Truncation of a Protein Disulfide Isomerase,PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance in Arabidopsis thaliana

Pollen tubes must navigate through different female tissues to deliver sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here, we show that certain T-DNA insertions in Arabidopsis thaliana PDIL2-1, a protein disulfide isomerase (PDI), have reduced seed set, due to delays in embryo sac maturation. Reciprocal crosses indicate that these mutations acted sporophytically, and aniline blue staining and scanning electron microscopy showed that funicular and micropylar pollen tube guidance were disrupted. A PDIL2-1-yellow fluorescent protein fusion was mainly localized in the endoplasmic reticulum and was expressed in all tissues examined. In ovules, expression in integument tissues was much higher in the micropylar region in later developmental stages, but there was no expression in embryo sacs. We show that reduced seed set occurred when another copy of full-length PDIL2-1 or when enzymatically active truncated versions were expressed, but not when an enzymatically inactive version was expressed, indicating that these T-DNA insertion lines are gain-of-function mutants. Our results suggest that these truncated versions of PDIL2-1 function in sporophytic tissues to affect ovule structure and impede embryo sac development, thereby disrupting pollen tube guidance.     

Chain collapse of an amyloidogenic intrinsically disordered protein

Natively unfolded or intrinsically disordered proteins (IDPs) are under intense scrutiny due to their involvement in both normal biological functions and abnormal protein misfolding disorders. Polypeptide chain collapse of amyloidogenic IDPs is believed to play a key role in protein misfolding, oligomerization, and aggregation leading to amyloid fibril formation, which is implicated in a number of human diseases. In this work, we used bovine κ-casein, which serves as an archetypal model protein for amyloidogenic IDPs. Using a variety of biophysical tools involving both prediction and spectroscopic techniques, we first established that monomeric κ-casein adopts a collapsed premolten-globule-like conformational ensemble under physiological conditions. Our time-resolved fluorescence and light-scattering data indicate a change in the mean hydrodynamic radius from ∼4.6 nm to ∼1.9 nm upon chain collapse. We then took the advantage of two cysteines separated by 77 amino-acid residues and covalently labeled them using thiol-reactive pyrene maleimide. This dual-labeled protein demonstrated a strong excimer formation upon renaturation from urea- and acid-denatured states under both equilibrium and kinetic conditions, providing compelling evidence of polypeptide chain collapse under physiological conditions. The implication of the IDP chain collapse in protein aggregation and amyloid formation is also discussed.     

The impact of COVID-19 on clinical research for Neglected Tropical Diseases (NTDs): A case study of bubonic plague

BackgroundAmong the many collaterals of the COVID-19 pandemic is the disruption of health services and vital clinical research. COVID-19 has magnified the challenges faced in research and threatens to slow research for urgently needed therapeutics for Neglected Tropical Diseases (NTDs) and diseases affecting the most vulnerable populations. Here we explore the impact of the pandemic on a clinical trial for plague therapeutics and strategies that have been considered to ensure research efforts continue.MethodsTo understand the impact of the COVID-19 pandemic on the trial accrual rate, we documented changes in patterns of all-cause consultations that took place before and during the pandemic at health centres in two districts of the Amoron’I Mania region of Madagascar where the trial is underway. We also considered trends in plague reporting and other external factors that may have contributed to slow recruitment.ResultsDuring the pandemic, we found a 27% decrease in consultations at the referral hospital, compared to an 11% increase at peripheral health centres, as well as an overall drop during the months of lockdown. We also found a nation-wide trend towards reduced number of reported plague cases.DiscussionCOVID-19 outbreaks are unlikely to dissipate in the near future. Declining NTD case numbers recorded during the pandemic period should not be viewed in isolation or taken as a marker of things to come. It is vitally important that researchers are prepared for a rebound in cases and, most importantly, that research continues to avoid NTDs becoming even more neglected.     

Identification of Novel Loci Regulating Interspecific Variation in Root Morphology and Cellular Development in Tomato

While the Arabidopsis (Arabidopsis thaliana) root has been elegantly characterized with respect to specification of cell identity, its development is missing a number of cellular features present in other species. We have characterized the root development of a wild and a domesticated tomato species, Solanum pennellii and Solanum lycopersicum ‘M82.’ We found extensive differences between these species for root morphology and cellular development including root length, a novel gravity set point angle, differences in cortical cell layer patterning, stem cell niche structure, and radial cell division. Using an introgression line population between these two species, we identified numerous loci that regulate these distinct aspects of development. Specifically we comprehensively identified loci that regulate (1) root length by distinct mechanisms including regulation of cell production within the meristem and the balance between cell division and expansion, (2) the gravity set point angle, and (3) radial cell division or expansion either in specific cell types or generally across multiple cell types. Our findings provide a novel perspective on the regulation of root growth and development between species. These loci have exciting implications with respect to regulation of drought resistance or salinity tolerance and regulation of root development in a family that has undergone domestication.The root system is of vital importance to plants because it anchors the plant and its cells absorb and transport water, nutrients, and solutes to the shoot. The root system has a complex branching architecture with numerous cell types whose development must be dynamic, plastic, and highly responsive to the environment to maximize plant fitness and yield. To optimize root system architecture for the specific environment in which the plant is growing, developmental programs associated with distinct developmental stages, and cell types are specifically and precisely regulated by both local and global signals. For instance, local nitrogen sources induce root hair tip growth and can regulate lateral root initiation (Malamy and Ryan, 2001; Bloch et al., 2011) while the search for water regulates primary root growth (Saucedo et al., 2012). This complex architecture and plasticity complicate the ability to enumerate and study root architecture concomitantly at all cellular and tissue levels. Because of this limitation, population-level studies are typically limited to measuring architecture-level variables such as root length, number, and branching, without focusing on the development of specific cell types that give rise to this architecture.Root cell type specification and development has been extensively studied using classical genetic methods in the model plant Arabidopsis (Arabidopsis thaliana). These studies revealed the elegant simplicity of the Arabidopsis root at the cellular level (Dolan et al., 1993). In Arabidopsis there is an invariant number of cells within the single cortical and endodermal layers of the primary root but variable cell numbers within lateral roots. The core of the root stem cell niche is formed by a set of four quiescent center (QC) cells, with a set of initial cells that give rise to all cell types in the root surrounding the QC. Developmental genetic studies in Arabidopsis have identified a variety of genes that regulate root length, lateral root number, and radial patterning (Benfey and Scheres, 2000; Mähönen et al., 2000; Schiefelbein et al., 2009). This includes the identification of genes that regulate vascular cell proliferation, endodermis and epidermis cell identity, and the asymmetric division of the cortex-endodermis initial (CEI).Arabidopsis has provided an excellent base model for root cellular development, yet as with any species there are unique cellular aspects that are present and/or missing within Arabidopsis that necessitate the study of other species. For instance, Arabidopsis is unusual as it contains only four QC cells, whereas most monocot and dicot species contain a greater number of QC cells (Jiang et al., 2003). To date, the regulatory mechanisms controlling this diversity in QC cell number are completely unknown. Additionally, most monocot and dicot species contain numerous cortex layers that are the product of repeated divisions of a CEI cell, whereas Arabidopsis only contains a single cortex layer (Dolan et al., 1993). The cell number in the cortex and the endodermis is invariant in the Arabidopsis primary root, but variable in many other plant species. Regulation of radial cell number variability in these cell types as well as the pericycle has never been addressed in any plant species. Furthermore, in 80% of flowering plant species, the outer layer of the root’s cortex, or exodermis, contains a suberinized cell wall to restrict passage of solutes from the outside of the root to the inside, but Arabidopsis does not contain a suberinized exodermis. The exodermis has been reported to be derived from an independent cortical initial, suggesting it is an independent specialized cell type whose genetics are not addressable within Arabidopsis (Heimsch and Seago, 2008). Genes regulating the specification of the exodermis and the production of multiple cortical layers have not been identified in monocots or dicots. Thus, classical genetic approaches have not addressed the genetic mechanisms regulating cell proliferation and patterning decisions within many cell types not present in Arabidopsis.One approach with significant potential to identify these unresolved genetic mechanisms and integrate them into the broader control of root system architecture is the use of natural variation within and between species (Shindo et al., 2007). The use of stable mapping populations such as a homozygous introgression line (IL) between two different species provides a stable genetic pool from which to repeatedly phenotype different cellular and morphological aspects of root architecture and integrate them into a common model. This quantitative genetic analysis is typically conducted using quantitative trait locus (QTL) mapping, which has identified loci or, in a small number of cases, genes that regulate root length in monocots and dicots (Bettey et al., 2000; Mouchel et al., 2004; Loudet et al., 2005; Fitz Gerald et al., 2006; Reymond et al., 2006; Fita et al., 2008; Khan et al., 2012). These studies however have typically been limited to the analysis of large-effect loci (Loudet et al., 2005; Reymond et al., 2006) and have not coordinately dissected root architecture at both the morphological and cellular levels.To determine how tomato (Solanum spp.) root morphogenesis is determined by cellular features including radial patterning, radial cell proliferation, radial cell expansion, and compensatory changes in cell expansion when cell proliferation is altered (Hemerly et al., 1995), we performed a detailed characterization of root development in two Solanum species. We used Solanum pennellii, a wild tomato species, and cv M82 of the domesticated species Solanum lycopersicum and their derived IL population. A wild species, S. pennellii is found in coastal deserts and rocky, arid soil and exhibits drought and salt tolerance and pathogen resistance in comparison with the domesticated cv M82 (Dehan and Tal, 1978; Koca et al., 2006; Easlon and Richards, 2009). In this study, we identified significant developmental differences between the two species by measuring a large range of root traits including the cell number within individual cell types, CEI spatiotemporal patterning differences, variability in cortex cell layer and QC cell number, root growth, and a novel gravity set point angle. To explore the link between the whole organ phenotype and cellular level using interspecific genetic variation we used the IL population derived from a cross between cv M82 and S. pennellii (Eshed and Zamir, 1995). This population comprises 76 segmental ILs with marker-defined genomic regions of S. pennellii substituting for homologous intervals of the cultivated variety cv M82 that partition the tomato genome into 107 bins. Measuring the above cellular and morphological phenotypes in these lines identified numerous major- and minor-effect loci for each phenotype, showing that interspecific variation in root development involves a complex suite of genetic changes, many of which display cell type-specific effects.     

Quantification of Campylobacter spp. in pig feces by direct real-time PCR with an internal control of extraction and amplification

The rapid and direct quantification of Campylobacter spp. in complex substrates like feces or environmental samples is crucial to facilitate epidemiological studies on Campylobacter in pig production systems. We developed a real-time PCR assay for detecting and quantifying Campylobacter spp. directly in pig feces with the use of an internal control. Campylobacter spp. and Yersinia ruckeri primers-probes sets were designed and checked for specificity with diverse Campylobacter, related organisms, and other bacterial pathogens before being used in field samples. The quantification of Campylobacter spp. by the real-time PCR then was realized on 531 fecal samples obtained from experimentally and naturally infected pigs; the numeration of Campylobacter on Karmali plate was done in parallel. Yersinia ruckeri, used as bacterial internal control, was added to the samples before DNA extraction to control DNA-extraction and PCR-amplification. The sensitivity of the PCR assay was 10 genome copies. The established Campylobacter real-time PCR assay showed a 7-log-wide linear dynamic range of quantification (R² = 0.99) with a detection limit of 200 Colony Forming Units of Campylobacter per gram of feces. A high correlation was found between the results obtained by real-time PCR and those by culture at both qualitative and quantitative levels. Moreover, DNA extraction followed by real-time PCR reduced the time needed for analysis to a few hours (within a working day). In conclusion, the real-time PCR developed in this study provides new tools for further epidemiological surveys to investigate the carriage and excretion of Campylobacter by pigs.     

Arabidopsis thaliana GEX1 has dual functions in gametophyte development and early embryogenesis

GEX1 is a plasma membrane protein that is conserved among plant species, and has previously been shown to be expressed in sperm cells and some sporophytic tissues. Here we show that GEX1 is also expressed in the embryo sac before cellularization, in the egg cell after cellularization, in the zygote/embryo immediately after fertilization and in the pollen vegetative cell. We functionally characterize GEX1 in Arabidopsis thaliana, and show that it is a versatile protein that performs functions during male and female gametophyte development, and during early embryogenesis. gex1-1/+ plants, which synthesize a truncated GEX1 mRNA encoding a protein lacking the predicted cytoplasmic domain, but still targeted to the plasma membrane, had embryos that arrested before the pre-globular stage. gex1-3/+ plants, carrying a null GEX1 allele, had defects during male and female gametophyte development, and during early embryogenesis. Using an antisense GEX1 transgenic line we demonstrate that the predicted GEX1 extracellular domain is sufficient and necessary for GEX1 function during the development of both gametophytes. The predicted cytoplasmic domain is necessary for correct early embryogenesis and mediates homodimer formation at the plasma membrane. We propose that dimerization of GEX1 in the zygote might be an upstream step in a signaling cascade regulating early embryogenesis.