Biology subjects

Nguyen, K. H.

Publications and source records attributed to Nguyen, K. H..

4 recordsLinked to original sources

bioRxiv2020

Targeted mutagenesis of the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family suggests competition of multiplexed sgRNAs for Cas9 apoprotein

Genome editing in plants typically relies on T-DNA plasmids that are mobilized by Agrobacterium-mediated transformation to deliver the CRISPR/Cas9 machinery. Here, we introduce a series of CRISPR/Cas9 T-DNA vectors for minimal lab settings, such as in the classroom or citizen science projects. Spacer sequences targeting genes of interest can be inserted as annealed short oligonucleotides in a single straightforward cloning step. Fluorescent markers expressed in mature seeds enable reliable selection of transgenic as well as transgene-free individuals using a combination of inexpensive LED lamps and colored-glass alternative filters. Testing these tools on the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family, we found that Cas9 expression from an EGG CELL1 (EC1) promoter resulted in tenfold lower mutation rates than expression from a UBIQUITIN10 (UBQ10) promoter. A collection of bona fide null mutations in all nine GRF genes could be established with little effort. Finally, we explored the effects of simultaneously targeting two, four and eight GRF genes on the rate of induced mutations at each target locus. Multiplexing caused strong interference effects: while mutation rates at some loci remained consistently high, mutation rates at other loci dropped dramatically with increasing number of single guide RNA species. Our results suggest potential detrimental genetic interaction between induced mutations as well as competition of CRISPR RNAs for a limiting amount of Cas9 apoprotein.

plant biology↗

bioRxiv2020

The histidine phosphotransfer AHP4 plays a negative role in Arabidopsis plant response to drought

Cytokinin plays an important role in plant stress responses via a multistep signaling pathway, involving the histidine phosphotransfer proteins (HPs). In Arabidopsis thaliana, the AHP2, AHP3 and AHP5 proteins are known to impact drought responses; however, the role of AHP4 in drought adaptation remains undetermined. In the present study, using a loss-of-function approach we showed that AHP4 possesses a negative regulatory role in Arabidopsiss response to drought. This is evidenced by both higher survival rates of ahp4 than wild-type (WT) plants under drought conditions, and the down-regulated AHP4 expression in WT during periods of dehydration. Comparative transcriptome analysis of ahp4 and WT plants revealed AHP4-mediated expression of several dehydration- and/or abscisic acid (ABA)-responsive genes involved in regulation of various physiological and biochemical processes important for plant drought acclimation. In comparison with WT, ahp4 plants showed increased wax crystal accumulation in stems, thicker cuticles in leaves, greater sensitivity to exogenous ABA at germination, narrow stomatal apertures, heightened leaf temperatures during dehydration, and longer root length under osmotic stress. Additionally, ahp4 plants showed greater photosynthetic efficiency, lower levels of reactive oxygen species (ROS), reduced electrolyte leakage and lipid peroxidation, and increased anthocyanin contents under drought, when compared with WT. These differences displayed in ahp4 plants are likely due to up-regulation of genes that encode enzymes involved in ROS-scavenging and non-enzymatic antioxidant metabolism. The role of AHP4 in negative regulation of multiple protective mechanisms associated with drought tolerance could make editing of AHP4 a promising approach for the production of drought-tolerant crop plants. Significance statementLoss-of-function analysis of the cytokinin signaling member AHP4 revealed its function in Arabidopsis adaptation to drought as a negative regulator, affecting various physiological and biochemical processes by modulating the expression of a large set of genes potentially in a crosstalk with ABA. AHP4 and its homologs are promising candidates for gene editing to develop drought-tolerant crop cultivars.

plant biology↗

bioRxiv2020

Copper nanoparticle application enhances plant growth and grain yield in maize under drought stress conditions

Abiotic stresses, including drought, detrimentally affect the growth and productivity of many economically important crop plants, leading to significant yield losses, which can result in food shortages and threaten the sustainability of agriculture. Balancing between plant growth and stress responses is one of the most important characters for agricultural application to maximize plant production. In this study, we initially report that copper nanoparticle priming positively regulates drought stress responses in maize. The copper nanoparticle priming plants displayed enhanced drought tolerance indicated by their higher leaf water content and plant biomass under drought as compared with water-treated plants. Moreover, our data showed that the treatment of copper nanoparticle on plants increased anthocyanin, chlorophyll and carotenoid contents compared to water-treated plants under drought stress conditions. Additionally, histochemical analyses with nitro blue tetrazolium and 3,3-diaminobenzidine revealed that reactive oxygen species accumulation of priming plants was decreased as a result of enhancement of reactive oxygen species scavenging enzyme activities under drought. Furthermore, our comparative yield analysis data indicated applying copper nanoparticle to plant increased total seed number and grain yield under drought stress conditions. Our data provided the evidences that copper nanoparticle regulates plant protective mechanisms associated with drought tolerance, which is a promising approach for the production of drought tolerant crop plants.

plant biology↗

bioRxiv2019

Effect of agrochemical exposure on Schistosoma mansoni cercariae survival and activity

Land conversion and agrochemical use has altered freshwater systems worldwide, introducing chemicals and pathogens (e.g., helminths) that threaten human health. In developing countries where stringent pesticide use and water treatment is limited, understanding how contaminants and pathogens interact is of particular importance. Schistosomiasis, a neglected tropical disease, is caused by the free-swimming cercariae of Schistosoma mansoni, a flatworm (trematode) that is transmitted from snails to humans. Schistosomiasis afflicts over 200 million people, reinforces poverty, and has an enormous impact on children. To investigate the effects of pesticide exposure on S. mansoni, we exposed cercariae to four insecticides (cypermethrin, deltamethrin, dimethoate, and methamidophos) at five concentrations above estimated environmental concentrations, and recorded survival and activity during a 24-hr time-to-death assay. To identify live, but paralyzed, cercariae from dead cercariae, we used Trypan blue dye, which is only expelled from live cells. We found no effect of cypermethrin, deltamethrin, or dimethoate exposure on the survival and activity of S. mansoni cercariae. Surprisingly, methamidophos exposure decreased activity and increased survival of cercariae compared to those in control treatments. This result is likely due to methamidophos causing paralysis of cercariae, which reduced energy consumption lengthening lifespan. Although methamidophos exposure increased survival time, the pesticide-induced paralysis left cercariae functionally dead, which could influence overall disease prevalence and thus human health. Future studies that examine the influence of agrochemicals on waterborne disease prevalence and transmission need to consider both the lethal and sublethal effects of exposure to fully understand the complexity of host-parasite interactions.\n\nAuthor SummaryPrevious methods used to investigate the effects of pesticide exposure on free-swimming life stages of trematode pathogens include 1) normal activity, 2) movement following stimuli, or 3) staining dyes. As pesticides commonly target motor function, the use of an individual metric to assign trematode survival might misidentify pesticide-induced paralysis as mortality, therefore underestimating trematode tolerance. In this study, we used activity assays in tandem with Trypan blue staining dye to assess the effects of four pesticides on Schistosoma mansoni cercariae. We found that cercariae are highly tolerant to pesticide levels far beyond environmentally relevant concentrations. Surprisingly, exposure to methamidophos increased the survival and decreased the activity of cercariae compared to those in control treatments. Reduced activity was presumably caused by methamidophos-induced paralysis of cercariae. Although we observed increased survival following methamidophos exposure, the pesticide-induced paralysis rendered cercariae functionally dead. Our results highlight the need for future assays examining trematode tolerance to contaminants to employ both activity assays and staining dye to discern cercarial paralysis from mortality. Understanding the effects of pesticide exposure on disease transmission is of vital importance as pesticide use and agricultural activities intensify in developing nations endemic to waterborne pathogens.

ecology↗