Genome-wide characterisation of microRNAs and their target genes in different durum wheat genotypes under water limiting conditions

Durum wheat (Triticum turgidum L. ssp. durum) is a tetraploid wheat species grown primarily in the North American Great Plains, Mediterranean Europe, Northern Africa, Mexico and Australia. An important limiting factor for durum production in Mediterranean environments like South Australia is water deficit immediately prior to and during anthesis, adversely affecting durum productivity and quality. Investigating water deficit response mechanisms and genotypic differences within a crop species is an important strategy for understanding the basis of water-deficit stress response and for selection of elite genotypes with improved stress tolerance. In plants, microRNAs (miRNAs), which are a class of small non coding RNAs, have been identified as important regulators of plant development and abiotic stress responses. While the miRNA transcriptome under water limiting conditions has been investigated in many crop species, it is poorly characterised in durum wheat. In this study, glasshouse experiments over two years evaluated 20 durum wheat genotypes for their variation in various morphological, physiological and yield responses to pre-anthesis water-deficit stress. Four Australian durum varieties with contrasting stress sensitivities were identified. High-throughput Illumina sequencing of 96 small RNA libraries constructed from the flag leaf and head tissues of these four genotypes detected 110 conserved miRNAs and 159 novel candidate miRNA hairpins. Statistical analysis of sequencing reads revealed the differential expression profiles of durum miRNAs associated with water-deficit stress treatment, tissue type and genotype. Most importantly, several conserved and novel miRNAs showed inverted regulatory profiles between the stress tolerant and sensitive varieties. Subsequent genome-wide in silico analysis identified 2055 putative targets for conserved durum miRNAs, and 131 targets for four novel durum miRNAs possibly contributing to genotypic stress tolerance. Predicted mRNA targets of the stress responsive miRNAs encode various transcription factors, binding proteins, and functional enzymes, which play vital roles in multiple biological pathways such as hormone signalling and metabolic processes, suggesting the extensive involvement of miRNA-target regulatory modules in water-deficit stress adaptation. Quantitative PCR profiling further characterised 50 target genes and 12 miRNAs with stress responsive and/or genotype-dependent expression profiles. A 5′ RLM-RACE approach subsequently validated the regulation of nine targets by water-deficit stress responsive miRNAs, providing the first experimental evidence that target mRNAs are genuinely cleaved by miRNAs in durum wheat. Characterisation of the individual miR160/Auxin Response Factors regulatory module further revealed their expression profile over different time points during water-deficit stress. The present study provides a comprehensive and comparative description of the miRNA transcriptome and their targets in durum wheat varieties with contrasting waterdeficit stress tolerance, providing new insights into the functional roles of miRNA-guided RNAi mechanisms. Results derived from this work could contribute to future research on the characterisation of individual miRNA regulatory modules and their specific biological functions, exploiting the potential of Triticum turgidum miRNA in developing RNAiimproved crops with stress tolerance.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food and Wine, 2016

Removal of a Conserved Disulfide Bond Does Not Compromise Mechanical Stability of a VHH Antibody Complex

Single-domain VHH antibodies are promising reagents for medical therapy. A conserved disulfide bond within the VHH framework region is known to be critical for thermal stability, however, no prior studies have investigated its influence on the stability of VHH antibody–antigen complexes under mechanical load. Here, we used single-molecule force spectroscopy to test the influence of a VHH domain’s conserved disulfide bond on the mechanical strength of the interaction with its antigen mCherry. We found that although removal of the disulfide bond through cysteine-to-alanine mutagenesis significantly lowered VHH domain denaturation temperature, it had no significant impact on the mechanical strength of the VHH:mCherry interaction with complex rupture occurring at ∼60 pN at 103–104 pN/sec regardless of disulfide bond state. These results demonstrate that mechanostable binding interactions can be built on molecular scaffolds that may be thermodynamically compromised at equilibrium

High force catch bond mechanism of bacterial adhesion in the human gut

Bacterial colonization of the human intestine requires firm adhesion of bacteria to insoluble substrates under hydrodynamic flow. Here we report the molecular mechanism behind an ultrastable protein complex responsible for resisting shear forces and adhering bacteria to cellulose fibers in the human gut. Using single-molecule force spectroscopy (SMFS), single-molecule FRET (smFRET), and molecular dynamics (MD) simulations, we resolve two binding modes and three unbinding reaction pathways of a mechanically ultrastable R. champanellensis (Rc) Dockerin:Cohesin (Doc:Coh) complex. The complex assembles in two discrete binding modes with significantly different mechanical properties, with one breaking at similar to 500 pN and the other at similar to 200 pN at loading rates from 1-100 nN s(-1). A neighboring X-module domain allosterically regulates the binding interaction and inhibits one of the low-force pathways at high loading rates, giving rise to a catch bonding mechanism that manifests under force ramp protocols. Multi-state Monte Carlo simulations show strong agreement with experimental results, validating the proposed kinetic scheme. These results explain mechanistically how gut microbes regulate cell adhesion strength at high shear stress through intricate molecular mechanisms including dual-binding modes, mechanical allostery and catch bonds

Mechanical Polyprotein Assembly Using Sfp and Sortase‐Mediated Domain Oligomerization for Single‐Molecule Studies

Single‐molecule force spectroscopy (SMFS) with the atomic force microscope (AFM) provides remarkable details on the energy landscapes governing protein (un)folding and intermolecular complex dissociation. In such experiments, multidomain polyproteins consisting of multiple copies of independently foldable domains provide internal controls identifiable by characteristic contour length increments, unfolding forces, and/or unfolding substeps. Here, a new approach to polyprotein synthesis is presented relying on posttranslational enzyme‐mediated oligomerization of domains. Mutant variants of immunoglobulin 27 (I27) and a bacterial cellulose binding module (CBM) fused to an Ig‐like X‐module (XMod), and a mechanostable receptor called Dockerin (Doc) are produced with complementary peptide tags. By utilizing 4′‐phosphopantetheinyl transferase and Sortase A, the system enables I27‐domain oligomerization into polyproteins of varying lengths followed by C‐terminal capping with mechanostable Doc. The number of oligomerized domains per molecule, the unfolding forces, and the complex rupture forces of posttranslationally assembled polyproteins are characterized using >40 h automated AFM–SMFS with a Cohesin (Coh)‐modified cantilever. Use of the Coh–Doc interaction to unfold polyproteins provides a high yield of ≈ 3800 specific single‐molecule interaction curves. This approach is advantageous for assembly of polyproteins from domains that lack proper folding or are insoluble in a polyprotein format

Fertility Enhancement but Premature Ovarian Failure in esr1-Deficient Female Zebrafish

It is well established that estrogens regulate female reproduction through estrogen receptors (ERs) in the ovary. However, the precise physiological role of estrogen/ER signaling in reproduction processes remains poorly defined in zebrafish. In this study, we successfully generated an ERα (esr1) mutant line in zebrafish via transcription activator-like effectors nucleases (TALENs). It was found in the mutant females that the fertility was enhanced and the ovarian histology was normal at 90 days post-fertilization (dpf). However, the number of fertile females decreased with age. By 180 dpf, esr1 mutant females were infertile with degenerated ovaries, while the age-matched wild-type females were still fertile. Additionally, few large vitellogenic granules can be found in full grown (FG) follicles at 90 dpf and the expression of vtg genes were down-regulated at both 90 and 180 dpf in esr1 mutant zebrafish. Moreover, steroidogenesis pathway and mTOR signaling pathway were over-activated at 90 dpf, but declined prematurely in esr1 mutant zebrafish by 180 dpf. Collectively, the present study provides evidence that esr1 is fundamental for ovarian maintenance in zebrafish

Steered molecular dynamics simulations reveal the role of Ca2+ in regulating mechanostability of cellulose-binding proteins

The conversion of cellulosic biomass into biofuels requires degradation of the biomass into fermentable sugars. The most efficient natural cellulase system for carrying out this conversion is an extracellular multi-enzymatic complex named the cellulosome. In addition to temperature and pH stability, mechanical stability is important for functioning of cellulosome domains, and experimental techniques such as Single Molecule Force Spectroscopy (SMFS) have been used to measure the mechanical strength of several cellulosomal proteins. Molecular dynamics computer simulations provide complementary atomic-resolution quantitative maps of domain mechanical stability for identification of experimental leads for protein stabilization. In this study, we used multi-scale steered molecular dynamics computer simulations, benchmarked against new SMFS measurements, to measure the intermolecular contacts that confer high mechanical stability to a family 3 Carbohydrate Binding Module protein (CBM3) derived from the archetypal Clostridium thermocellum cellulosome. Our data predicts that electrostatic interactions in the calcium binding pocket modulate the mechanostability of the cellulose-binding module, which provides an additional design rule for the rational re-engineering of designer cellulosomes for biotechnology. Our data offers new molecular insights into the origins of mechanostability in cellulose binding domains and gives leads for synthesis of more robust cellulose-binding protein modules. On the other hand, simulations predict that insertion of a flexible strand can promote alternative unfolding pathways and dramatically reduce the mechanostability of the carbohydrate binding module, which gives routes to rational design of tailormade fingerprint complexes for force spectroscopy experiments

AFM-based experimental investigation, numerical simulation and theoretical modeling of mechanics of cell adhesion

Cell-extracellular matrix and cell-cell adhesion are essential for biological processes such as cell motility, signaling, proliferation, cytoskeletal organization and gene expression. For this reason, extensive effort has been devoted in the past few decades to measure cell adhesion as well as identify key molecules involved. This thesis focuses on two outstanding problems in this area, namely, how to quantitatively characterize the adhesion between neural cells and the substrate and how to model the turnover of adhesions in the intriguing phenomenon of stretch-induced cell realignment. First of all, using a combined atomic force (AFM) and total internal reflection fluorescence microscope (TIRFM) system a novel method was developed to systematically and quantitatively examine the adhesion between neurite branches and the extracellular matrix. Specifically, a tipless AFM cantilever was used to penetrate between a well-developed neurite and the functionalized substrate and then gradually peel the neurite from the surface. At the same time, a laser TIRFM was utilized to monitor the activities of different adhesion molecules during the detaching process. This approach provides a solution to the long-standing problem of how to quantitatively measure neuron-extracellular matrix interactions while, simultaneously, identify the roles of various adhesion proteins in the process. Besides heathy neurons, testes have also been conducted on cells affected by the Alzheimer's disease (AD) where the influence of such disease on the mechanical response of neural cells was demonstrated. Secondly, to better understand the observed peeling response of the neurite, as well as extract key information from it, finite element (FEM) simulation was carried out using ABAQUS. It was shown that a good fit between the simulation results and experimental data can be achieved by representing the adhesion between two surfaces with simple cohesive interactions. In particular, it was found that the apparent adhesion energy density, a quantity of central interest in cell adhesion studies, is in the range of 0.2-0.8 mj/m^2. Last but not the least, a mechanochemical modeling framework was developed to investigate the mechanism of cell reorientation induced by cyclic stretching on the substrate. It was shown that the final alignment of cells reflects the competition between stress fiber assembly or disassembly, focal adhesion growth or disruption, substrate stiffening and whole-cell rotation. Predictions from the model are consistent with a variety of experimental observations, suggesting that the main physics of this intriguing phenomenon may have been well captured.published_or_final_versionMechanical EngineeringDoctoralDoctor of Philosoph

Biomaterial nanotopography-mediated cell responses: experiment and modeling

The rapid development of fabrication and processing technologies in the past two decades has enabled researchers to introduce nanoscale features into materials which, interestingly, have been shown to greatly regulate the behavior and fate of biological cells. In particular, important cell responses (such as adhesion, proliferation, differentiation, migration, and filopodial growth) have all been correlated with material nanotopography. Given its great potential, intensive efforts have been made, both experimentally and theoretically, to understand why and how cells respond to nanoscale surface features, and this article reviews recent progress in this field. Specifically, a brief overview on the fabrication and modification techniques to create nanotopography on different materials is given first. After that, a summary of important experimental findings on the mediation of nanoscale surface topography on the behavior of various cells, as well as the underlying mechanism, is provided. Finally, both classical and recently developed approaches for modeling nanotopography-mediated cell adhesion and filopodial growth are reviewed

Multi-Omics Analysis of Small RNA, Transcriptome, and Degradome in T. turgidum—Regulatory Networks of Grain Development and Abiotic Stress Response

Crop reproduction is highly sensitive to water deficit and heat stress. The molecular networks of stress adaptation and grain development in tetraploid wheat (Triticum turgidum durum) are not well understood. Small RNAs (sRNAs) are important epigenetic regulators connecting the transcriptional and post-transcriptional regulatory networks. This study presents the first multi-omics analysis of the sRNAome, transcriptome, and degradome in T. turgidum developing grains, under single and combined water deficit and heat stress. We identified 690 microRNAs (miRNAs), with 84 being novel, from 118 sRNA libraries. Complete profiles of differentially expressed miRNAs (DEMs) specific to genotypes, stress types, and different reproductive time-points are provided. The first degradome sequencing report for developing durum grains discovered a significant number of new target genes regulated by miRNAs post-transcriptionally. Transcriptome sequencing profiled 53,146 T. turgidum genes, swith differentially expressed genes (DEGs) enriched in functional categories such as nutrient metabolism, cellular differentiation, transport, reproductive development, and hormone transduction pathways. miRNA–mRNA networks that affect grain characteristics such as starch synthesis and protein metabolism were constructed on the basis of integrated analysis of the three omics. This study provides a substantial amount of novel information on the post-transcriptional networks in T. turgidum grains, which will facilitate innovations for breeding programs aiming to improve crop resilience and grain quality