scholarly journals Gene Disruption by Homologous Recombination in the Xylella fastidiosa Citrus Variegated Chlorosis Strain

2002 ◽  
Vol 68 (9) ◽  
pp. 4658-4665 ◽  
Author(s):  
Patrice Gaurivaud ◽  
Leonardo C. A. Souza ◽  
Andrea C. D. Virgílio ◽  
Anelise G. Mariano ◽  
Renê R. Palma ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Disruption ◽  
Target Gene ◽  
Xylella Fastidiosa ◽  
Chloramphenicol Acetyltransferase ◽  
Model Integration ◽  
Citrus Variegated Chlorosis ◽  
Endogenous Gene ◽  
Gene Coding ◽  
Chloramphenicol Acetyltransferase Gene

ABSTRACT Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for β-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to β-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant.

Targeted gene insertion and replacement in the basidiomycete Ganoderma lucidum by inactivation of non-homologous end joining using CRISPR/Cas9

2021 ◽  
Author(s):  
Jun-Liang Tu ◽  
Xin-Yuan Bai ◽  
Yong-Liang Xu ◽  
Na Li ◽  
Jun-Wei Xu
Keyword(s):  
Homologous Recombination ◽  
Functional Genomics ◽  
Ganoderma Lucidum ◽  
Gene Disruption ◽  
Target Gene ◽  
End Joining ◽  
Gene Insertion ◽  
Replacement Method ◽  
Low Efficiency ◽  
Non Homologous End Joining

Targeted gene insertion or replacement is a promising genome editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum , targeted gene insertion and replacement remains a serious challenge due to the low efficiency of homologous recombination (HR) in these species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the non-homologous end joining pathway (NHEJ) at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma , we first inactivated the NHEJ pathway via disruption of the Ku70 gene ( ku70 ) using a dual sgRNA-directed gene deletion method. Disruption of the ku70 significantly decreased NHEJ activity in G. lucidum . Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of a targeted gene insertion and replacement when target DNA orotidine 5’-monophosphate decarboxylase gene ( ura3 ) with 1.5 kb 5’ and 3’ homologous flanking sequences were used as a donor template, compared to 3.3% and 0% for a control strain (Cas9 strain) at these targeted sites, respectively. Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum . Importance Functional genomic studies have been hindered in Ganoderma by the absence of adequate genome engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum , targeted gene insertion and replacement has remained a serious challenge due to the low efficiency of homologous recombination in these species, although such precise genome modifications including site mutations, site-specific integrations and allele or promoter replacements would be incredibly valuable. In this work, we inactivated the non-homologous end joining repair mechanism in G. lucidum by disrupting the ku70 using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70 -disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.

scholarly journals Stable Transformation of the Xylella fastidiosa Citrus Variegated Chlorosis Strain withoriC Plasmids

2001 ◽  
Vol 67 (5) ◽  
pp. 2263-2269 ◽  
Author(s):  
Patrı́cia B. Monteiro ◽  
Diva C. Teixeira ◽  
Renê R. Palma ◽  
Monique Garnier ◽  
Joseph-Marie Bové ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Sequence ◽  
Promoter Region ◽  
Xylella Fastidiosa ◽  
Severe Disease ◽  
Kanamycin Resistance ◽  
Stable Transformation ◽  
Specific Gene ◽  
Host Chromosome ◽  
Citrus Variegated Chlorosis

ABSTRACT Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain ofX. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kanr). In plasmid p16KdAori, the oriCfragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori theoriC fragment was restricted to thednaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kanr gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.

Artificial microRNA (amiRNA) induced gene silencing in alfalfa (Medicago sativa)

Botany ◽  
2013 ◽  
Vol 91 (2) ◽  
pp. 117-122 ◽  
Author(s):  
Julian C. Verdonk ◽  
Michael L. Sullivan
Keyword(s):  
Gene Silencing ◽  
Medicago Sativa ◽  
Target Gene ◽  
Target Genes ◽  
Mirna Precursor ◽  
Crop Species ◽  
Forage Crop ◽  
Ribonucleic Acids ◽  
Endogenous Gene ◽  
Medicago Sativa L

Gene silencing is a powerful technique that allows the study of the function of specific genes by selectively reducing their transcription. Several different approaches can be used, however they all have in common the artificial generation of single stranded small ribonucleic acids (RNAs) that are utilized by the endogenous gene silencing machinery of the organism. Artificial microRNAs (amiRNA) can be used to very specifically target genes for silencing because only a short sequence of 21 nucleotides of the gene of interest is used. Gene silencing via amiRNA has been developed for Arabidopsis thaliana (L.) Heynh. and rice using endogenous microRNA (miRNA) precursors and has been shown to also work effectively in other dicot species using the arabidopsis miRNA precursor. Here, we demonstrate that the arabidopsis miR319 precursor can be used to silence genes in the important forage crop species alfalfa (Medicago sativa L.) by silencing the expression of a transgenic beta-glucuronidase (GUSPlus) target gene.

scholarly journals A direct role for C/EBP and the AP-I-binding site in gene expression linked to adipocyte differentiation.

1989 ◽  
Vol 9 (12) ◽  
pp. 5331-5339 ◽  
Author(s):  
R Herrera ◽  
H S Ro ◽  
G S Robinson ◽  
K G Xanthopoulos ◽  
B M Spiegelman
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Adipocyte Differentiation ◽  
Binding Protein ◽  
Point Mutations ◽  
Chloramphenicol Acetyltransferase ◽  
Lipid Binding ◽  
Direct Role ◽  
New Genes ◽  
Chloramphenicol Acetyltransferase Gene

Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including the gene encoding adipocyte P2 (aP2), an intracellular lipid-binding protein. Using specific deletions and point mutations, we have shown that at least two distinct sequence elements in the aP2 promoter contribute to the expression of the chloramphenicol acetyltransferase gene in chimeric constructions transfected into adipose cells. An AP-I site at -120, shown earlier to bind Jun- and Fos-like proteins, serves as a positive regulator of chloramphenicol acetyltransferase gene expression in adipocytes but is specifically silenced by adjacent upstream sequences in preadipocytes. Sequences upstream of the AP-I site at -140 (termed AE-1) can function as an enhancer in both cell types when linked to a viral promoter but can stimulate expression only in fat cells in the intact aP2 promoter. The AE-1 sequence binds an adipocyte protein identical or very closely related to an enhancer-binding protein (C/EBP) that has been previously implicated in the regulation of several liver-specific genes. A functional role for C/EBP in the regulation of the aP2 gene is indicated by the facts that C/EBP mRNA is induced during adipocyte differentiation and the aP2 promoter is transactivated by cotransfection of a C/EBP expression vector into preadipose cells. These results indicate that sequences that bind C/EBP and the Fos-Jun complex play major roles in the expression of the aP2 gene during adipocyte differentiation and demonstrate that C/EBP can directly regulate cellular gene expression.

scholarly journals Transmission of the Citrus Variegated Chlorosis Bacterium Xylella fastidiosa with the Sharpshooter Oncometopia nigricans

Plant Disease ◽  
2002 ◽  
Vol 86 (11) ◽  
pp. 1237-1239 ◽  
Author(s):  
R. H. Brlansky ◽  
V. D. Damsteegt ◽  
J. S. Hartung
Keyword(s):  
United States ◽  
Polymerase Chain Reaction ◽  
Xylella Fastidiosa ◽  
The United States ◽  
Reliable Indicator ◽  
Symptom Development ◽  
Chain Reaction ◽  
Citrus Variegated Chlorosis ◽  
Specific Polymerase Chain Reaction ◽  
Polymerase Chain

Citrus variegated chlorosis (CVC) is an economically important, destructive disease in Brazil and is caused by the bacterium Xylella fastidiosa Wells. The bacterium has been found to be transmitted in Brazil by sharpshooter leafhoppers (Cicadellidae). Sharpshooters are present in most citrus growing areas of the United States. The sharpshooter leafhopper, Oncometopia nigricans Walker, frequently is found feeding on citrus in Florida. This sharpshooter transmits the X. fastidiosa strains that cause Pierce's disease of grape and ragweed stunt. Research was initiated to determine if O. nigricans was capable of vectoring the X. fastidiosa that causes CVC. In 59 different transmission tests, using 1 to 57 insects per test, transmission of the bacterium was observed 12 times (20.3%). Symptom development in the greenhouse was not a reliable indicator of transmission. Transmission was verified by specific polymerase chain reaction-based assays. Individual insects were able to transmit the bacterium. This information on sharpshooter transmission of CVC is needed to assess the threat posed by the CVC disease to the citrus industries in the United States.

Generation of infectious Moloney murine leukemia viruses with deletions in the U3 portion of the long terminal repeat

1986 ◽  
Vol 6 (12) ◽  
pp. 4634-4640
Author(s):  
R Hanecak ◽  
S Mittal ◽  
B R Davis ◽  
H Fan
Keyword(s):  
Long Terminal Repeat ◽  
Transient Expression ◽  
Leukemia Virus ◽  
Chloramphenicol Acetyltransferase ◽  
Terminal Repeat ◽  
Tata Box ◽  
Moloney Murine Leukemia Virus ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Leukemia Viruses ◽  
Murine Leukemia

Deletional analysis within the long terminal repeat (LTR) of Moloney murine leukemia virus (M-MuLV) was performed. By molecular cloning, deletions were made in the vicinity of the XbaI site at -150 base pairs (bp) in the U3 region, between the tandemly repeated enhancers and the TATA box. The effects of the deletions on LTR function were measured in two ways. First, deleted LTRs were fused to the bacterial chloramphenicol acetyltransferase gene and used in transient expression assays. Second, infectious M-MuLVs were generated by transfection of M-MuLV proviruses containing the deleted LTRs, and the relative infectivity of the mutant viruses was assessed by XC-syncytial assay. Most of the deleted LTRs examined showed relatively high promoter activity in the transient chloramphenicol acetyltransferase assays, with values ranging from 20 to 50% of the wild-type M-MuLV LTR. Thus, the sequences between the enhancers and the TATA box were not absolutely required for transient expression. However, infectivity of viruses carrying the same deleted LTRs showed more pronounced effects. Deletion of sequences from -195 to -174 bp reduced infectivity 20- to 100-fold. Deletion of sequences within the region from -174 to -122 bp did not affect infectivity, indicating that this region is dispensable. On the other hand, deletion of sequences from -150 to -40 bp reduced infectivity from 5 to 6 logs, although the magnitude of the reduction partly may have reflected threshold envelope protein requirements for positive XC assays. The reduced infectivity did not appear to result from a failure of proviral DNA synthesis or integration by the mutant. Thus, the infectivity measurements identified three functional domains in the region between the enhancers and the TATA box.

Homology dependence of targeted recombination at the Chinese hamster APRT locus

1994 ◽  
Vol 14 (10) ◽  
pp. 6663-6673
Author(s):  
J B Scheerer ◽  
G M Adair
Keyword(s):  
Homologous Recombination ◽  
Sequence Homology ◽  
Chinese Hamster Ovary ◽  
Target Gene ◽  
Gene Deletion ◽  
Chinese Hamster ◽  
Adenine Phosphoribosyltransferase ◽  
Aprt Gene ◽  
Logarithmic Relationship ◽  
Targeted Recombination

Using simple linear fragments of the Chinese hamster adenine phosphoribosyltransferase (APRT) gene as targeting vectors, we have investigated the homology dependence of targeted recombination at the endogenous APRT locus in Chinese hamster ovary (CHO) cells. We have examined the effects of varying either the overall length of targeting sequence homology or the length of 5' or 3' flanking homology on both the frequency of targeted homologous recombination and the types of recombination events that are obtained. We find an exponential (logarithmic) relationship between length of APRT targeting homology and the frequency of targeted recombination at the CHO APRT locus, with the frequency of targeted recombination dependent upon both the overall length of targeting homology and the length of homology flanking each side of the target gene deletion. Although most of the APRT+ recombinants analyzed reflect simple targeted replacement or conversion of the target gene deletion, a significant fraction appear to have arisen by target gene-templated extension and correction of the targeting fragment sequences. APRT fragments with limited targeting homology flanking one side of the target gene deletion yield proportionately fewer target gene conversion events and proportionately more templated extension and vector correction events than do fragments with more substantial flanking homology.

Gene replacement with one-sided homologous recombination

1992 ◽  
Vol 12 (1) ◽  
pp. 360-367
Author(s):  
N Berinstein ◽  
N Pennell ◽  
C A Ottaway ◽  
M J Shulman
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Target Gene ◽  
Gene Replacement ◽  
Immunoglobulin Genes ◽  
Nonhomologous Recombination ◽  
Immunoglobulin Locus ◽  
Chromosomal Sequences ◽  
Dna Vectors

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.

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