Transcriptional Variability Associated With CRISPR-Mediated Gene Replacements at the Phytophthora sojae Avr1b-1 Locus

Transcriptional plasticity enables oomycetes to rapidly adapt to environmental challenges including emerging host resistance. For example, the soybean pathogen Phytophthora sojae can overcome resistance conferred by the host resistance gene Rps1b through natural silencing of its corresponding effector gene, Avr1b-1. With the Phytophthora CRISPR/Cas9 genome editing system, it is possible to generate site-specific knock-out (KO) and knock-in (KI) mutants and to investigate the biological functions of target genes. In this study, the Avr1b-1 gene was deleted from the P. sojae genome using a homology-directed recombination strategy that replaced Avr1b-1 with a gene encoding the fluorescent protein mCherry. As expected, all selected KO transformants gained virulence on Rps1b plants, while infection of plants lacking Rps1b was not compromised. When a sgRNA-resistant version of Avr1b-1 was reintroduced into the Avr1b-1 locus of an Avr1b KO transformant, KI transformants with a well-transcribed Avr1b-1 gene were unable to infect Rps1b-containing soybeans. However, loss of expression of the incoming Avr1b-1 gene was frequently observed in KI transformants, which resulted in these transformants readily infecting Rps1b soybeans. A similar variability in the expression levels of the incoming gene was observed with AVI- or mCherry-tagged Avr1b-1 constructs. Our results suggest that Avr1b-1 may be unusually susceptible to transcriptional variation.

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The Non-Canonical Aspects of MicroRNAs: Many Roads to Gene Regulation

Cells ◽

10.3390/cells8111465 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1465 ◽

Cited By ~ 30

Author(s):

Christiaan J. Stavast ◽

Stefan J. Erkeland

Keyword(s):

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Small Rnas ◽

Target Genes ◽

Biological Functions ◽

Rnase Iii ◽

Mrna Targets ◽

Argonaute Proteins ◽

Epigenetic Modifiers ◽

Encoding Genes

MicroRNAs (miRNAs) are critical regulators of gene expression. As miRNAs are frequently deregulated in many human diseases, including cancer and immunological disorders, it is important to understand their biological functions. Typically, miRNA-encoding genes are transcribed by RNA Polymerase II and generate primary transcripts that are processed by RNase III-endonucleases DROSHA and DICER into small RNAs of approximately 21 nucleotides. All miRNAs are loaded into Argonaute proteins in the RNA-induced silencing complex (RISC) and act as post-transcriptional regulators by binding to the 3′- untranslated region (UTR) of mRNAs. This seed-dependent miRNA binding inhibits the translation and/or promotes the degradation of mRNA targets. Surprisingly, recent data presents evidence for a target-mediated decay mechanism that controls the level of specific miRNAs. In addition, several non-canonical miRNA-containing genes have been recently described and unexpected functions of miRNAs have been identified. For instance, several miRNAs are located in the nucleus, where they are involved in the transcriptional activation or silencing of target genes. These epigenetic modifiers are recruited by RISC and guided by miRNAs to specific loci in the genome. Here, we will review non-canonical aspects of miRNA biology, including novel regulators of miRNA expression and functions of miRNAs in the nucleus.

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An FYVE-Domain-Containing Protein, PsFP1, Is Involved in Vegetative Growth, Oxidative Stress Response and Virulence of Phytophthora sojae

International Journal of Molecular Sciences ◽

10.3390/ijms22126601 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6601

Author(s):

Jinhui Zhang ◽

Xiaoran Du ◽

Xin Zhou ◽

Duo Jin ◽

Jianqiang Miao ◽

...

Keyword(s):

Oxidative Stress ◽

Membrane Trafficking ◽

Vegetative Growth ◽

Genome Mining ◽

Gene Replacement ◽

Wide Distribution ◽

Stress Sensitivity ◽

Phytophthora Sojae ◽

Knock Out ◽

Fyve Domain

Proteins that contain the FYVE zinc-finger domain are recruited to PtdIns3P-containing membranes, participating in numerous biological processes such as membrane trafficking, cytoskeletal regulation, and receptor signaling. However, the genome-wide distribution, evolution, and biological functions of FYVE-containing proteins are rarely reported for oomycetes. By genome mining of Phytophthora sojae, two proteins (PsFP1 and PsFP2) with a combination of the FYVE domain and the PX domain (a major phosphoinositide binding module) were found. To clarify the functions of PsFP1 and PsFP2, the CRISPR/Cas9-mediated gene replacement system was used to knock out the two genes respectively. Only heterozygous deletion mutants of PsFP1 were recovered, and the expression level of PsFP1 in the heterozygous knockout transformants was significantly down-regulated. These PsFP1 mutants showed a decrease in mycelial growth and pathogenicity and were more sensitive to hydrogen peroxide. These phenotypes were recovered to the level of wild-type by overexpression PsFP1 gene in the PsFP1 heterozygous knockout transformant. In contrast, deletion of PsFP2 had no significant effect on vegetative growth, asexual and sexual reproduction, pathogenicity, or oxidative stress sensitivity. PsFP1 was primarily localized in vesicle-like structures and both the FYVE and PX domains are important for its localization. Overall, our results indicate that PsFP1 plays an important role in the vegetative growth and virulence of P. sojae.

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A PPR Protein ACM1 Is Involved in Chloroplast Gene Expression and Early Plastid Development in Arabidopsis

International Journal of Molecular Sciences ◽

10.3390/ijms22052512 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2512

Author(s):

Xinwei Wang ◽

Yaqi An ◽

Ye Li ◽

Jianwei Xiao

Keyword(s):

Fluorescent Protein ◽

Chloroplast Development ◽

Nuclear Gene ◽

Pentatricopeptide Repeat ◽

Plastid Development ◽

Chloroplast Gene Expression ◽

Knock Out ◽

Rnai Line ◽

Ppr Protein ◽

P Type

Chloroplasts cannot develop normally without the coordinated action of various proteins and signaling connections between the nucleus and the chloroplast genome. Many questions regarding these processes remain unanswered. Here, we report a novel P-type pentatricopeptide repeat (PPR) factor, named Albino Cotyledon Mutant1 (ACM1), which is encoded by a nuclear gene and involved in chloroplast development. Knock-down of ACM1 transgenic plants displayed albino cotyledons but normal true leaves, while knock-out of the ACM1 gene in seedlings was lethal. Fluorescent protein analysis showed that ACM1 was specifically localized within chloroplasts. PEP-dependent plastid transcript levels and splicing efficiency of several group II introns were seriously affected in cotyledons in the RNAi line. Furthermore, denaturing gel electrophoresis and Western blot experiments showed that the accumulation of chloroplast ribosomes was probably damaged. Collectively, our results indicate ACM1 is indispensable in early chloroplast development in Arabidopsis cotyledons.

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RNA Interference of Genes Encoding the Vacuolar-ATPase in Liriomyza trifolii

Insects ◽

10.3390/insects12010041 ◽

2021 ◽

Vol 12 (1) ◽

pp. 41

Author(s):

Ya-Wen Chang ◽

Yu-Cheng Wang ◽

Xiao-Xiang Zhang ◽

Junaid Iqbal ◽

Yu-Zhou Du

Keyword(s):

Rna Interference ◽

Expression Analysis ◽

Target Genes ◽

Fluorescent Protein ◽

Control Strategies ◽

Vacuolar Atpase ◽

Invasive Pest ◽

Liriomyza Trifolii ◽

Horticultural Crops ◽

Genes Encoding

The leafminer fly, Liriomyza trifolii, is an invasive pest of vegetable and horticultural crops in China. In this study, a microinjection method based on dsRNA was developed for RNA interference (RNAi) in L. trifolii using genes encoding vacuolar-ATPase (V-ATPase). Expression analysis indicated that V-ATPase B and V-ATPase D were more highly expressed in L. trifolii adults than in larvae or pupae. Microinjection experiments with dsV-ATPase B and dsV-ATPase D were conducted to evaluate the efficacy of RNAi in L. trifolii adults. Expression analysis indicated that microinjection with 100 ng dsV-ATPase B or dsV-ATPase led to a significant reduction in V-ATPase transcripts as compared to that of the dsGFP control (dsRNA specific to green fluorescent protein). Furthermore, lower dsRNA concentrations were also effective in reducing the expression of target genes when delivered by microinjection. Mortality was significantly higher in dsV-ATPase B- and dsV-ATPase D-treated insects than in controls injected with dsGFP. The successful deployment of RNAi in L. trifolii will facilitate functional analyses of vital genes in this economically-important pest and may ultimately result in new control strategies.

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The role of m6A modification in the biological functions and diseases

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-020-00450-x ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Xiulin Jiang ◽

Baiyang Liu ◽

Zhi Nie ◽

Lincan Duan ◽

Qiuxia Xiong ◽

...

Keyword(s):

Cancer Progression ◽

Molecular Mechanisms ◽

Target Genes ◽

Rna Modification ◽

Biological Functions ◽

Rna Methylation ◽

Downstream Target ◽

Different Types ◽

M6a Rna Methylation

AbstractN6-methyladenosine (m6A) is the most prevalent, abundant and conserved internal cotranscriptional modification in eukaryotic RNAs, especially within higher eukaryotic cells. m6A modification is modified by the m6A methyltransferases, or writers, such as METTL3/14/16, RBM15/15B, ZC3H3, VIRMA, CBLL1, WTAP, and KIAA1429, and, removed by the demethylases, or erasers, including FTO and ALKBH5. It is recognized by m6A-binding proteins YTHDF1/2/3, YTHDC1/2 IGF2BP1/2/3 and HNRNPA2B1, also known as “readers”. Recent studies have shown that m6A RNA modification plays essential role in both physiological and pathological conditions, especially in the initiation and progression of different types of human cancers. In this review, we discuss how m6A RNA methylation influences both the physiological and pathological progressions of hematopoietic, central nervous and reproductive systems. We will mainly focus on recent progress in identifying the biological functions and the underlying molecular mechanisms of m6A RNA methylation, its regulators and downstream target genes, during cancer progression in above systems. We propose that m6A RNA methylation process offer potential targets for cancer therapy in the future.

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Differential activation mechanisms of two isoforms of Gcr1 transcription factor generated from spliced and un-spliced transcripts in Saccharomyces cerevisiae

Nucleic Acids Research ◽

10.1093/nar/gkaa1221 ◽

2020 ◽

Author(s):

Seungwoo Cha ◽

Chang Pyo Hong ◽

Hyun Ah Kang ◽

Ji-Sook Hahn

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Target Genes ◽

Gene Encoding ◽

Metabolic Switch ◽

Differential Activation ◽

N Terminus ◽

Activation Mechanisms ◽

In Trans ◽

Separate Activation

Abstract Gcr1, an important transcription factor for glycolytic genes in Saccharomyces cerevisiae, was recently revealed to have two isoforms, Gcr1U and Gcr1S, produced from un-spliced and spliced transcripts, respectively. In this study, by generating strains expressing only Gcr1U or Gcr1S using the CRISPR/Cas9 system, we elucidate differential activation mechanisms of these two isoforms. The Gcr1U monomer forms an active complex with its coactivator Gcr2 homodimer, whereas Gcr1S acts as a homodimer without Gcr2. The USS domain, 55 residues at the N-terminus existing only in Gcr1U, inhibits dimerization of Gcr1U and even acts in trans to inhibit Gcr1S dimerization. The Gcr1S monomer inhibits the metabolic switch from fermentation to respiration by directly binding to the ALD4 promoter, which can be restored by overexpression of the ALD4 gene, encoding a mitochondrial aldehyde dehydrogenase required for ethanol utilization. Gcr1U and Gcr1S regulate almost the same target genes, but show unique activities depending on growth phase, suggesting that these isoforms play differential roles through separate activation mechanisms depending on environmental conditions.

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CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)

Scientific Reports ◽

10.1038/s41598-021-95547-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yoshihiko Nanasato ◽

Masafumi Mikami ◽

Norihiro Futamura ◽

Masaki Endo ◽

Mitsuru Nishiguchi ◽

...

Keyword(s):

Genome Editing ◽

Cryptomeria Japonica ◽

Fluorescent Protein ◽

Japanese Cedar ◽

Targeted Mutagenesis ◽

Embryogenic Tissue ◽

Breeding Period ◽

Single Mutation ◽

Knock Out ◽

Guide Rna

AbstractCryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

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Phase Variation of Campylobacter jejuni 81-176 Lipooligosaccharide Affects Ganglioside Mimicry and Invasiveness In Vitro

Infection and Immunity ◽

10.1128/iai.70.2.787-793.2002 ◽

2002 ◽

Vol 70 (2) ◽

pp. 787-793 ◽

Cited By ~ 154

Author(s):

Patricia Guerry ◽

Christine M. Szymanski ◽

Martina M. Prendergast ◽

Thomas E. Hickey ◽

Cheryl P. Ewing ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Phase Variation ◽

Outer Core ◽

Gene Encoding ◽

Site Specific ◽

Reading Frame ◽

Specific Mutation ◽

Normal Human ◽

A Site

ABSTRACT The outer cores of the lipooligosaccharides (LOS) of many strains of Campylobacter jejuni mimic human gangliosides in structure. A population of cells of C. jejuni strain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM2 and GM3 gangliosides, with minor amounts of structures mimicking GD1b and GD2. Genetic analyses of genes involved in the biosynthesis of the outer core of C. jejuni 81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, an N-acetylgalactosaminyltransferase. Variation in the number of G residues within cgtA affected the length of the open reading frame, and these changes in cgtA corresponded to a change in LOS structure from GM2 to GM3 ganglioside mimicry. Site-specific mutation of cgtA in 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM3 ganglioside. Compared to wild-type 81-176, the cgtA mutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of the neuC1 gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.

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Abstract 016: Cells Programmed for the Renin Phenotype Contribute to Vascular Pathology in Renin Deficient Mice

Hypertension ◽

10.1161/hyp.68.suppl_1.016 ◽

2016 ◽

Vol 68 (suppl_1) ◽

Author(s):

Masafumi Oka ◽

Silvia Medrano ◽

Maria Luisa S Sequeira-Lopez ◽

R A Gomez

Keyword(s):

Arterial Wall ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Renin Angiotensin System ◽

Arterial Disease ◽

Vascular Pathology ◽

Wall Thickening ◽

Knock Out ◽

Reporter Mice ◽

Specific Proteins

Deletions of the renin-angiotensin system genes or pharmacological inhibition in early life result in a distinctive renal pathology: concentric and disorganized intra-renal arteriolar thickening. The origin and distribution of the cells contributing to the arterial disease are not known. Because the arteriolar thickening disappears with ablation of renin cells, we hypothesized that renin cell precursors contribute to the arterial pathology. To reveal the origin and distribution of the cells responsible for the arterial thickening we generated several mouse lines for fate tracing and also stained for cell identity specific proteins. Kidneys from Ren1c-/- (n=6) and Ren1c+/- (n=6) mice were immunostained for renin, αSMA and PECAM1. Arterial wall thickness was measured using a light microscope and the Leica MM AF ® version1.5 software. Renin cells (unable to produce renin because of the knock out) were identified using Ren1c-/-; Ren1c-YFP mice, where the yellow fluorescent protein is expressed by the Ren1c-YFP transgene designed to label all cells with an active renin promoter. In addition, we tracked the expression and distribution of aldo-keto reductase 1b7, AKR1b7, which mark cells programmed for the renin phenotype even when renin is absent. As expected, Ren1c-/- kidneys showed no renin and thicker intra-renal arteries (Arterioles: Ren1c+/- , 8.26 ± 2.5 μm vs. Ren1c-/- , 14.3 ± 3.8 μm, P<0.0001 , larger arteries: Ren1c+/- , 29.2 ± 11.1 μm vs. Ren1c-/- , 42.1 ± 11.1 μm, P<0.0001 ) AKR1b7+ and YFP+ cells were retained and observed throughout the renal arterioles. To investigate the fate and distribution of cells from the renin lineage, we used Ren1c-Cre and R26R.LacZ or mT/mG reporter mice (6 knock out and 6 control mice per strain). Cells from the renin lineage surrounded arterioles and persisted within larger arterial walls whereas PECAM1+ endothelial cells did not contribute to the arterial wall thickening. In control mice, renin cells were confined to the juxtaglomerular area. We conclude that precursor cells programmed for the renin phenotype maintain their molecular program and together with vascular smooth muscle cells contribute to nephro-vascular disease.

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Autoregulated Expression of Schizosaccharomyces pombe Meiosis-Specific Transcription Factor Mei4 and a Genome-Wide Search for Its Target Genes

Genetics ◽

10.1093/genetics/154.4.1497 ◽

2000 ◽

Vol 154 (4) ◽

pp. 1497-1508 ◽

Cited By ~ 4

Author(s):

Hiroko Abe ◽

Chikashi Shimoda

Keyword(s):

Transcription Factor ◽

Schizosaccharomyces Pombe ◽

Target Genes ◽

Northern Blotting ◽

Forkhead Transcription Factor ◽

Gene Encoding ◽

Putative Promoter Region ◽

Cis Acting ◽

A Genome ◽

Genome Wide Search

Abstract The Schizosaccharomyces pombe mei4+ gene encoding a forkhead transcription factor is necessary for the progression of meiosis and sporulation. We searched for novel meiotic genes, the expression of which is dependent on Mei4p, since only the spo6+ gene has been assigned to its targets. Six known genes responsible for meiotic recombination were examined by Northern blotting, but none were Mei4 dependent for transcription. We determined the important cis-acting element, designated FLEX, to which Mei4p can bind. The S. pombe genome sequence database (The Sanger Centre, UK) was scanned for the central core heptamer and its flanking 3′ sequence of FLEX composed of 17 nucleotides, and 10 candidate targets of Mei4 were selected. These contained a FLEX-like sequence in the 5′ upstream nontranslatable region within 1 kb of the initiation codon. Northern blotting confirmed that 9 of them, named mde1+ to mde9+, were transcriptionally induced during meiosis and were dependent on mei4+. Most mde genes have not been genetically defined yet, except for mde9+, which is identical to spn5+, which encodes one of the septin family of proteins. mde3+ and a related gene pit1+ encode proteins related to Saccharomyces cerevisiae Ime2. The double disruptant frequently produced asci having an abnormal number and size of spores, although it completed meiosis. We also found that the forkhead DNA-binding domain of Mei4p binds to the FLEX-like element in the putative promoter region of mei4 and that the maximum induction level of mei4 mRNA required functional mei4 activity. Furthermore, expression of a reporter gene driven by the authentic mei4 promoter was induced in vegetative cells by ectopic overproduction of Mei4p. These results suggest that mei4 transcription is positively autoregulated.

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