Intraspecific adaptive response of Periconia macrospinosa, a ubiquitous root-associated fungus, to persistent salinity stress provides evidence for its adaptive variability and high-osmolarity glycerol pathway mediated adaptive response

Master of ScienceDepartment of BiologyAri M JumpponenRoot associated fungi are critical to environmental success of their plant hosts. Whether through their interactions with hosts, nutrient recycling, decomposition, or even contribution to soil structure, soil-dwelling fungi play a pivotal role in environmental preservation and function. As climate change continues to affect and change patterns in climate, researchers are increasingly concerned about the impacts our global environments may endure as a result. In a terrestrial ecosystem, mean annual precipitation (MAP) is an important control of biological productivity. As water leaves a system, via evapotranspiration, mass flow, surface flow, or other means, salts remain and concentrate in soils causing salinity stress for organisms that remain. This increasing salt concentration is a major stressor for organisms and requires specialized osmoadaptive responses. Plant communities as well as those of soil-dwelling fungi and bacteria change along precipitation gradients, indicating their responses to water availability. However, it is less certain if conspecific organisms also differ across similar gradients, especially in their abilities to tolerate salt stress. To investigate the inter- and intraspecific adaptive responses of root-associated ascomycetes to salinity, we devised an experiment wherein conspecific isolates representing five ascomycete species were subjected to increasing concentrations of salinity in an effort to quantify and compare the effective dose of salt (NaCl) necessary to limit colony growth by 50% (ED50). For each of the five species, we selected three conspecifics originating from drier “arid” environments and compared those to three conspecifics originating from wetter “mesic” environments. We hypothesized that (1) ascomycete species differ in their growth response to salinity across species; (2) conspecific strains from drier sites have greater salt tolerance than those from more mesic sites. Each isolate was tested three times in triplicate and exposed to four levels of NaCl concentrations in a quad-plate. We measured colony growth and used regression analyses to estimate the isolates’ ED50. In support of our first hypothesis, we observed that species differed in their growth response to salinity according to ED50. However, we observed no consistent, strong evidence to support our second hypothesis. Still, we observed non-significant differences in ED50 within three species observed, partial support in Periconia macrospinosa isolates and the most significant difference across sites within Fusarium cf. equiseti isolates. While isolate results within F. cf. equiseti do support our second hypothesis, Periconia macrospinosa appear to demonstrate an inverse response. This coupled with the non-significant differences between ED50 results across site conditions of the other three species – our second hypothesis was rejected. To further investigate the underlying mechanisms and differences in adaptive response among conspecific isolates, we designed an experiment in which two isolates, DS 1091 and DS 0982, of Periconia macrospinosa that differed most in their salt tolerance as inferred from NaCl ED50 estimated in the experiment described above were subjected to prolonged salinity stress followed by proteomic analysis. The selection of Periconia macrospinosa isolates over Fusarium equiseti isolates was based on the species’ critical importance globally in ecosystems experiencing low nutrient and MAP inputs as well as the availability of a recently annotated genome. We hypothesized that (3) the highest and lowest performing strains within a species will differ in their proteomic profiles; (4) colony expansion under saline stress (ED50) will correlate with cell wall related protein abundances under salt stress (signaling cell wall modifications); (5) similarly to the cell wall associated proteins, colony growth (ED50) will correlate with plasma membrane related protein abundances under salt stress (signaling cell membrane alterations); (6) the mitogen-activated protein kinase (MAPK) signaling pathway will be one strategy for tolerating prolonged salinity amongst the conspecifics (suggesting “compatible-solute” strategy); and, (7) conspecifics associated with the highest ED50 will demonstrate greater abundance of proteins involved in salt-tolerant strategies such as cell wall, plasma membrane, and MAPK-related proteins than their counterparts originating from more mesic sites. Our third hypothesis was supported as the two isolates demonstrated unique proteomic profiles both with and without salinity induced responses. Our fourth and fifth hypotheses, which proposed correlation between isolate colony growth and the abundance profiles of proteins involved in cell walls and cell membranes respectively, were also supported by our proteomic data. The fourth hypothesis was reinforced by our proteomic analysis of our high-ED50 isolate, DS 0982, through the upregulated chitin synthase coupled with downregulation of chitinase demonstrating an investment in cell wall maintenance. The fifth hypothesis was supported through the evident DS 0982 responses to salinity where indicators of sphingolipid biosynthetic processing and ergosterol biosynthesis were observed. Specifically, support for the cell membrane related protein increase correlated with the colony growth was through ergosterol biosynthesis contributors CDP-diacylglycerol synthase, sterol C-14 reductase-like protein, and sterol 24-C methyltransferase. The sixth hypothesis, which focused on the positive correlation between colony growth and MAPK signaling interactions was also supported. The MAPK high-osmolarity glycerol HOG pathway regulates and initiates osmoadaptive responses within the cell, the most critical of which being the production of the compatible-solute – glycerol. This hypothesis was strongly supported due to observations of abundance differences in response to salinity of several key HOG pathway MAPK proteins, most notably Gdp1 the downstream initiator of glycerol production. Finally, our seventh hypothesis was supported by the observed inverse responses between our two conspecific isolates. As per our hypothesis, the isolate with greatest ED50, DS 0982, did indeed have the greatest abundance of proteins involved with osmoadaptive strategy across the board from those involved in cell wall modifications such as chitin synthase, to proteins involved in plasma membrane amendments such as including such observations as C-14 reductase-like protein, and even the critical MAPK compatible osmolyte production initiator protein Gdp1. Understanding soil-inhabiting and root-associated fungi is critical to ecosystem health. Whether through providing nutrient cycling and ecosystem services, or direct interactions with host plants, fungal impacts cannot be ignored. Through the effects of climate change and corresponding changes in precipitation, soil salinization is a major threat to plant and animal health as well as the overall function of our natural world. This research aims to fill the gap in our knowledge on the impact of salinity toward root-associated fungi and their limits in salt-tolerance both within and across species. Further, this study aids in identifying varying functional adaptations soil-dwelling fungi depend on for survival in response to salt stress in soil matrices

Understanding the mechanisms of robustness in intracellular protein signalling cascades and gene expression

We seek to understand the structural as well as the mechanistic basis of robustness in intracellular protein signalling cascades and in transcriptional regulation of gene expression. For protein signalling cascades, we employ a comparison based study involving a single, a double and a cascade of two double phosphorylation-dephosphorylation (PD) cycles. Using deterministic modelling approaches based on ordinary differential equations (ODE), we observe that the cascade of two double PD cycles exhibits robust output behaviour compared to that of a single and a double PD cycle upon constant as well as time- varying input perturbations. Furthermore, a system theoretic analysis reveals that the protein phosphorylation cascades act as an efficient low-pass filter that attenuates the noise mimicked as high-frequency input signals. Afterwards, we extend the study for a stochastic environment. Simulation results based on the stochastic simulation algorithm (SSA) reveal a novel phenomenon called dynamic sequestration that plays an ambivalent role as an intrinsic noise filter. Overall, the analysis indicates that complexity can be one of the basic principles of robust biological designs such as intracellular protein signalling cascades. A major function of intracellular signalling cascades is to transmit the extracellular signal to the nucleus to initiate the process of gene expression. Gene expression is an intrinsically stochastic process that results into cell-to-cell variability in protein and messenger RNA (mRNA) levels, often termed as the expression noise. In spite of such noise, how cells achieve robustness is therefore a fundamental biological problem. We conclude the thesis by introducing a rule-based modelling approach based on the Kappa (κ) platform with the goal to understand the underlying mechanisms that ensure robust cellular functioning during gene expression. In particular, we introduce a gene expression model that keeps the process of transcription and excludes the process of translation. Therefore, we quantify the expression noise using mRNA which is the end product of transcription. Besides, the motivation behind adopting a rule-based modelling approach is that unlike the ODE-based approach, the former subsumes the combinatorial complexity arises due to various binding configurations of transcription factors (TF) for regulation of gene expression and offers a compact graphical representation of the same. Afterwards, the representation is transformed into an equivalent set of executable κ rules that are simulated using the SSA to obtain distributions of mRNA copy numbers corresponding to different regulatory mechanisms.Wir wollen sowohl die strukturellen als auch die mechanistischen Grundlagen der Robustheit in intrazellulären Proteinsignalkaskaden und in der transkriptionellen Regulation der Genexpression verstehen. Für die Untersuchung von Proteinsignalkaskaden verwenden wir eine vergleichsbasierte Studie mit einer Einzelphosphorylierung, einer Doppelphosphorylierung und einer Kaskade von zwei Doppelphosphorylierungs-Dephosphorylierungs-(PD)-Zyklen. Zur Modellierung verwenden wir deterministische Ansätze, die auf gewöhnlichen Differentialgleichungen (ODE) basieren. Im Gegensatz zu einem einzelnen und einem doppelten PD-Zyklus weist die Kaskade von zwei doppelten PD-Zyklen ein robustes Ausgabeverhalten bei konstanten sowie zeitvariablen Eingangsstörungen auf. Darüber hinaus zeigt eine systemtheoretische Analyse, dass die Proteinphosphorylierungskaskaden als effizienter Tiefpassfilter wirken, der hochfrequente Eingangssignale dämpft. Anschließend erweitern wir die Studie mit einer stochastischen Umgebung. Simulationsergebnisse, die auf dem stochastischen Simulationsalgorithmus (SSA) basieren, zeigen ein neuartiges Phänomen namens "Dynamic Sequestration", das eine ambivalente Rolle als intrinsischer Rauschfilter spielt. Insgesamt zeigt die Analyse, dass Komplexität eines der Grundprinzipien robuster biologischer Systeme wie intrazellulärer Proteinsignalkaskaden sein kann. Eine der Hauptfunktionen intrazellulärer Signalkaskaden besteht darin das extrazelluläre Signal an den Kern zu übertragen, um den Prozess der Genexpression einzuleiten. Die Genexpression ist ein intrinsisch stochastischer Prozess, der zu einer Variabilität der Protein- und Messenger-RNA (mRNA)-Menge von Zelle zu Zelle führt, die oft als Expressionsrauschen bezeichnet wird. Trotz des Rauschens ist es daher ein grundlegendes biologisches Problem, wie Zellen ihre Robustheit erreichen. Um zugrunde liegende Mechanismen zu verstehen, die eine robuste zelluläre Funktion während der Genexpression gewährleisten, schließen wir die Arbeit mit der Einüfhrung eines regelbasierten Modellierungsansatzes auf Basis der Kappa (κ)-Plattform ab. Insbesondere stellen wir ein Genexpressionsmodell vor, das den Prozess der Transkription beibehält und den Prozess der Translation ausschließt. Daher quantifizieren wir das Expressionsrauschen mit Hilfe der mRNA, die das Endprodukt der Transkription ist. Darüber hinaus ist die Motivation für die Verwendung eines regelbasierten Modellierungsansatzes, dass im Gegensatz zum ODE-basierten Ansatz die kombinatorische Komplexität durch verschiedene Bindungskonfigurationen von Transkriptionsfaktoren (TF) zur Regulierung der Genexpression abgebildet wird und eine kompakte grafische Darstellung derselben geboten wird. Anschließend wird die Darstellung in einen äquivalenten Satz von ausführbaren κ-Regeln umgewandelt, die mit Hilfe der SSA simuliert werden, um Verteilungen von mRNA-Molekülen zu erhalten, die verschiedenen Regulationsmechanismen entsprechen. Wir wollen sowohl die strukturellen als auch die mechanistischen Grundlagen der Robustheit in intrazellulären Proteinsignalkaskaden und in der transkriptionellen Regulation der Genexpression verstehen. Für die Untersuchung von Proteinsignalkaskaden verwenden wir eine vergleichsbasierte Studie mit einer Einzelphosphorylierung, einer Doppelphosphorylierung und einer Kaskade von zwei Doppelphosphorylierungs-Dephosphorylierungs-(PD)-Zyklen. Zur Modellierung verwenden wir deterministische Ansätze, die auf gewöhnlichen Differentialgleichungen (ODE) basieren. Im Gegensatz zu einem einzelnen und einem doppelten PD-Zyklus weist die Kaskade von zwei doppelten PD-Zyklen ein robustes Ausgabeverhalten bei konstanten sowie zeitvariablen Eingangsstörungen auf. Darüber hinaus zeigt eine systemtheoretische Analyse, dass die Proteinphosphorylierungskaskaden als effizienter Tiefpassfilter wirken, der hochfrequente Eingangssignale dämpft. Anschließend erweitern wir die Studie mit einer stochastischen Umgebung. Simulationsergebnisse, die auf dem stochastischen Simulationsalgorithmus (SSA) basieren, zeigen ein neuartiges Phänomen namens "Dynamic Sequestration", das eine ambivalente Rolle als intrinsischer Rauschfilter spielt. Insgesamt zeigt die Analyse, dass Komplexität eines der Grundprinzipien robuster biologischer Systeme wie intrazellulärer Proteinsignalkaskaden sein kann. Eine der Hauptfunktionen intrazellulärer Signalkaskaden besteht darin das extrazelluläre Signal an den Kern zu übertragen, um den Prozess der Genexpression einzuleiten. Die Genexpression ist ein intrinsisch stochastischer Prozess, der zu einer Variabilität der Protein- und Messenger-RNA (mRNA)-Menge von Zelle zu Zelle führt, die oft als Expressionsrauschen bezeichnet wird. Trotz des Rauschens ist es daher ein grundlegendes biologisches Problem, wie Zellen ihre Robustheit erreichen. Um zugrunde liegende Mechanismen zu verstehen, die eine robuste zelluläre Funktion während der Genexpression gewährleisten, schließen wir die Arbeit mit der Einüfhrung eines regelbasierten Modellierungsansatzes auf Basis der Kappa (κ)-Plattform ab. Insbesondere stellen wir ein Genexpressionsmodell vor, das den Prozess der Transkription beibehält und den Prozess der Translation ausschließt. Daher quantifizieren wir das Expressionsrauschen mit Hilfe der mRNA, die das Endprodukt der Transkription ist. Darüber hinaus ist die Motivation für die Verwendung eines regelbasierten Modellierungsansatzes, dass im Gegensatz zum ODE-basierten Ansatz die kombinatorische Komplexität durch verschiedene Bindungskonfigurationen von Transkriptionsfaktoren (TF) zur Regulierung der Genexpression abgebildet wird und eine kompakte grafische Darstellung derselben geboten wird. Anschließend wird die Darstellung in einen äquivalenten Satz von ausführbaren κ-Regeln umgewandelt, die mit Hilfe der SSA simuliert werden, um Verteilungen von mRNA-Molekülen zu erhalten, die verschiedenen Regulationsmechanismen entsprechen

Robust network structure of the Sln1-Ypd1-Ssk1 three-component phospho-relay prevents unintended activation of the HOG MAPK pathway in Saccharomyces cerevisiae

Background: The yeast Saccharomyces cerevisiae relies on the high-osmolarity glycerol (HOG) signaling pathway to respond to increases in external osmolarity. The HOG pathway is rapidly activated under conditions of elevated osmolarity and regulates transcriptional and metabolic changes within the cell. Under normal growth conditions, however, a three-component phospho-relay consisting of the histidine kinase Sln1, the transfer protein Ypd1, and the response regulator Ssk1 represses HOG pathway activity by phosphorylation of Ssk1. This inhibition of the HOG pathway is essential for cellular fitness in normal osmolarity. Nevertheless, the extent to and mechanisms by which inhibition is robust to fluctuations in the concentrations of the phospho-relay components has received little attention. Results: We established that the Sln1-Ypd1-Ssk1 phospho-relay is robust—it is able to maintain inhibition of the HOG pathway even after significant changes in the levels of its three components. We then developed a biochemically realistic mathematical model of the phospho-relay, which suggested that robustness is due to buffering by a large excess pool of Ypd1. We confirmed experimentally that depletion of the Ypd1 pool results in inappropriate activation of the HOG pathway. Conclusions: We identified buffering by an intermediate component in excess as a novel mechanism through which a phospho-relay can achieve robustness. This buffering requires multiple components and is therefore unavailable to two-component systems, suggesting one important advantage of multi-component relays. Electronic supplementary material The online version of this article (doi:10.1186/s12918-015-0158-y) contains supplementary material, which is available to authorized users