mutations in the DNA binding domain (DEAF-ADWA) and nuclear localization signal of DEAF-1 abolished this increase. DEAF-1 was shown to bind sequences between -339 and -233. Rabdomyosarcoma (RD) stable cell lines with inducible expression of DEAF-1 and DEAF-1-ADWA were produced. Increased DEAF-1 expression had no significant effects on PTEN RNA expression or cell proliferation when compared to controls, but did increase susceptibility to UV-induced apoptosis. These studies suggest that DEAF-1 may contribute to the regulation of PTEN gene expression, but other factors may play a more significant role.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18477号 / 農博第2077号 / 新制||農||1025(附属図書館) / 学位論文||H26||N4861(農学部図書室) / 31355 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 左子 芳彦, 教授 澤山 茂樹, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Recently, through reporter gene studies, the novel BTB/POZ protein, Kaiso, has been identified as a transcriptional repressor. The purpose of this study was to determine if Kaiso recruited the Histone Deacetylase Complex to mediate repression and if the previously identified Kaiso Binding Site (KBS; TCCTGCNA) is a physiological target regulated by Kaiso. The two objectives are complementary because an HDAC interaction identifies the mechanism of transcriptional regulation used by Kaiso and regulation of the KBS element identifies a novel, non-methylation dependent, physiological target under transcriptional regulation by Kaiso. Through coimmunoprecipitation and Western blot analyses, Kaiso does not interact with HDAC1, HDAC2 or mSIN3A. These results were surprising since all three of these proteins are common to a variety of repression complexes. mSIN3A is a common component of SIN3 mediated repression and HDAC1/HDAC2 are part of various repression complexes including SIN3, NuRD and CtBP. Although the remaining HDAC proteins were not assayed for an interaction, Kaiso transcriptional activity was demonstrated to be insensitive to the HDAC inhibiting drug, Trichostatin A (TSA). These results indicate either a non-HDAC mechanism of action or alternatively, transcriptional activation. Complementary to the observations of no Kaiso-HDAC interaction and TSA insensitivity was the findings that Kaiso activates transcription of the KBS cis-element in HCT116, HCA-7 and 293 cells, but not MOCK cells in reporter gene assays. Taken together, these results indicate that Kaiso is a dual functioning protein capable of both transcriptional activation and repression and that the mechanism of repression is not through the direct recruitment of HDAC proteins. / Thesis / Master of Science (MSc)

Quantitative resistance is controlled by multiple genes and has been shown to be a durable form of resistance to pathogens affecting cultivated crops including soybeans (Glycine max L. Merr). Root rot of soybean caused by Phytophthora sojae ranks among the most damaging soybean diseases. Quantitative resistance has proven durable in soybean against P. sojae, however the molecular mechanisms underlying this form of resistance are still unknown. The objective of this project is to gain insight into molecular basis of quantitative resistance in the soybean-P. sojae pathosystem. The approach was to use global transcriptional profiling based on microarray technology to identify genes that were differentially expressed in four cultivars of soybeans with varying levels of quantitative resistance at different time points during infection by P. sojae. Our results provide a better understanding of the potential regulatory factors that may contribute to quantitative resistance during early hours of P. sojae infection. / Ph. D.