Human health is increasingly threatened by the emergence of multiply drug resistant malignant organisms. Yet, our understanding of the numerous ways by which such resistance arises is modest. Here, we present evidence of a bet hedging strategy in the budding yeast, Saccharomyces cerevisiae, to counter the effects of cytotoxic drugs through the action of Pdr5, an ATP-binding cassette transporter. We have employed flow cytometry and fluorescent activated cell sorting to probe the expression levels of a GFP-tagged version of PDR5 in individual cells. The results obtained from these experiments demonstrate that each yeast population is variable in the levels of Pdr5 production, and a small subpopulation of cells produces this efflux pump at much higher quantities than the population average. Consequently, cells with high and low levels of Pdr5 grow differentially in presence and absence of cycloheximide, a cytotoxic drug. These properties are highly suggestive of a bet hedging strategy mediated by Pdr5 levels. We further link this bet hedging strategy to the transcriptional regulatory network of PDR5 consisting of two major transcription factors, Pdr1 and Pdr3. Our analysis suggests that a self-activating feedback loop acting on Pdr3 plays an important role in generation of the aforementioned subpopulation. Furthermore, our results point to a large difference in the activity of these two regulators wherein Pdr3 is notably stronger than Pdr1. The disparity in their activity could indicate a mechanism for generation of the observed proportions of subpopulations with regards to the level of Pdr5.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18326号 / 農博第2051号 / 新制||農||1022(附属図書館) / 学位論文||H26||N4833(農学部図書室) / 31184 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 喜多 恵子, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Little is still known about what allows mature Arabidopsis thaliana (Arabidopsis) plants to respond with an Age-Related Resistance response (ARR). To better understand how mature plants initiate and establish ARR, gene expression in the leaves of young and mature plants responding to Pseudomonas syringae pv. tomato (Pst) was investigated using RNA-sequencing analysis. Genes involved in the biosynthesis of N-hydroxypipecolic acid (NHP) were upregulated in ARR-responding leaves leading to the idea that NHP, a signaling molecule in Systemic AcquiredResistance (SAR) may also be required for ARR. The ARR response was examined in NHP biosynthesis mutants and revealed that NHP biosynthesis is required for ARR. During ARR, NHP biosynthesis mutants were also shown to accumulate less salicylic acid (SA) compared to wild-type leaves in response to Pst. Healthy untreated leaves had modest accumulation of NHP and modest expression of several cell-surface receptors was observed compared to the healthy untreated leaves of young plants, suggesting that ARR competence in mature untreated plants involves a primed/immune ready state similar to what is observed in systemic leaves of plants induced for SAR in a local leaf. The ARR response also requires the accumulation of intercellular SA which is involved in inhibiting biofilm-like aggregate formation of Pst in mature plant leaves. To understand how SA is transported from the cytosol to the intercellular space during ARR, the ARR response of the PDR-type transporter mutants pdr8-4 and pdr12-3 was examined. The pdr8-4 pdr12-3 double mutant was partially ARR-defective and SA accumulation in leaf intercellular spaces was reduced by ~50% compared to wild-type mature plants during ARR, demonstrating that PDR8 and PDR12 are required for the intercellular localisation of SA during ARR. To obtain evidence that PDR8 and PDR12 act as transporters of SA, SA transporter assays were performed with yeast expressing PDR8 and PDR12. PDR8- and PDR12-expressing yeast accumulated less intracellular SA than empty vector containing yeast cells, suggesting that PDR8 and PDR12 act as SA transporters. Together, this work found ARR shared signaling components with SAR and found additional support for SA as an antimicrobial and signaling molecule during ARR. / Thesis / Doctor of Science (PhD)