Analysis of Arabidopsis thaliana glucosinolate profiles in response to a native root fungal endophyte

Plants host communities of microbes termed the microbiota, which contributes to plant nutrition and stress relief, notably in roots. With diverse microbiota fungi, plants can accept invasive hyphal growth inside their cells. Which immune signaling pathways regulate colonization of root tissues and cells by fungal endophytes is unclear. However, at the metabolite level, Tryptophan (Trp)-derived compounds, including indolic-glucosinolates (GLS), prevent excessive fungal endophyte growth and associated harm in roots of Arabidopsis thaliana (Arabidopsis). This PhD thesis aims to provide i) knowledge on immune pathways relevant to general fungal colonization and ii) insights into the regulation of aliphatic- and indolic-GLS by phosphate (Pi). In the first chapter, I explored whether the broad host range Sebacinales fungi showed enhanced or reduced growth in roots of diverse established Arabidopsis immunity mutants but detected no differences compared to wild-type Col-0 plants. In the second chapter, I investigated whether the Phosphate Starvation Response (PSR) system modulates aliphatic and indolic-GLS accumulation as important immune metabolic outputs. As fungal endophyte test organism, I selected a strain of Truncatella angustata (F73) isolated from Arabidopsis roots in nature and known to relieve long-term Pi limitation stress in an agar-based system. Using confocal microscopy, I determined that the root differentiation zone is more permissive to rapid F73 intracellular colonization, compared to the root apical meristem or the elongation zone. A fluorescent transcriptional reporter of the Trp-derived metabolites pathway showed increased signal width in the differentiation zone upon F73 inoculation, suggesting Arabidopsis responds to fungal colonization by spatially expanding Trp-derived metabolite production. I then performed time course RNA-seq and GLS quantification experiments to reveal how Pi and immune signaling are integrated at the transcriptional and metabolomics levels. Pi availability did not impact GLS-related gene expression but governed the number of F73-responsive genes in Col-0 in a temporal manner, suggesting that the PSR regulates general immune dynamics. Yet, amounts of long chained aliphatic-GLS in roots and shoots were constitutively higher at low Pi. Furthermore, F73 induced greater accumulation of most GLS at high Pi, hinting at PSR dependent modulation of GLS synthesis. F73 displayed hallmarks of increased growth rate and virulence at a transcriptional level, but not deleterious infection, in the cyp79b2/b3 mutant depleted in Trp-derived metabolites. Altogether, my work provides spatio-temporal insights into how Pi stress influences interactions with fungi and suggests that the PSR fine-tunes aliphatic- and indolic-GLS accumulation. This hypothesis warrants further exploration using PSR mutants

Scientific Utopia III: crowdsourcing science

Most scientific research is conducted by small teams of investigators who together formulate hypotheses, collect data, conduct analyses, and report novel findings. These teams operate independently as vertically integrated silos. Here we argue that scientific research that is horizontally distributed can provide substantial complementary value, aiming to maximize available resources, promote inclusiveness and transparency, and increase rigor and reliability. This alternative approach enables researchers to tackle ambitious projects that would not be possible under the standard model. Crowdsourced scientific initiatives vary in the degree of communication between project members from largely independent work curated by a coordination team to crowd collaboration on shared activities. The potential benefits and challenges of large-scale collaboration span the entire research process: ideation, study design, data collection, data analysis, reporting, and peer review. Complementing traditional small science with crowdsourced approaches can accelerate the progress of science and improve the quality of scientific research

Voluntary workplace genomic testing: wellness benefit or Pandora\u27s box?

Consumer interest in genetic and genomic testing is growing rapidly, with more than 26 million Americans having purchased direct-to-consumer genetic testing services. Capitalizing on the increasing comfort of consumers with genetic testing outside the clinical environment, commercial vendors are expanding their customer base by marketing genetic and genomic testing services, including testing for pharmacogenomic and pathogenic variants, to employers for inclusion in workplace wellness programs. We describe the appeal of voluntary workplace genomic testing (wGT) to employers and employees, how the ethical, legal, and social implications literature has approached the issue of genetic testing in the workplace in the past, and outline the relevant legal landscape. Given that we are in the early stages of development of the wGT market, now is the time to identify the critical interests and concerns of employees and employers, so that governance can develop and evolve along with the wGT market, rather than behind it, and be based on data, rather than speculative hopes and fears

Investigation on shape deviation of horizontal interior circular channels fabricated by laser powder bed fusion

The fabrication of horizontal interior circular channels poses some unique challenges to the laser powder bed fusion (L-PBF) process. The engineering challenge is to be able to print horizontal interior channels using L-PBF without using support structures, while the scientific challenge is to predict the shape deviation in the horizontal channel. This paper studies the geometric fidelity (roundness and shape deviation) of L-PBF printed horizontal interior circular channels (diameters 1−3 mm) by developing experiment-based regression models and a preliminary computational fluid dynamics (CFD) simulation model. The roundness error is found to be affected by the shape/size of the melt pool, thermal stresses, beam offset, and the slicing algorithm. It is recommended that to decrease the roundness error, in addition to choosing a proper beam offset, the width/depth of the melt pool should be minimized by minimizing the volumetric energy density (smaller laser power or higher scanning speed). Shape deviation in overhanging structures is determined by the thermo-mechanical driven molten flow in the melt pool. Hanging structures with irregular profiles (dross) are formed due to the sinking of the melt pool on an unconsolidated powder bed under the effect of gravity, surface tension, and poor thermal conductivity. (Partially) unmelted powder randomly adheres to the edges of the melt pool enlarging the hanging structure and roughening the profile. Small laser power or large scanning speed benefits reducing the roundness error and hang-diameter ratio. 0° or 45° rotational linear scanning strategy can be selected for minimizing the roundness error or the hang-diameter ratio, respectively

Effects of Thermal and Pressure Histories on the Chemical Strengthening of Sodium Aluminosilicate Glass

Glasses can be chemically strengthened through the ion exchange process, wherein smaller ions in the glass (e.g., Na+) are replaced by larger ions from a salt bath (e.g., K+). This develops a compressive stress (CS) on the glass surface, which, in turn, improves the damage resistance of the glass. The magnitude and depth of the generated CS depends on the thermal and pressure histories of the glass prior to ion exchange. In this study, we investigate the ion exchange-related properties (mutual diffusivity, CS, and hardness) of a sodium aluminosilicate glass, which has been densified through annealing below the initial fictive temperature of the glass or through pressure-quenching from the glass transition temperature at 1 GPa prior to ion exchange. We show that the rate of alkali interdiffusivity depends only on the density of the glass, rather than on the applied densification method. However, we also demonstrate that for a given density, the increase in CS and increase in hardness induced by ion exchange strongly depends on the densification method. Specifically, at constant density, the CS and hardness values achieved through thermal annealing are larger than those achieved through pressure-quenching. These results are discussed in relation to the structural changes in the environment of the network-modifier and the overall network densification

Determining the phyllochron and final leaf pair number in on-farm cut dahlia cultivars

Dahlia is an important ornamental crop and widely used as a garden plant in beds and mixed borders as well as a cut flower in bouquets and flower arrangements. Understanding the factors that support sustainable flower production is essential for dahlia growers to increase their profits. Two key variables that define leaf development during the vegetative phase of a crop are the  rate of appearance of leaves on the main stem and the final number of leaves. The objective in this study was to determine the phyllochron and the final leaf pair number (FLPN) in cut dahlias cultivars grown in different locations. Three on farm experiments varying from two to eight cut dahlia cultivars were conducted during two years (2021/2022) in six locations in Rio Grande do Sul State, Southern Brazil. The number of unfolded leaf pairs (NLP - an unfolded leaf was assumed when the foliolates edges were not touching anymore) on each plant was counted once or twice a week, depending on the farm, until the last leaf pair was unfolded. The NLP was linearly regressed against TT (thermal time °C day-1) and the phyllochron (oC day leaf pair-1) was calculated as the slope of the linear regression. The results indicated that the phyllochron of dahlia cultivars varied between 45.7 to 95.6 °C pair of leaves-1 and 8 to 14 of final number of pair of leaves in the first experiment, 27.2 to 97.4 °C pair of leaves-1 and 6 to 15 of final leaf pair number in the second experiment and 46.8 at 106.4 °C leaf pair-1 and 6 to 13 the final leaf pair number in the third. Thus, there was no significant difference between the sites in the phyllochron and NFP variables

Variation in plant Toll/Interleukin-1 receptor domain protein dependence on ENHANCED DISEASE SUSCEPTIBILITY 1

Toll/Interleukin-1 receptor (TIR) domains are integral to immune systems across all kingdoms. In plants, TIRs are present in nucleotide-binding leucine-rich repeat (NLR) immune receptors, NLR-like, and TIR-only proteins. Although TIR-NLR and TIR signaling in plants require the ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) protein family, TIRs persist in species that have no EDS1 members. To assess whether particular TIR groups evolved with EDS1, we searched for TIR-EDS1 co-occurrence patterns. Using a large-scale phylogenetic analysis of TIR domains from 39 algal and land plant species, we identified 4 TIR families that are shared by several plant orders. One group occurred in TIR-NLRs of eudicots and another in TIR-NLRs across eudicots and magnoliids. Two further groups were more widespread. A conserved TIR-only group co-occurred with EDS1 and members of this group elicit EDS1-dependent cell death. In contrast, a maize (Zea mays) representative of TIR proteins with tetratricopeptide repeats was also present in species without EDS1 and induced EDS1-independent cell death. Our data provide a phylogeny-based plant TIR classification and identify TIRs that appear to have evolved with and are dependent on EDS1, while others have EDS1-independent activity

Fragilities Caused by Dosage Imbalance in Regulation of the Budding Yeast Cell Cycle

Cells can maintain their functions despite fluctuations in intracellular parameters, such as protein activities and gene expression levels. This commonly observed biological property of cells is called robustness. On the other hand, these parameters have different limitations, each reflecting the property of the subsystem containing the parameter. The budding yeast cell cycle is quite fragile upon overexpression of CDC14, but is robust upon overexpression of ESP1. The gene products of both CDC14 and ESP1 are regulated by 1∶1 binding with their inhibitors (Net1 and Pds1), and a mathematical model predicts the extreme fragility of the cell cycle upon overexpression of CDC14 and ESP1 caused by dosage imbalance between these genes. However, it has not been experimentally shown that dosage imbalance causes fragility of the cell cycle. In this study, we measured the quantitative genetic interactions of these genes by performing combinatorial “genetic tug-of-war” experiments. We first showed experimental evidence that dosage imbalance between CDC14 and NET1 causes fragility. We also showed that fragility arising from dosage imbalance between ESP1 and PDS1 is masked by CDH1 and CLB2. The masking function of CLB2 was stabilization of Pds1 by its phosphorylation. We finally modified Chen's model according to our findings. We thus propose that dosage imbalance causes fragility in biological systems

Ybp2 Associates with the Central Kinetochore of Saccharomyces cerevisiae and Mediates Proper Mitotic Progression

The spindle checkpoint ensures the accurate segregation of chromosomes by monitoring the status of kinetochore attachment to microtubules. Simultaneous mutations in one of several kinetochore and cohesion genes and a spindle checkpoint gene cause a synthetic-lethal or synthetic-sick phenotype. A synthetic genetic array (SGA) analysis using a mad2Δ query mutant strain of yeast identified YBP2, a gene whose product shares sequence similarity with the product of YBP1, which is required for H2O2-induced oxidation of the transcription factor Yap1. ybp2Δ was sensitive to benomyl and accumulated at the mitotic stage of the cell cycle. Ybp2 physically associates with proteins of the COMA complex (Ctf19, Okp1, Mcm21, and Ame1) and 3 components of the Ndc80 complex (Ndc80, Nuf2, and Spc25 but not Spc24) in the central kinetochore and with Cse4 (the centromeric histone and CENP-A homolog). Chromatin-immunoprecipitation analyses revealed that Ybp2 associates specifically with CEN DNA. Furthermore, ybp2Δ showed synthetic-sick interactions with mutants of the genes that encode the COMA complex components. Ybp2 seems to be part of a macromolecular kinetochore complex and appears to contribute to the proper associations among the central kinetochore subcomplexes and the kinetochore-specific nucleosome