Elucidating the molecular genetics of host and nonhost resistance in barley to stripe rust

Plants have a remarkable ability to resist the majority of pathogenic microbes they encounter. As such, they are described as nonhosts. Nonhost resistance is often conceptualised as a qualitative separation from host resistance. Classification into these two states is generally facile, as they fail to fully describe the range of states that exist in the transition from host to nonhost. This poses a problem when studying pathosystems that cannot be classified into either of these categories due to their intermediate status relative to the two extremes. Therefore, the terms intermediate host and intermediate nonhost have been proposed to describe pathosystems in the evolutionary transition between host and nonhost status. At present, a significant amount of research exists into the molecular genetics of host and nonhost pathosystems but very little is known about intermediate systems. The work in this Ph. D. thesis focuses on the interaction of barley with Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, as an intermediate host pathosystem. The first research chapter describes the development of two microscopic phenotypic assays used to quantify P. striformis f. sp. tritici in barley leaves challenged with the pathogen. These assays are then used to screen a large panel of barley accessions to define the intermediate host status of barley relative to a host pathosystem. Subsequently, these assays play a key role in determining that the genetic architecture of resistance in barley is underpinned by three major effect resistance loci: Rpst1, Rpst2, and Rpst3. Using a combination of classical map-based genetics and contemporary genomics information I identify a candidate NLR gene underlying Rpst2 resistance on chromosome 7HL. Furthermore, I show that distinct genes condition host and nonhost resistance in barley by mapping the host resistance gene, rps2 to chromosome 2HL

Structural Analysis and Activity Correlation of Amphiphilic Cyclic Antimicrobial Peptides Derived from the [W\u3csub\u3e4\u3c/sub\u3eR\u3csub\u3e4\u3c/sub\u3e] Scaffold

In our ongoing quest to design effective antimicrobial peptides (AMPs), this study aimed to elucidate the mechanisms governing cyclic amphiphilic AMPs and their interactions with membranes. The objective was to discern the nature of these interactions and understand how peptide sequence and structure influence antimicrobial activity. We introduced modifications into the established cyclic AMP peptide, [W4R4], incorporating an extra aromatic hydrophobic residue (W), a positively charged residue (R), or the unique 2,5-diketopiperazine (DKP). This study systematically explored the structure–activity relationships (SARs) of a series of cyclic peptides derived from the [W4R4] scaffold, including the first synthesis and evaluation of [W4R4(DKP)]. Structural, dynamic, hydrophobic, and membrane-binding properties of four cyclic peptides ([W4R4], [W5R4], [W4R5], [W4R4(DKP)]) were explored using molecular dynamics simulations within a DOPC/DOPG lipid bilayer that mimics the bacterial membrane. The results revealed distinct SARs linking antimicrobial activity to parameters such as conformational plasticity, immersion depth in the bilayer, and population of the membrane binding mode. Notably, [W4R5] exhibited an optimal “activity/binding to the bacterial membrane” pattern. This multidisciplinary approach efficiently decoded finely regulated SAR profiles, laying a foundation for the rational design of novel antimicrobial peptides

Optical nanoscopy

AbstractThis article deals with the developments of optical microscopy towards nanoscopy. Basic concepts of the methods implemented to obtain spatial super-resolution are described, along with concepts related to the study of biological systems at the molecular level. Fluorescence as a mechanism of contrast and spatial resolution will be the starting point to developing a multi-messenger optical microscope tunable down to the nanoscale in living systems. Moreover, the integration of optical nanoscopy with scanning probe microscopy and the charming possibility of using artificial intelligence approaches will be shortly outlined

Protein regulation in Trichodesmium and other marine bacteria: observational and interpretive biomarkers of biogeochemical processes

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical Oceanography and Microbial Biogeochemistry at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February 2020.Marine microbes play key roles in global biogeochemistry by mediating chemical transformations and linking nutrient cycles to one another. A major goal in oceanography is to predict the activity of marine microbes across disparate ocean ecosystems. Towards this end, molecular biomarkers are important tools in chemical oceanography because they allow for both the observation and interpretation of microbial behavior. In this thesis, I use molecular biomarkers to develop a holistic, systems biology approach to the study of marine microbes. I begin by identifying unique patterns in the biochemical sensory systems of marine bacteria and suggest that these represent a specific adaptation to the marine environment. Building from this, I focus on the prevalent marine nitrogen fixer Trichodesmium, whose activity affects global nitrogen, carbon, phosphorus, and trace metal cycles. A metaproteomic survey of Trichodesmium populations identified simultaneous iron and phosphate co-stress throughout the tropical and subtropical oceans, demonstrating that this is caused by the biophysical limits of membrane space and nutrient diffusion. Tackling the problem at a smaller scale, I investigated the metaproteomes of individual Trichodesmium colonies captured from a single field site, and identified significant variability related to iron acquisition from mineral particles. Next, I investigated diel proteomes of cultured Trichodesmium erythraeum sp. IMS101 to highlight its physiological complexity and understand how and why nitrogen fixation occurs in the day, despite the incompatibly of the nitrogenase enzyme with oxygen produced in photosynthesis. This thesis develops a fundamental understanding of how Trichodesmium and other organisms affect, and are affected by, their surroundings. It indicates that a reductionist approach in which environmental drivers are considered independently may not capture the full complexity of microbechemistry interactions. Future work can focus on benchmarking and calibration of the protein biomarkers identified here, as well as continued connection of systems biology frameworks to the study of ocean chemistry.This work was supported by an MIT Walter A. Rosenblith Presidential Fellowship and a National Science Foundation Graduate Research Program Fellowship under grant number 1122274 [N.Held]. This work was also supported by the WHOI Ocean Ventures fund [N.Held], Gordon and Betty Moore Foundation grant number 3782 [M.Saito], National Science Foundation grant numbers OCE-1657766 [M.Saito], EarthCube-1639714 [M.Saito], OCE-1658030 [M.Saito], and OCE-1260233 [M.Saito], and funding from the UK Natural Environment Research Council (NERC) under grants awarded to C.M. (NE/N001079/1) and M.L. (NE/N001125/1). This thesis was completed during a writing residency at the Turkeyland Cove Foundation

A genome-wide association study of resistance to the yellow rust pathogen (Puccinia striiformis f. sp. tritici) in elite UK wheat germplasm

ABSTRACT Pauline G. M. Bansept-Basler, 2013 A genome wide association study of resistance to the yellow rust pathogen (Puccinia striiformis f. sp. tritici) in elite UK wheat germplasm Identification of marker-trait associations (MTA) in germplasm relevant to breeding program via association mapping (AM) can be an effective way to identify loci useful for selection. This approach does not require the generation of specific mapping populations and takes advantage of historical phenotypic data. In the present study, an association panel of 327 bread wheat varieties have been assembled and genotyped with 1806 DArT markers. Genetic structure analysis revealed a low stratification of the panel based on geographical origin (UK versus mixed European varieties) and a close relatedness between lines, which is confirmed by pedigree information. Historical evaluations against the yellow rust pathogen (Puccinia striiformis f.sp. tritici (Pst)) carried in the United Kingdom between 1990 and 2009, as well as de novo evaluations against recent Pst races have been collected and analysed for MTAs. Association scans considering historical data focused on specific Pst pathotypes and de novo seedling tests identified markers linked to known racespecific Yr genes Yr6, Yr7, Yr9, Yr17 and Yr32. When evaluated against current Pst races in the field, 35% of the lines from the panel presented repeatedly a high level of resistance (Area Under the Disease Progress Curve relative<0.2) which is due to the presence of seedling resistances as well as adult plant resistances within the lines. AM with de novo phenotypes revealed 23 MTA groups pointing to potential resistance loci, 14 of them were also identified with historical data and six seemed to point to adult plant resistance loci on chromosomes 2A, 2B, 3A, 6A, 6B and 7A. These results confirm the value of AM using historical data for QTL discovery and suggest the availability of diverse sources of yellow rust resistances within wheat elite UK germplasm

Strategies for wheat stripe rust pathogenicity identified by "omics" technologies

Stripe rust is a major constraint to wheat production worldwide. The causal agent is the fungus Puccinia striiformis f.sp. tritici (Pst). During infection, the fungus creates a specialized cellular structure within host cells called the haustorium which allows Pst to obtain the nutrients necessary for development and reproduction. The haustorium is also thought to secrete virulence molecules called 'effectors', which are suspected to manipulate the physiological and immune responses of host cells. Despite this broad outline, the molecular events that underlie host colonization and the produced effectors proteins are largely unknown. In my PhD, I extensively investigated Pst using transcriptomics and proteomics techniques to obtain a better understanding of how the pathogen establishes a compatible interaction with its host, and to identify the effector proteins that are synthesised and secreted during infection. First, by the use of next generation sequencing (454 and Illumina) the transcriptomes of two contrasting pathogenic stages (germinated spores and haustoria) were generated, de novo assembled and extensively annotated. A digital gene expression analysis revealed many differentially expressed genes which highlight key metabolic differences between these cell types, and provide insight into their different roles during infection. Spores turn on the metabolic pathways to derive energy from non carbohydrate sources, required to sustain growth and development. Conversely, haustoria deploy all the necessary machinery to take advantage of the abundant nutrients derived from the host nutrients and focus on energy production and biosynthetic pathways to support fungal growth and spore production. Further analysis of the haustoria transcriptome, allowed me to identify the first set of potential effector candidate genes of Pst, comprised of 437 genes, with two thirds of these up-regulated in haustoria compared to germinated spores. Using a bacterial system to synthesise and deliver proteins encoded by effector gene candidates, a small subset of these genes was cloned and used to establish two functional characterization methods. The first one aimed to test if these proteins could be recognised by wheat resistance genes and the second one tested their capacity to inhibit cell death triggered by a necrotic toxin. From the later one two effector gene candidates were found to partially inhibit plant cell death. In parallel, I have developed a method to isolate highly purified haustoria combining density gradients and flow cytometry. Haustoria purified by this method were successfully used for proteomics analysis. Proteomics data from haustoria, germinated and ungerminated spores were generated and analyzed preliminarily to determine the presence of effector candidates as well as non-effector proteins in each tissue. More than 3,000 proteins were validated by proteomic data, including 150 effector candidates. The correlation of transcriptomic and proteomic data suggested that the synthesis and deployment of some effector proteins could occur at different spatiotemporal sites and even could have destinations other than the host cell cytoplasm. Together, these studies have substantially increased our knowledge of Pst effectors and have provided insights into the pathogenic strategies of this important organism, opening new avenues of research with immense potential in the design of novel disease control strategies

Cross-talk between Dps proteins triggers manganese distribution as a defense strategy against oxidative stress

"Deinococcus radiodurans is a radiation resistant bacterium. For this reason, it has been a focus of several studies over the years. The aim has been to understand what makes this organism so resistant to different extreme conditions. Several protection mechanisms are present, such as enzymatic and non-enzymatic systems, namely Mn2+-Pi complexes. These mechanisms work synergistically, thereby conferring higher protection to this extraordinary organism.(...)

Carbohydrate-Active Enzymes

Carbohydrate-active enzymes are responsible for both biosynthesis and the breakdown of carbohydrates and glycoconjugates. They are involved in many metabolic pathways; in the biosynthesis and degradation of various biomolecules, such as bacterial exopolysaccharides, starch, cellulose and lignin; and in the glycosylation of proteins and lipids. Carbohydrate-active enzymes are classified into glycoside hydrolases, glycosyltransferases, polysaccharide lyases, carbohydrate esterases, and enzymes with auxiliary activities (CAZy database, www.cazy.org). Glycosyltransferases synthesize a huge variety of complex carbohydrates with different degrees of polymerization, moieties and branching. On the other hand, complex carbohydrate breakdown is carried out by glycoside hydrolases, polysaccharide lyases and carbohydrate esterases. Their interesting reactions have attracted the attention of researchers across scientific fields, ranging from basic research to biotechnology. Interest in carbohydrate-active enzymes is due not only to their ability to build and degrade biopolymers—which is highly relevant in biotechnology—but also because they are involved in bacterial biofilm formation, and in glycosylation of proteins and lipids, with important health implications. This book gathers new research results and reviews to broaden our understanding of carbohydrate-active enzymes, their mutants and their reaction products at the molecular level

Theoretical-experimental study on protein-ligand interactions based on thermodynamics methods, molecular docking and perturbation models

The current doctoral thesis focuses on understanding the thermodynamic events of protein-ligand interactions which have been of paramount importance from traditional Medicinal Chemistry to Nanobiotechnology. Particular attention has been made on the application of state-of-the-art methodologies to address thermodynamic studies of the protein-ligand interactions by integrating structure-based molecular docking techniques, classical fractal approaches to solve protein-ligand complementarity problems, perturbation models to study allosteric signal propagation, predictive nano-quantitative structure-toxicity relationship models coupled with powerful experimental validation techniques. The contributions provided by this work could open an unlimited horizon to the fields of Drug-Discovery, Materials Sciences, Molecular Diagnosis, and Environmental Health Sciences