Biology subjects

Guan, C.

Publications and source records attributed to Guan, C..

6 recordsLinked to original sources

bioRxiv2018

Temporal correlation of elevated PRMT1 gene expression with mushroom body neurogenesis during bumblebee brain development

Proper neural development in insects depends on the controlled proliferation and differentiation of neural precursors. In holometabolous insects, these processes must be coordinated during larval and pupal development. Recently, protein arginine methylation has come into focus as an important mechanism of controlling neural stem cell proliferation and differentiation in mammals. Whether a similar mechanism is at work in insects is unknown. We investigated this possibility by determining the expression pattern of three protein arginine methyltransferase mRNAs (PRMT1, 4 and 5) in the developing brain of bumblebees by in situ hybridisation. We detected expression in neural precursors and neurons in functionally important brain areas throughout development. We found markedly higher expression of PRMT1, but not PRMT4 and PRMT5, in regions of mushroom bodies containing dividing cells during pupal stages at the time of active neurogenesis within this brain area. At later stages of development, PRMT1 expression levels were found to be uniform and did not correlate with actively dividing cells. Our study suggests a role for PRMT1 in regulating neural precursor divisions in the mushroom bodies of bumblebees during the period of neurogenesis.

developmental biology↗

bioRxiv2018

Evaluation of InSeq to Identify Genes Essential for Pseudomonas aeruginosa PGPR2 Corn Root Colonization

The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the TnSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5-to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in P. aeruginosa PGPR2 for root colonization.

genomics↗

bioRxiv2018

Uniform distribution of 35S promoter-driven mDII auxin control sensor in leaf primordia

The DII sensor has been an invaluable tool to map spatiotemporal auxin response and distribution in the model plant Arabidopsis thaliana. The DII sensor and mDII control sensor are driven by the widely used constitutive 35S promoter. However, the reliability of DII sensor has recently been questioned (Bhatia and Heisler, 2018). Here we provide additional evidence to show that the mDII control sensor is indeed uniformly distributed in early leaf primordia, which echoes the original reports (Vernoux et al., 2011; Brunoud et al., 2012). We also use DII/mDII and the PRS5A promoter-driven R2D2 sensors to confirm asymmetric auxin signaling in early leaf primordia. On the other hand, we provide evidence that light penetration may lead to artifacts during whole-mount imaging.

plant biology↗

bioRxiv2018

Improved sgRNA design in bacteria via genome-wide activity profiling

CRISPR/Cas9 is a promising tool in prokaryotic genome engineering, but its success is limited by the widely varying on-target activity of single guide RNAs (sgRNAs). Based on the association of CRISPR/Cas9-induced DNA cleavage with cellular lethality, we systematically profiled sgRNA activity by co-expressing a genome-scale library (~70,000 sgRNAs) with Cas9 or its specificity-improved mutant in E. coli. Based on this large-scale dataset, we constructed a comprehensive and high-density sgRNA activity map, which enables selecting highly active sgRNAs for any locus across the genome in this model organism. We also identified resistant genomic loci with respect to CRISPR/Cas9 activity, notwithstanding the highly accessible DNA in bacterial cells. Moreover, we found that previous sgRNA activity prediction models that were trained on mammalian cell datasets were inadequate when coping with our results, highlighting the key limitations and biases of previous models. We hence developed an integrated algorithm to accurately predict highly effective sgRNAs, aiming to facilitate the design of CRISPR/Cas9-based genome engineering or screenings in bacteria. We also isolated the important sgRNA features that contribute to DNA cleavage and characterized their key differences among wild type Cas9 and its mutant, shedding light on the biophysical mechanisms of the CRISPR/Cas9 system.

microbiology↗

bioRxiv2017

NRT1.1-dependent NH4+ toxicity in Arabidopsis is associated with disturbed balance between NH4+ uptake and assimilation

A high concentration of a sole ammonium (NH4+) source in growth media is often toxic to plants. The nitrate transporter NRT1.1 is involved in plant NH4+ toxicity; however, its mechanism remains undefined. In this study, wild-type Arabidopsis (Col-0) and NRT1.1 mutants (chl1-1 and chl1-5) were grown hydroponically in NH4NO3 and (NH4)2SO4 media to evaluate NRT1.1 function in NH4+ stress responses. All plants grew normally in mixed N sources, but Col-0 displayed more chlorosis, and lower biomass and photosynthesis than the NRT1.1 mutants in the (NH4)2SO4 condition. Grafting experiments between Col-0 and chl1-5 further confirmed that NH4+ toxicity is NRT1.1-dependent. In (NH4)2SO4 medium, NRT1.1 facilitated the higher expression of NH4+ transporters, increasing NH4+ uptake. Additionally, glutamine synthetase (GS) and glutamate synthetase (GOGAT) in roots of Col-0 plants decreased and soluble sugar accumulated significantly, whereas pyruvate kinase (PK)-mediated glycolysis was not affected, all of which contributed to NH4+ accumulation. In contrast, the NRT1.1 mutants reduced NH4+ accumulation and enhanced NH4+ assimilation through glutamate dehydrogenase (GDH) and glutamate-oxaloacetate transamination (GOT) activity. In addition, the upregulation of genes involved in senescence in Col-0 plants treated with (NH4)2SO4 suggests that ethylene could be involved in NH4+ toxicity responses. Our results indicate that NH4+ toxicity is dependent on NRT1.1 in Arabidopsis, characterized by enhanced NH4+ accumulation and by perturbed NH4+ metabolism, which stimulated ethylene-induced plant senescence.\n\nHighlight: Nitrate transporter NRT1.1 enhances NH4+ accumulation, disturbs the NH4+ metabolism, and aggravates NH4+ toxicity in Arabidopsis when grown under sole NH4+ condition.

plant biology↗

bioRxiv2017

Pooled CRISPR interference screens enable high-throughput functional genomics study and elucidate new rules for guide RNA library design in Escherichia coli

Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology provides potential advantages in high-throughput functional genomics analysis in prokaryotes over previously established platforms based on recombineering or transposon mutagenesis. In this work, as a proof-of-concept to adopt CRISPR/Cas9 method as a pooled functional genomics analysis platform in prokaryotes, we developed a CRISPR interference (CRISPRi) library consisting of 3,148 single guide RNAs (sgRNAs) targeting the open reading frame (ORF) of 67 genes with known knockout phenotypes and performed pooled screens under two stressed conditions (minimal and acidic medium) in Escherichia coli. Our approach confirmed most of previously described gene-phenotype associations while maintaining < 5% false positive rate, suggesting that CRISPRi screen is both sensitive and specific. Our data also supported the ability of this method to narrow down the candidate gene pool when studying operons, a unique structure in prokaryotic genome. Meanwhile, assessment of multiple loci across treatments enables us to extract several guidelines for sgRNA design for such pooled functional genomics screen. For instance, sgRNAs locating at the first 5% upstream region within ORF exhibit enhanced activity and 10 sgRNAs per gene is suggested to be enough for robust identification of gene-phenotype associations. We also optimized the hit-gene calling algorithm to identify target genes more robustly with even fewer sgRNAs. This work showed that CRISPRi could be adopted as a powerful functional genomics analysis tool in prokaryotes and provided the first guideline for the construction of sgRNA libraries in such applications.\n\nImportanceTo fully exploit the valuable resource of explosive sequenced microbial genomes, high-throughput experimental platform is needed to associate genes and phenotypes at the genome level, giving microbiologists the insight about the genetic structure and physiology of a microorganism. In this work, we adopted CRISPR interference method as a pooled high-throughput functional genomics platform in prokaryotes with Escherichia coli as the model organism. Our data suggested that this method was highly sensitive and specific to map genes with previously known phenotypes, potent to act as a new strategy for high-throughput microbial genetics study with advantages over previously established methods. We also provided the first guideline for the sgRNA library design by comprehensive analysis of the screen data. The concept, gRNA library design rules and open-source scripts of this work should benefit prokaryotic genetics community to apply high-throughput mapping of defined gene set with phenotypes in a broad spectrum of microorganisms.

microbiology↗