scholarly journals Development of Spontaneous Hygromycin B Resistance in Monilinia fructicola and Its Impact on Growth Rate, Morphology, Susceptibility to Demethylation Inhibitor Fungicides, and Sporulation

2003 ◽  
Vol 93 (11) ◽  
pp. 1354-1359 ◽  
Author(s):  
Qun Dai ◽  
Zhihuan Sun ◽  
Guido Schnabel
Keyword(s):  
Insertional Mutagenesis ◽  
Selectable Marker ◽  
Fungal Spores ◽  
Potato Dextrose Agar ◽  
Monilinia Fructicola ◽  
Hygromycin B ◽  
Phenotypic Characters ◽  
Hygromycin B Resistance ◽  
Selection Of ◽  
Demethylation Inhibitor

Agrobacterium tumefaciens-mediated transformation with plasmids carrying the hygromycin B resistance gene hph frequently is being used for inserting genes into fungal spores and mycelial cells and for conducting insertional mutagenesis to identify genes connected to a particular phenotype. In this article, we report that stable hygromycin B resistance can develop spontaneously in germinating conidia from Monilinia fructicola and that the mutants exhibit altered phenotypes. One spontaneously developing hygromycin B-resistant colony developed per 2.5 × 105 germinating conidia. Mutants grew significantly slower on potato dextrose agar, were 2.4- to 3.1-fold more sensitive to demethylation inhibitor fungicides, lacked melanization, and did not produce spores. The mode of action of hygromycin B resistance in the mutants seemed to be different from the hph transgene-mediated hygromycin B resistance based on different phenotypic characters. The ability of M. fructicola and possibly other fungi to spontaneously develop hygromycin B resistance associated with an altered phenotype may interfere with the selection of true transformants if hygromycin B is used as selective agent. This is particularly confounding if the hph gene is used as selectable marker in insertional mutagenesis experiments conducted for the identification of genes involved in melanization, sporulation, or fungicide resistance.

Hygromycin B resistance as dominant selectable marker in yeast

1984 ◽  
Vol 8 (5) ◽  
pp. 353-358 ◽  
Author(s):  
Kevin R. Kaster ◽  
Stanley G. Burgett ◽  
Thomas D. Ingolia
Keyword(s):  
Selectable Marker ◽  
Hygromycin B ◽  
Hygromycin B Resistance ◽  
Dominant Selectable Marker

scholarly journals Use of a bacterial hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation

1989 ◽  
Vol 36 (1) ◽  
pp. 79 ◽  
Author(s):  
C. Staben ◽  
B. Jensen ◽  
M. Singer ◽  
J. Pollock ◽  
M. Schechtman ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Resistance Gene ◽  
Selectable Marker ◽  
Hygromycin B ◽  
Hygromycin B Resistance ◽  
Dominant Selectable Marker

scholarly journals Nourseothricin N-acetyl transferase (NAT), a new selectable marker for nuclear gene expression in Chlamydomonas

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Xinjia Yang ◽  
Jialin Peng ◽  
Junmin Pan
Keyword(s):  
Resistance Genes ◽  
Selectable Marker ◽  
Ectopic Expression ◽  
Nuclear Gene ◽  
Expression Vectors ◽  
Cross Resistance ◽  
Hygromycin B ◽  
Selectable Markers ◽  
Hygromycin B Resistance ◽  
Transgenic Strains

Abstract Background Chlamydomonas reinhardtii is a unicellular green alga, which is a most commonly used model organism for basic research and biotechnological applications. Generation of transgenic strains, which usually requires selectable markers, is instrumental in such studies/applications. Compared to other organisms, the number of selectable markers is limited in this organism. Nourseothricin (NTC) N-acetyl transferase (NAT) has been reported as a selectable marker in a variety of organisms but not including C. reinhardtii. Thus, we investigated whether NAT was useful and effective for selection of transgenic strains in C. reinhardtii. The successful use of NAT would provide alterative choice for selectable markers in this organism and likely in other microalgae. Results C. reinhardtii was sensitive to NTC at concentrations as low as 5 µg/ml. There was no cross-resistance to nourseothricin in strains that had been transformed with hygromycin B and/or paromomycin resistance genes. A codon-optimized NAT from Streptomyces noursei was synthesized and assembled into different expression vectors followed by transformation into Chlamydomonas. Around 500 transformants could be obtained by using 50 ng DNA on selection with 10 µg/ml NTC. The transformants exhibited normal growth rate and were stable at least for 10 months on conditions even without selection. We successfully tested that NAT could be used as a selectable marker for ectopic expression of IFT54-HA in strains with paromomycin and hygromycin B resistance markers. We further showed that the selection rate for IFT54-HA positive clones was greatly increased by fusing IFT54-HA to NAT and processing with the FMDV 2A peptide. Conclusions This work represents the first demonstration of stable expression of NAT in the nuclear genome of C. reinhardtii and provides evidence that NAT can be used as an effective selectable marker for transgenic strains. It provides alterative choice for selectable markers in C. reinhardtii. NAT is compatible with paromomycin and hygromycin B resistance genes, which allows for multiple selections.

scholarly journals Instability of Propiconazole Resistance and Fitness in Monilinia fructicola

2007 ◽  
Vol 97 (4) ◽  
pp. 448-453 ◽  
Author(s):  
K. D. Cox ◽  
P. K. Bryson ◽  
G. Schnabel
Keyword(s):  
Growth Rate ◽  
Cold Storage ◽  
Spore Production ◽  
Potato Dextrose Agar ◽  
Monilinia Fructicola ◽  
Relative Growth ◽  
Germination And Growth ◽  
Time Of Collection ◽  
Demethylation Inhibitor

The fitness and the dynamics of demethylation inhibitor fungicide (DMI) sensitivity in isolates of Monilinia fructicola sensitive (no growth at 0.3 mg/liter propiconazole) and resistant (≥50% relative growth at 0.3 mg/liter propiconazole) to propiconazole were investigated. Overall, there was no considerable compromise in the fitness of resistant isolates compared to sensitive isolates of M. fructicola at the time of collection. Resistant and sensitive isolates differed in their sensitivity to propiconazole (P < 0.001) and incubation period (P = 0.044), but not in latent period, growth rate, spore production, and spore germination frequency (P > 0.05). Consecutive transferring on potato dextrose agar had an impact on conidia production, conidial germination, and growth rate (P < 0.0001). Consecutive transferring also had an impact on propiconazole sensitivity in resistant isolates. In the resistant isolates, sensitivity to propiconazole increased (R2 = 0.960, P = 0.0034) within the first eight transfers. Similarly, sensitivity to propiconazole increased by 273% over the course of 34 months in cold storage in propiconazole-resistant isolates. Our results show that propiconazole resistance is unstable in vitro and that standard subculturing and cold storage procedures impact propiconazole sensitivity of resistant isolates. The instability of propiconazole resistance in M. fructicola may have important implications for disease management in that a reversion to propiconazole sensitivity could potentially occur in the absence of DMI fungicide pressure in the field.

scholarly journals Agrobacterium-Mediated Transformation of Fusarium oxysporum: An Efficient Tool for Insertional Mutagenesis and Gene Transfer

2001 ◽  
Vol 91 (2) ◽  
pp. 173-180 ◽  
Author(s):  
E. D. Mullins ◽  
X. Chen ◽  
P. Romaine ◽  
R. Raina ◽  
D. M. Geiser ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fusarium Oxysporum ◽  
Fungal Pathogens ◽  
Insertional Mutagenesis ◽  
Genetic Manipulation ◽  
Fungal Spores ◽  
Hygromycin B ◽  
Fungal Transformation ◽  
Efficient Tool ◽  
Binary Vectors

Agrobacterium tumefaciens-mediated transformation (ATMT) has long been used to transfer genes to a wide variety of plants and has also served as an efficient tool for insertional mutagenesis. In this paper, we report the construction of four novel binary vectors for fungal transformation and the optimization of an ATMT protocol for insertional mutagenesis, which permits an efficient genetic manipulation of Fusarium oxysporum and other phytopathogenic fungi to be achieved. Employing the binary vectors, carrying the bacterial hygromycin B phosphotrans-ferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 300 to 500 hygromycin B resistant transformants per 1 × 106 conidia of F. oxysporum, which is at least an order of magnitude higher than that previously accomplished. Transformation efficiency correlated strongly with the duration of cocultivation of fungal spores with Agrobacterium tumefaciens cells and significantly with the number of Agrobacteruium tumefaciens cells present during the cocultivation period (r = 0.996; n = 3; P < 0.01). All transformants tested remained mitotically stable, maintaining their hygromycin B resistance. Growing Agrobacterium tumefaciens cells in the presence of acetosyringone (AS) prior to cocultivation shortened the time required for the formation of transformants but decreased to 53% the percentage of transformants containing a single T-DNA insert per genome. This increased to over 80% when Agrobacterium tumefaciens cells grown in the absence of AS were used. There was no correlation between the average copy number of T-DNA per genome and the colony diameter of the transformants, the period of cocultivation or the quantity of Agrobacterium tumefaciens cells present during cocultivation. To isolate the host sequences flanking the inserted T-DNA, we employed a modified thermal asymmetric interlaced PCR (TAIL-PCR) technique. Utilizing just one arbitrary primer resulted in the successful amplification of desired products in 90% of those transformants analyzed. The insertion event appeared to be a random process with truncation of the inserted T-DNA, ranging from 1 to 14 bp in size, occurring on both the right and left border sequences. Considering the size and design of the vectors described here, coupled with the efficiency and flexibility of this ATMT protocol, it is suggested that ATMT should be regarded as a highly efficient alternative to other DNA transfer procedures in characterizing those genes important for the pathogenicity of F. oxysporum and potentially those of other fungal pathogens.

Insertional mutagenesis in Coprinus cinereus : use of a dominant selectable marker to generate tagged, sporulation-defective mutants

1999 ◽  
Vol 36 (6) ◽  
pp. 371-382 ◽  
Author(s):  
W. Jason Cummings ◽  
Martina Celerin ◽  
Jennifer Crodian ◽  
Linda K. Brunick ◽  
Miriam E. Zolan
Keyword(s):  
Insertional Mutagenesis ◽  
Selectable Marker ◽  
Coprinus Cinereus ◽  
Dominant Selectable Marker

Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells

1984 ◽  
Vol 4 (12) ◽  
pp. 2929-2931 ◽  
Author(s):  
K Blochlinger ◽  
H Diggelmann
Keyword(s):  
Selectable Marker ◽  
Dna Transfer ◽  
Direct Selection ◽  
Regulatory Sequences ◽  
Hygromycin B ◽  
Dominant Marker ◽  
Dna Coding ◽  
Sarcoma Virus ◽  
Moloney Sarcoma Virus ◽  
Selection For

The DNA coding sequence for the hygromycin B phosphotransferase gene was placed under the control of the regulatory sequences of a cloned long terminal repeat of Moloney sarcoma virus. This construction allowed direct selection for hygromycin B resistance after transfection of eucaryotic cell lines not naturally resistant to this antibiotic, thus providing another dominant marker for DNA transfer in eucaryotic cells.

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