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BioRxiv : the Preprint Server For... Jul 2023Over the past three decades, researchers have isolated plant mutants that display constitutively activated defense responses in the absence of pathogen infection. These...
Over the past three decades, researchers have isolated plant mutants that display constitutively activated defense responses in the absence of pathogen infection. These mutants are called autoimmune mutants and are typically dwarf and/or bearing chlorotic/necrotic lesions. From a genetic screen for genes involved in maintaining a normal leaf microbiota, we identified (), which encodes a S-acyltransferase, as a key player in guarding leaves against abnormal microbiota level and composition under high humidity conditions. The mutant has several characteristic phenotypes of classical autoimmune mutants, including a dwarf stature, displaying lesions, and having a high basal level of defense gene expression. Gnotobiotic experiments revealed that the autoimmune phenotypes of the mutant are largely dependent on the presence of microbiota as axenic plants have markedly reduced autoimmune phenotypes. We found that the microbiota dependency of autoimmune phenotypes is shared by several "lesion mimic"-type autoimmune mutants in . Interestingly, autoimmune phenotypes caused by mutations in genes do not require the presence of microbiota and can even be partially alleviated by microbiota. Our results therefore suggest the existence of two classes of autoimmunity (microbiota-dependent vs. microbiota-independent) in plants. The observed interplay between autoimmunity and microbiota in the lesion mimic class of autoimmunity is reminiscent of the interactions between autoimmunity and dysbiosis in the animal kingdom.
PubMed: 36945461
DOI: 10.1101/2023.03.06.531303 -
Plants (Basel, Switzerland) Oct 2023Flavonoids are antioxidant phytochemicals that confer a beneficial effect on human health. We have previously developed and characterized eight lettuce ( L.) lines that...
Flavonoids are antioxidant phytochemicals that confer a beneficial effect on human health. We have previously developed and characterized eight lettuce ( L.) lines that accumulated high levels of diverse flavonoids and their precursors in controlled environment conditions. Three Rutgers Scarlet lettuce (RSL) lines selected in tissue culture for deep-red color (RSL-NAR, RSL-NBR, RSL-NFR) accumulate anthocyanins and quercetin, three lines identified in a chemically mutagenized red lettuce population accumulate kaempferol (KfoA and KfoB) or naringenin chalcone (Nco), and two lines that were spontaneous green mutants derived from the red line RSL-NAR (GSL, GSL-DG) accumulate quercetin. These eight lines were field-grown in the Salinas Valley of California for four years together with seven control accessions of varying colors (light green, dark green, red, and dark red). At market maturity, a substantial variation in plant composition was observed, but the three RSL lines consistently accumulated high levels of cyanidin, GSL and GSL-DG accumulated the highest levels of quercetin, KfoA and KfoB accumulated kaempferol, and Nco amassed naringenin chalcone, confirming that these mutant lines produce high levels of beneficial phytochemicals under field conditions. Mutant lines and control accessions were also assessed for their biomass production (plant weight, height, and width), overall content of pigments (leaf chlorophyll and anthocyanins), resistance to diseases (downy mildew, lettuce drop, and Impatiens necrotic spot virus), postharvest quality of processed tissue (deterioration and enzymatic discoloration), and composition of 23 mineral elements. All but one mutant line had a fresh plant weight at harvest comparable to commercial leaf cultivars; only Nco plants were significantly ( < 0.05) smaller. Therefore, except for Nco, the new, flavonoid hyperaccumulating lines can be considered for field cultivation.
PubMed: 37836207
DOI: 10.3390/plants12193467 -
ACS Pharmacology & Translational Science Jul 2023Mutations in connexin 26 (Cx26) cause hearing disorders of a varying degree. Herein, to identify compounds capable of restoring the function of mutated Cx26, a novel...
Mutations in connexin 26 (Cx26) cause hearing disorders of a varying degree. Herein, to identify compounds capable of restoring the function of mutated Cx26, a novel miniaturized microarray-based screening system was developed to perform an optical assay of Cx26 functionality. These molecules were identified through a viability assay using HeLa cells expressing wild-type (WT) Cx26, which exhibited sensitivity toward the HSP90 inhibitor radicicol in the submicromolar concentration range. Open Cx26 hemichannels are assumed to mediate the passage of molecules up to 1000 Da in size. Thus, by releasing radicicol, WT Cx26 active hemichannels in HeLa cells contribute to a higher survival rate and lower cell viability when Cx26 is mutated. HeLa cells expressing Cx26 mutations exhibited reduced viability in the presence of radicicol, such as the mutants F161S or R184P. Next, molecules exhibiting chemical chaperoning activity, suspected of restoring channel function, were assessed regarding whether they induced superior sensitivity toward radicicol and increased HeLa cell viability. Through a viability assay and microarray-based flux assay that uses Lucifer yellow in HeLa cells, compounds and were identified to restore mutant functionality. Furthermore, thermophoresis experiments revealed that only (VRT-534) exhibited dose-responsive binding to recombinant WT Cx26 and mutant Cx26K188N with half maximal effective concentration values of 19 and ∼5 μM, respectively. The findings of this study reveal that repurposing compounds already being used to treat other diseases, such as cystic fibrosis, in combination with functional bioassays and binding tests can help identify novel potential candidates that can be used to treat hearing disorders.
PubMed: 37470015
DOI: 10.1021/acsptsci.3c00056 -
Bio-protocol Aug 2023Pectin is a complex polysaccharide present in the plant cell wall, whose composition is constantly remodelled to adapt to environmental or developmental changes. Mutants...
Pectin is a complex polysaccharide present in the plant cell wall, whose composition is constantly remodelled to adapt to environmental or developmental changes. Mutants with altered pectin composition have been reported to exhibit altered stress or pathogen resistance. Understanding the link between mutant phenotypes and their pectin composition requires robust analytical methods to detect changes in the relative monosaccharide composition. Here, we describe a quick and efficient gas chromatography-mass spectrometry (GC-MS)-based method that allows the differential analysis of pectin monosaccharide composition in plants under different conditions or between mutant plants and their respective wild types. Pectin is extracted from seed mucilage or from the alcohol-insoluble residue prepared from leaves or other organs and is subsequently hydrolysed with trifluoracetic acid. The resulting acidic and neutral monosaccharides are then derivatised and measured simultaneously by GC-MS. Key features Comparative analysis of monosaccharide content in -derived pectin between different genotypes or different treatments. Procedures for two sources of pectin are shown: seed coat mucilage and alcohol-insoluble residue. Allows quick analyses of neutral and acidic monosaccharides simultaneously. Graphical overview.
PubMed: 37638300
DOI: 10.21769/BioProtoc.4746 -
Nucleic Acids Research Oct 2023Gene expression is a multistep process and crosstalk among regulatory layers plays an important role in coordinating gene expression. To identify functionally relevant...
Gene expression is a multistep process and crosstalk among regulatory layers plays an important role in coordinating gene expression. To identify functionally relevant gene expression coordination, we performed a systematic reverse-genetic interaction screen in C. elegans, combining RNA binding protein (RBP) and transcription factor (TF) mutants to generate over 100 RBP;TF double mutants. We identified many unexpected double mutant phenotypes, including two strong genetic interactions between the ALS-related RBPs, fust-1 and tdp-1, and the homeodomain TF ceh-14. Losing any one of these genes alone has no effect on the health of the organism. However, fust-1;ceh-14 and tdp-1;ceh-14 double mutants both exhibit strong temperature-sensitive fertility defects. Both double mutants exhibit defects in gonad morphology, sperm function, and oocyte function. RNA-Seq analysis of double mutants identifies ceh-14 as the main controller of transcript levels, while fust-1 and tdp-1 control splicing through a shared role in exon inhibition. A skipped exon in the polyglutamine-repeat protein pqn-41 is aberrantly included in tdp-1 mutants, and genetically forcing this exon to be skipped in tdp-1;ceh-14 double mutants rescues their fertility. Together our findings identify a novel shared physiological role for fust-1 and tdp-1 in promoting C. elegans fertility and a shared molecular role in exon inhibition.
PubMed: 37587694
DOI: 10.1093/nar/gkad665 -
International Journal of Molecular... Oct 2023The emergence of phage-resistant bacterial strains is one of the biggest challenges for phage therapy. However, the emerging phage-resistant bacteria are often...
The emergence of phage-resistant bacterial strains is one of the biggest challenges for phage therapy. However, the emerging phage-resistant bacteria are often accompanied by adaptive trade-offs, which supports a therapeutic strategy called "phage steering". The key to phage steering is to guide the bacterial population toward an evolutionary direction that is favorable for treatment. Thus, it is important to systematically investigate the impacts of phages targeting different bacterial receptors on the fitness of the bacterial population. Herein, we employed 20 different phages to impose strong evolutionary pressure on the host PAO1 and examined the genetic and phenotypic responses of their phage-resistant mutants. Among these strains with impaired adsorptions, four types of mutations associated with bacterial receptors were identified, namely, lipopolysaccharides (LPSs), type IV pili (T4Ps), outer membrane proteins (OMPs), and exopolysaccharides (EPSs). PAO1, responding to LPS- and EPS-dependent phage infections, mostly showed significant growth impairment and virulence attenuation. Most mutants with T4P-related mutations exhibited a significant decrease in motility and biofilm formation ability, while the mutants with OMP-related mutations required the lowest fitness cost out of the bacterial populations. Apart from fitness costs, PAO1 strains might lose their resistance to antibiotics when counteracting with phages, such as the presence of large-fragment mutants in this study, which may inspire the usage of phage-antibiotic combination strategies. This work provides methods that leverage the merits of phage resistance relative to obtaining therapeutically beneficial outcomes with respect to phage-steering strategies.
Topics: Bacteriophages; Virulence; Lipopolysaccharides; Biological Evolution; Anti-Bacterial Agents; Pseudomonas aeruginosa
PubMed: 37958578
DOI: 10.3390/ijms242115594 -
Frontiers in Plant Science 2023Cuticular wax is a characteristic feature of land plants that provides protection against both biotic and abiotic stresses. In this study, a glossy mutant lacking an...
Cuticular wax is a characteristic feature of land plants that provides protection against both biotic and abiotic stresses. In this study, a glossy mutant lacking an epicuticular wax layer was identified in the γ-irradiated M mutant population of the onion cultivar Bhima Super. The inheritance of the mutant's glossy phenotype was determined to be recessive and single locus. Scanning electron microscopy analysis showed poor accumulation of wax crystals in the glossy mutant, concentrated near the stomata. The plant height, number of leaves per plant, and stomatal parameters of the mutant were similar to the wild-type. RNA-seq was used to comprehend the expression variations of waxy cuticle-related genes in the glossy mutant and its wild-type waxy cultivars. Differential gene expression analysis of the RNA-seq data revealed that the genes involved in wax biosynthesis, such as , and , were downregulated by 2.72, 1.74, 2.59 and 2.12-fold, respectively, in the glossy mutant respectively. The expression patterns of these four unigenes were validated using semi-quantitative RT-PCR. The glossy mutant displayed a substantial 3.5-fold reduction in cuticular wax load compared to the wild-type due to the significant downregulation of these wax biosynthesis genes. These findings represent early advancements in understanding the molecular mechanisms of wax biosynthesis in onions. Furthermore, they provide a foundation for utilizing the glossy mutant trait in breeding programmes to enhance stress and pest resilience.
PubMed: 37680361
DOI: 10.3389/fpls.2023.1245308 -
Proceedings of the National Academy of... Nov 2023Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies...
Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies have shown that phosphoregulation of PIN1 by AGC kinases including PID directs auxin flux to drive organ initiation. Here, we report unexpected findings on the genetic interactions between these two genes. We deleted the first 2/3 of the coding sequence using CRISPR/Cas9, and the resulting mutant () was a strong allele. Surprisingly, heterozygous suppressed two independent null mutants, whereas homozygous enhanced the phenotypes of the mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous mutants were also capable of rescuing nulls. Moreover, we inserted green fluorescent protein (GFP) into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the line are similar to those in the previously reported transgenic lines. Interestingly, the line also rescued various null mutant alleles in a semidominant fashion. We conclude that decreasing the number of functional copies is sufficient to suppress the mutant phenotype, suggesting that PIN1 is likely part of a larger protein complex required for organogenesis.
Topics: Arabidopsis Proteins; Arabidopsis; Protein Serine-Threonine Kinases; Green Fluorescent Proteins; Indoleacetic Acids; Mutation; Phenotype; Gene Expression Regulation, Plant; Membrane Transport Proteins
PubMed: 37983505
DOI: 10.1073/pnas.2312918120 -
Nature Communications Feb 2024Mutations in rhodopsin can cause it to misfold and lead to retinal degeneration. A distinguishing feature of these mutants in vitro is that they mislocalize and...
Mutations in rhodopsin can cause it to misfold and lead to retinal degeneration. A distinguishing feature of these mutants in vitro is that they mislocalize and aggregate. It is unclear whether or not these features contribute to retinal degeneration observed in vivo. The effect of P23H and G188R misfolding mutations were examined in a heterologous expression system and knockin mouse models, including a mouse model generated here expressing the G188R rhodopsin mutant. In vitro characterizations demonstrate that both mutants aggregate, with the G188R mutant exhibiting a more severe aggregation profile compared to the P23H mutant. The potential for rhodopsin mutants to aggregate in vivo was assessed by PROTEOSTAT, a dye that labels aggregated proteins. Both mutants mislocalize in photoreceptor cells and PROTEOSTAT staining was detected surrounding the nuclei of photoreceptor cells. The G188R mutant promotes a more severe retinal degeneration phenotype and greater PROTEOSTAT staining compared to that promoted by the P23H mutant. Here, we show that the level of PROTEOSTAT positive cells mirrors the progression and level of photoreceptor cell death, which suggests a potential role for rhodopsin aggregation in retinal degeneration.
Topics: Animals; Mice; Disease Models, Animal; Mutation; Photoreceptor Cells; Retinal Degeneration; Retinitis Pigmentosa; Rhodopsin; Protein Aggregates
PubMed: 38365903
DOI: 10.1038/s41467-024-45748-4 -
Neurobiology of Disease Jul 2023PRRT2 is a neuronal protein that controls neuronal excitability and network stability by modulating voltage-gated Na channel (Nav). PRRT2 pathogenic variants cause...
PRRT2 is a neuronal protein that controls neuronal excitability and network stability by modulating voltage-gated Na channel (Nav). PRRT2 pathogenic variants cause pleiotropic syndromes including epilepsy, paroxysmal kinesigenic dyskinesia and episodic ataxia attributable to loss-of-function pathogenetic mechanism. Based on the evidence that the transmembrane domain of PRRT2 interacts with Nav1.2/1.6, we focused on eight missense mutations located within the domain that show expression and membrane localization similar to the wild-type protein. Molecular dynamics simulations showed that the mutants do not alter the structural stability of the PRRT2 membrane domain and preserve its conformation. Using affinity assays, we found that the A320V and V286M mutants displayed respectively decreased and increased binding to Nav1.2. Accordingly, surface biotinylation showed an increased Nav1.2 surface exposure induced by the A320V mutant. Electrophysiological analysis confirmed the lack of modulation of Nav1.2 biophysical properties by the A320V mutant with a loss-of-function phenotype, while the V286M mutant displayed a gain-of-function with respect to wild-type PRRT2 with a more pronounced left-shift of the inactivation kinetics and delayed recovery from inactivation. The data confirm the key role played by the PRRT2-Nav interaction in the pathogenesis of the PRRT2-linked disorders and suggest an involvement of the A320 and V286 residues in the interaction site. Given the similar clinical phenotype caused by the two mutations, we speculate that circuit instability and paroxysmal manifestations may arise when PRRT2 function is outside the physiological range.
Topics: Mutation, Missense; NAV1.2 Voltage-Gated Sodium Channel; Membrane Proteins; Nerve Tissue Proteins; Mutation
PubMed: 37271286
DOI: 10.1016/j.nbd.2023.106177