Nodulation Compatibility and Symbiotic Performance of Rhizobia spp. With Different Landraces of Bambara Groundnut (Vigna Subterranea (L.) Verdc.) Collections

The symbiosis of the legume bambara groundnut (Vigna subterranean L.Verde) with its rhizobial partners has not been studied sufficiently compared to several other legumes throughout Africa. In this study, a nodulation compatibility screening was conducted on 16 different landraces of this legume using five Rhizobia strains previously isolated from active nodules of Desmodium uncinatum, Arachis hypogaea, Cyamopsis tetragonoloba, Glycine max and Phaseolus vulgaris and deposited at the South African Rhizobium Culture Collection (SARCC). A screening assay was conducted under glasshouse to select compatible rhizobia strains that nodulate and enhance growth in one or more genotypes of V. subterranean (L.) Verdc. Pre-germinated seeds of each landraces planted in sterile river sand medium were inoculated with 108 cfu ml-1 of the rhizobial strains (2ml/seed) and monitored with regular watering for six weeks. Parameters such as nodule number, nodule color and positions, plant biomass were determined in test genotypes. Significant differences were observed among landraces in nodule number and plant biomass, and among rhizobial strains in nodule number. Principal component analysis (PCA) showed that root nodule rhizobia strains SARCC-388 and SARCC-578 characterized as Bradyrhizobium zhangiangens and Bradyrhizobium centrosematis, respectively exhibited the highest nodulation compatibility with one or more bambara groundnut landraces. This study demonstrated that many of the bambara landraces did not show nodulation preference to a unique group of rhizobia, confirming that V. subterranean (L) Verdc can be nodulated by more than one species of rhizobia, especially by rhizobia belonging to the cowpea miscellany cross inoculation group.

Quantitative Trait Loci Mapping and Development of KASP Marker Smut Screening Assay Using High-Density Genetic Map and Bulked Segregant RNA Sequencing in Sugarcane (Saccharum spp.)

Sugarcane is one of the most important industrial crops globally. It is the second largest source of bioethanol, and a major crop for biomass-derived electricity and sugar worldwide. Smut, caused by Sporisorium scitamineum, is a major sugarcane disease in many countries, and is managed by smut-resistant varieties. In China, smut remains the single largest constraint for sugarcane production, and consequently it impacts the value of sugarcane as an energy feedstock. Quantitative trait loci (QTLs) associated with smut resistance and linked diagnostic markers are valuable tools for smut resistance breeding. Here, we developed an F1 population (192 progeny) by crossing two sugarcane varieties with contrasting smut resistance and used for genome-wide single nucleotide polymorphism (SNP) discovery and mapping, using a high-throughput genotyping method called “specific locus amplified fragment sequencing (SLAF-seq) and bulked-segregant RNA sequencing (BSR-seq). SLAF-seq generated 148,500 polymorphic SNP markers. Using SNP and previously identified SSR markers, an integrated genetic map with an average 1.96 cM marker interval was produced. With this genetic map and smut resistance scores of the F1 individuals from four crop years, 21 major QTLs were mapped, with a phenotypic variance explanation (PVE) > 8.0%. Among them, 10 QTLs were stable (repeatable) with PVEs ranging from 8.0 to 81.7%. Further, four QTLs were detected based on BSR-seq analysis. aligning major QTLs with the genome of a sugarcane progenitor Saccharum spontaneum, six markers were found co-localized. Markers located in QTLs and functional annotation of BSR-seq-derived unigenes helped identify four disease resistance candidate genes located in major QTLs. 77 SNPs from major QTLs were then converted to Kompetitive Allele-Specific PCR (KASP) markers, of which five were highly significantly linked to smut resistance. The co-localized QTLs, candidate resistance genes, and KASP markers identified in this study provide practically useful tools for marker-assisted sugarcane smut resistance breeding.

3-oxo-C12:2-HSL, Quorum Sensing Molecule from Human Intestinal Microbiota, Inhibits Pro-inflammatory Pathways in Immune Cells via Bitter Taste Receptors

In the gut ecosystem, microorganisms regulate group behaviour and interplay with the host via a molecular system called quorum sensing (QS). The QS molecule 3-oxo-C12:2-HSL, first identified in human gut microbiota, exerts anti-inflammatory effects and could play a role in inflammatory bowel diseases where dysbiosis has been described. Our aim was to identify which signalling pathways are involved in this effect. We observed that 3-oxo-C12:2-HSL decreases expression of pro-inflammatory cytokines such as, Interleukine-1β (-3 %) and Tumor Necrosis Factor-α (TNFα) (40 %) by stimulated immune RAW264.7 cells and decreased TNF secretion by stimulated PBMC in a dose-dependent manner, between 25 µM to 100 µM. Transcriptomic analysis of RAW264.7 cells exposed to 3-oxo-C12:2-HSL, in a pro-inflammatory context, highlighted JAK-STAT, NF-κB and TFN signalling pathways and we confirmed that 3-oxo-C12:2-HSL inhibited JAK1 and STAT1 phosphorylation. We also showed through a screening assay that 3-oxo-C12:2-HSL interacted with several human bitter taste receptors. Its anti-inflammatory effect involved TAS2R38 as shown by pharmacologic inhibition and led to an increase in intracellular calcium levels. We thus unravelled the involvement of several cellular pathways in the anti-inflammatory effects exerted by the QS molecule 3-oxo-C12:2-HSL.

Transcription Factor CitERF16 Is Involved in Citrus Fruit Sucrose Accumulation by Activating CitSWEET11d

Sugars are the primary products of photosynthesis and play an important role in plant growth and development. They contribute to sweetness and flavor of fleshy fruits and are pivotal to fruit quality, and their translocation and allocation are mainly dependent on sugar transporters. Genome-wide characterization of Satsuma mandarin identified eighteen SWEET family members that encode transporters which facilitate diffusion of sugar across cell membranes. Analysis of the expression profiles in tissues of mandarin fruit at different developmental stages showed that CitSWEET11d transcripts were significantly correlated with sucrose accumulation. Further studies indicated that overexpression of CitSWEET11d in citrus callus and tomato fruit showed a higher sucrose level compared to wild-type, suggesting that CitSWEET11d could enhance sucrose accumulation. In addition, we identified an ERF transcription factor CitERF16 by yeast one-hybrid screening assay which could directly bind to the DRE cis-element on the promoter of CitSWEET11d. Overexpression of CitERF16 in citrus callus significantly induced CitSWEET11d expression and elevated sucrose content, suggesting that CitERF16 acts as a positive regulator to promote sucrose accumulation via trans-activation of CitSWEET11d expression.

Constitutive, Basal, and β-Alanine-Mediated Activation of the Human Mas-Related G Protein-Coupled Receptor D Induces Release of the Inflammatory Cytokine IL-6 and Is Dependent on NF-κB Signaling

G protein-coupled receptors (GPCRs) have emerged as key players in regulating (patho)physiological processes, including inflammation. Members of the Mas-related G protein coupled receptors (MRGPRs), a subfamily of GPCRs, are largely expressed by sensory neurons and known to modulate itch and pain. Several members of MRGPRs are also expressed in mast cells, macrophages, and in cardiovascular tissue, linking them to pseudo-allergic drug reactions and suggesting a pivotal role in the cardiovascular system. However, involvement of the human Mas-related G-protein coupled receptor D (MRGPRD) in the regulation of the inflammatory mediator interleukin 6 (IL-6) has not been demonstrated to date. By stimulating human MRGPRD-expressing HeLa cells with the agonist β-alanine, we observed a release of IL-6. β-alanine-induced signaling through MRGPRD was investigated further by probing downstream signaling effectors along the Gαq/Phospholipase C (PLC) pathway, which results in an IkB kinases (IKK)-mediated canonical activation of nuclear factor kappa-B (NF-κB) and stimulation of IL-6 release. This IL-6 release could be blocked by a Gαq inhibitor (YM-254890), an IKK complex inhibitor (IKK-16), and partly by a PLC inhibitor (U-73122). Additionally, we investigated the constitutive (ligand-independent) and basal activity of MRGPRD and concluded that the observed basal activity of MRGPRD is dependent on the presence of fetal bovine serum (FBS) in the culture medium. Consequently, the dynamic range for IL-6 detection as an assay for β-alanine-mediated activation of MRGPRD is substantially increased by culturing the cells in FBS free medium before treatment. Overall, the observation that MRGPRD mediates the release of IL-6 in an in vitro system, hints at a role as an inflammatory mediator and supports the notion that IL-6 can be used as a marker for MRGPRD activation in an in vitro drug screening assay.

The Effects of Environmental Contaminants on Freshwater Cyanobacteria

<p>Cyanobacteria are photosynthetic prokaryotes with a cosmopolitan distribution and are key contributors to Earth’s primary production. In favourable conditions, some taxa have the ability to form dense algal blooms. When blooms are created by toxin-producing strains they can become a public health threat and may lead to deaths of wild and domestic animals. Different species and strains respond differently to environmental conditions and contaminants, and this thesis investigates the impacts of natural (microcystin, a hepatotoxic cyanotoxin) and anthropogenic (glyphosate) contaminants on cyanobacteria. Some non-microcystin-producing cyanobacteria have the ability to sequester microcystin, one of them being the strain Cuspidothrix issatschenkoi CAWBG02, and the reason for this ability is unknown. This strain was supplemented with microcystin in a range of environmental conditions (temperature/light intensity combinations) to determine any impacts on fitness by measuring photosynthetic performance, growth, and pigment content (chlorophyll-a and phycocyanin). Microcystin supplementation at a concentration of 25 μg L-1 did not have an effect on growth or pigment content in C. issatschenkoi, and photosynthetic performance was also largely unaffected. Based on the results from this thesis, C. issatschenkoi does not acquire competitive advantages from microcystin sequestration due to the lack of effects on growth and photosynthetic functioning. Previous research has suggested that microcystin is used to suppress competitors, and a 33% decrease in maximum quantum yield was detected in one environmental condition (low temperature, intermediate light), however, there was no significant difference in all of the other conditions used. Therefore, the results from this study do not support the idea that microcystin is used to suppress competitors. Microcystin sequestration may be more beneficial to non-producing-strains within the same species as the toxin-producer (e.g. non-toxic M. aeruginosa). Glyphosate and MCPA are two of the most commonly used herbicides in New Zealand and globally. These herbicides enter waterways through leaching and come into contact with non- target species. To determine whether these herbicides have an effect on freshwater cyanobacteria in New Zealand, a screening assay was carried out on fourteen strains. Further, the impacts on pigment content (chlorophyll-a and phycocyanin), growth, and photosynthetic performance in two toxic (Nodularia spumigena, Microcystis aeruginosa) and two non-toxic (Dolichospermum lemmermannii, Microcystis wesenbergii) strains were quantified. Growth rates were not impacted by MCPA in any of the fourteen cyanobacterial strains. Glyphosate impacted growth in seven of the fourteen strains, and response differed between strains even within a single species. In general, strains employing a colonial growth strategy were more tolerant to glyphosate in the screening assay. N. spumigena and M. wesenbergii were unaffected by glyphosate at the concentrations used in the functional analysis, and D. lemmermannii had decreased photosynthetic performance from glyphosate. Intermediate concentrations of glyphosate resulted in promotion of PSII functioning in M. aeruginosa, and high concentrations inhibited maximum quantum yield. The lack of significant impacts on M.wesenbergii and N. spumigena, as well as negative effects of PSII functioning in D. lemmermannii, suggest that glyphosate contamination in freshwaters may benefit toxic M. aeruginosa more than competing toxic and non-toxic species or strains.</p>

The Effects of Environmental Contaminants on Freshwater Cyanobacteria

<p>Cyanobacteria are photosynthetic prokaryotes with a cosmopolitan distribution and are key contributors to Earth’s primary production. In favourable conditions, some taxa have the ability to form dense algal blooms. When blooms are created by toxin-producing strains they can become a public health threat and may lead to deaths of wild and domestic animals. Different species and strains respond differently to environmental conditions and contaminants, and this thesis investigates the impacts of natural (microcystin, a hepatotoxic cyanotoxin) and anthropogenic (glyphosate) contaminants on cyanobacteria. Some non-microcystin-producing cyanobacteria have the ability to sequester microcystin, one of them being the strain Cuspidothrix issatschenkoi CAWBG02, and the reason for this ability is unknown. This strain was supplemented with microcystin in a range of environmental conditions (temperature/light intensity combinations) to determine any impacts on fitness by measuring photosynthetic performance, growth, and pigment content (chlorophyll-a and phycocyanin). Microcystin supplementation at a concentration of 25 μg L-1 did not have an effect on growth or pigment content in C. issatschenkoi, and photosynthetic performance was also largely unaffected. Based on the results from this thesis, C. issatschenkoi does not acquire competitive advantages from microcystin sequestration due to the lack of effects on growth and photosynthetic functioning. Previous research has suggested that microcystin is used to suppress competitors, and a 33% decrease in maximum quantum yield was detected in one environmental condition (low temperature, intermediate light), however, there was no significant difference in all of the other conditions used. Therefore, the results from this study do not support the idea that microcystin is used to suppress competitors. Microcystin sequestration may be more beneficial to non-producing-strains within the same species as the toxin-producer (e.g. non-toxic M. aeruginosa). Glyphosate and MCPA are two of the most commonly used herbicides in New Zealand and globally. These herbicides enter waterways through leaching and come into contact with non- target species. To determine whether these herbicides have an effect on freshwater cyanobacteria in New Zealand, a screening assay was carried out on fourteen strains. Further, the impacts on pigment content (chlorophyll-a and phycocyanin), growth, and photosynthetic performance in two toxic (Nodularia spumigena, Microcystis aeruginosa) and two non-toxic (Dolichospermum lemmermannii, Microcystis wesenbergii) strains were quantified. Growth rates were not impacted by MCPA in any of the fourteen cyanobacterial strains. Glyphosate impacted growth in seven of the fourteen strains, and response differed between strains even within a single species. In general, strains employing a colonial growth strategy were more tolerant to glyphosate in the screening assay. N. spumigena and M. wesenbergii were unaffected by glyphosate at the concentrations used in the functional analysis, and D. lemmermannii had decreased photosynthetic performance from glyphosate. Intermediate concentrations of glyphosate resulted in promotion of PSII functioning in M. aeruginosa, and high concentrations inhibited maximum quantum yield. The lack of significant impacts on M.wesenbergii and N. spumigena, as well as negative effects of PSII functioning in D. lemmermannii, suggest that glyphosate contamination in freshwaters may benefit toxic M. aeruginosa more than competing toxic and non-toxic species or strains.</p>