scholarly journals A Peroxidase Gene Promoter Induced by Phytopathogens and Methyl Jasmonate in Transgenic Plants

1997 ◽  
Vol 10 (3) ◽  
pp. 326-338 ◽  
Author(s):  
Mark D. Curtis ◽  
Anne L. Rae ◽  
Anca G. Rusu ◽  
Stuart J. Harrison ◽  
John M. Manners
Keyword(s):  
Transgenic Plants ◽  
Methyl Jasmonate ◽  
Fungal Pathogen ◽  
Gus Expression ◽  
Histochemical Staining ◽  
Gus Activity ◽  
Microbial Pathogens ◽  
Foreign Gene ◽  
Coding Region ◽  
Peroxidase Gene

The expression of two closely related peroxidase isogenes, Shpx6a and Shpx6b, of the legume Stylosanthes humilis was studied using isogene-specific reverse transcriptase PCR techniques. Results indicated that transcripts of both genes were rapidly induced following inoculation with the fungal pathogen Colletotrichum gloeosporioides, wounding and treatment with the defense regulator methyl jasmonate (MeJA). In contrast, treatment of leaves of S. humilis with abscisic acid (ABA) and salicylic acid (SA) did not induce transcripts of either isogene. A genomic clone containing the Shpx6b gene was isolated and 594 bp of 5′ sequence upstream of the translation start was fused in frame to the coding region of the uidA reporter gene and introduced into tobacco. Expression from the Shpx6b promoter in transgenic plants was determined by histochemical staining and quantitative assays of β-glucuronidase (GUS). In transgenic tobacco, GUS expression was detected in cotyledons, vascular cells of young leaves, anthers, pollen, and the stigma and style. Wounding of the tobacco plants produced very localized GUS staining. Much more extensive staining for GUS was observed following inoculation of tobacco leaves with conidia of the fungal pathogen Cercospora nicotianae and the inoculation of wound sites with mycelium of the Oomycete pathogen Phytophthora parasitica var. nicotianae. Treatment of mature leaves with methyl jasmonate induced GUS activity while treatment with ABA, SA, and H2O2 had no effect. A similar strong induction of GUS activity was measured in young transgenic seedlings germinated on MeJA while some, but much weaker, induction of GUS activity was observed in seedlings treated with SA. The sequence of the promoter contained motifs homologous to putative cis elements in other plant genes responsive to MeJA. The Shpx6b gene is the first plant peroxidase gene shown to be induced by both microbial pathogens and MeJA and its promoter will be useful for investigations of signaling processes during fungal infection and for the expression of foreign gene products at infection sites.

scholarly journals Activation of Latent Transgenes in Arabidopsis Using a Hybrid Transcription Factor

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 633-639 ◽  
Author(s):  
Dave Guyer ◽  
Ann Tuttle ◽  
Sabrina Rouse ◽  
Sandra Volrath ◽  
Marie Johnson ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Activation ◽  
Gus Expression ◽  
Histochemical Staining ◽  
Gus Activity ◽  
Synthetic Promoter ◽  
Cis Acting ◽  
Transcription Activation Domain ◽  
Adenylosuccinate Synthetase ◽  
Transgenic Lines

Abstract A hybrid transcription factor comprising a fusion of the DNA-binding domain of Saccharomyces cerevisiae GAL4 and the transcription activation domain of maize C1 was expressed in stably transformed Arabidopsis. Additional transgenic lines were created containing test genes controlled by a synthetic promoter consisting of concatemeric copies of the cis-acting site recognized by GAL4 (UASG) fused to a minimal promoter. The GAL4/C1 effector line was crossed to two lines containing a synthetic promoter/GUS fusion. Both histochemical staining and GUS activity assays indicate strong activation of GUS expression was achieved only after crossing. The GAL4/C1 effector line was also crossed to 15 lines containing a synthetic promoter/antisense adenylosuccinate synthetase gene. Severely retarded growth, and in some cases lethality, was observed in 40% of the F1 lines. This system of activation by crossing is generally useful for activating expression of test transgenes.

Improved electroporation buffer enhances transient gene expression in Arachis hypogaea protoplasts

Genome ◽  
1995 ◽  
Vol 38 (5) ◽  
pp. 858-863 ◽  
Author(s):  
Zhijian Li ◽  
Ming Cheng ◽  
James W. Demski ◽  
Robert L. Jarret
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Arachis Hypogaea ◽  
Transient Gene Expression ◽  
Gus Expression ◽  
Histochemical Staining ◽  
Media Analysis ◽  
Gus Activity ◽  
Arachis Hypogaea L ◽  
Protoplast Viability

An electroporation medium containing 50 mM glycine or 10 mM glycylglycine (glygly), 70 mM potassium glutamate, and 0.4 M mannitol was evaluated for its ability to improve transient β-glucuronidase (GUS) expression in immature cotyledonary protoplasts of Arachis hypogaea L. GUS activity in electroporated protoplasts was 8- to 430-fold greater than that obtained using any of other four commonly employed poration media. Analysis of viability and histochemical staining of protoplasts indicated that electroporation using the glycine- or glygly-based poration medium resulted in increased protoplast viability and GUS expression when compared with other poration media. Replacement of glygly with MES or HEPES buffers significantly reduced the level of GUS expression in electroporated protoplasts.Key words: transient expression, electroporation, Arachis, protoplasts, GUS.

scholarly journals Overexpression of a Redox-Regulated Cutinase Gene, MfCUT1, Increases Virulence of the Brown Rot Pathogen Monilinia fructicola on Prunus spp.

2010 ◽  
Vol 23 (2) ◽  
pp. 176-186 ◽  
Author(s):  
Miin-Huey Lee ◽  
Chiu-Min Chiu ◽  
Tatiana Roubtsova ◽  
Chien-Ming Chou ◽  
Richard M. Bostock
Keyword(s):  
Gene Expression ◽  
Redox Regulation ◽  
Brown Rot ◽  
Gus Expression ◽  
Apple Fruit ◽  
Gus Activity ◽  
Monilinia Fructicola ◽  
Gus Reporter ◽  
Flanking Regions ◽  
Prunus Spp

A 4.5-kb genomic DNA containing a Monilinia fructicola cutinase gene, MfCUT1, and its flanking regions were isolated and characterized. Sequence analysis revealed that the genomic MfCUT1 carries a 63-bp intron and a promoter region with several transcription factor binding sites that may confer redox regulation of MfCUT1 expression. Redox regulation is indicated by the effect of antioxidants, shown previously to inhibit MfCUT1 gene expression in cutin-induced cultures, and in the present study, where H2O2 enhanced MfCUT1 gene expression. A β-glucuronidase (GUS) reporter gene (gusA) was fused to MfCUT1 under the control of the MfCUT1 promoter, and this construct was then used to generate an MfCUT1-GUS strain by Agrobacterium spp.-mediated transformation. The appearance of GUS activity in response to cutin and suppression of GUS activity by glucose in cutinase-inducing medium verified that the MfCUT1-GUS fusion protein was expressed correctly under the control of the MfCUT1 promoter. MfCUT1-GUS expression was detected following inoculation of peach and apple fruit, peach flower petals, and onion epidermis, and during brown rot symptom development on nectarine fruit at a relatively late stage of infection (24 h postinoculation). However, semiquantitative reverse-transcriptase polymerase chain reaction provided sensitive detection of MfCUT1 expression within 5 h of inoculation in both almond and peach petals. MfCUT1-GUS transformants expressed MfCUT1 transcripts at twice the level as the wild type and caused more severe symptoms on Prunus flower petals, consistent with MfCUT1 contributing to the virulence of M. fructicola.

Agrobacterium tumefaciens-mediated Transformation of Double Haploid Canola (Brassica napus) Lines

1997 ◽  
Vol 24 (1) ◽  
pp. 97 ◽  
Author(s):  
K. Kazan ◽  
M. D. Curtis ◽  
K. C. Goulter ◽  
J. M. Manners
Keyword(s):  
Brassica Napus ◽  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Genetic Transformation ◽  
Double Haploid ◽  
Gene Promoter ◽  
Root Explants ◽  
Hypocotyl Explants ◽  
Peroxidase Gene ◽  
Hypocotyl Segments

Double haploid (DH) genotypes of canola (Brassica napus L.) have a high level of genetic uniformity but have not been previously tested for genetic transformation. Transgenic plants from three of four DH genotypes derived from cv. Westar were obtained by inoculation of either hypocotyl segments or root explants with Agrobacterium tumefaciens. For hypocotyl transformation, A. tumefaciens strain LBA4404 containing a binary plasmid with the neomycin phosphotransferase gene (nptII) and a CaMV 35S-peroxidase gene cassette was co-cultivated with hypocotyl segments taken from the 5–6-day-old seedlings. Transformation frequencies for hypocotyl explants of two DH genotypes were 0.3–3%. Direct evidence for genetic transformation of hypocotyl explants was obtained through molecular hybridisation analysis. Using this protocol, mature transformed plants were obtained within 4–6 months of co-cultivation. A method of root transformation was successfully modified for one DH genotype of canola and transgenic plants were obtained at a frequency of 2%. Using this protocol, a peroxidase gene promoter–GUS fusion construct was introduced into a DH genotype. Tissue specific GUS expression driven by the peroxidase gene promoter in transgenic plants was analysed by GUS staining. Transformation systems for double haploid canola lines will permit the assessment of introduced genes for their effect on agronomic and physiological traits.

scholarly journals Overexpression of Plasma Membrane H+-ATPase in Guard Cells Enhances Light-Induced Stomatal Opening, Photosynthesis, and Plant Growth in Hybrid Aspen

2021 ◽  
Vol 12 ◽  
Author(s):  
Shigeo Toh ◽  
Naoki Takata ◽  
Eigo Ando ◽  
Yosuke Toda ◽  
Yin Wang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transgenic Plants ◽  
Woody Plants ◽  
Stem Elongation ◽  
Guard Cells ◽  
Gus Expression ◽  
Stomatal Opening ◽  
Wild Type ◽  
Exchange System ◽  
Gas Exchange System

Stomata in the plant epidermis open in response to light and regulate CO2 uptake for photosynthesis and transpiration for uptake of water and nutrients from roots. Light-induced stomatal opening is mediated by activation of the plasma membrane (PM) H+-ATPase in guard cells. Overexpression of PM H+-ATPase in guard cells promotes light-induced stomatal opening, enhancing photosynthesis and growth in Arabidopsis thaliana. In this study, transgenic hybrid aspens overexpressing Arabidopsis PM H+-ATPase (AHA2) in guard cells under the strong guard cell promoter Arabidopsis GC1 (AtGC1) showed enhanced light-induced stomatal opening, photosynthesis, and growth. First, we confirmed that AtGC1 induces GUS expression specifically in guard cells in hybrid aspens. Thus, we produced AtGC1::AHA2 transgenic hybrid aspens and confirmed expression of AHA2 in AtGC1::AHA2 transgenic plants. In addition, AtGC1::AHA2 transgenic plants showed a higher PM H+-ATPase protein level in guard cells. Analysis using a gas exchange system revealed that transpiration and the photosynthetic rate were significantly increased in AtGC1::AHA2 transgenic aspen plants. AtGC1::AHA2 transgenic plants showed a>20% higher stem elongation rate than the wild type (WT). Therefore, overexpression of PM H+-ATPase in guard cells promotes the growth of perennial woody plants.

Effect of UTRs from TMV-RNA on the expression of foreign gene in transgenic plants

2000 ◽  
Vol 45 (18) ◽  
pp. 1681-1685
Author(s):  
Hongmin Qin ◽  
Hongnian Guo ◽  
Yantao Jia ◽  
Lihong Li ◽  
Yingchuan Tian
Keyword(s):  
Transgenic Plants ◽  
Foreign Gene

scholarly journals Differential Colonization of Tomato Roots by Nonpathogenic and Pathogenic Fusarium oxysporum Strains May Influence Fusarium Wilt Control

2001 ◽  
Vol 91 (5) ◽  
pp. 449-456 ◽  
Author(s):  
Jian R. Bao ◽  
George Lazarovits
Keyword(s):  
Fusarium Oxysporum ◽  
Pathogenic Fungus ◽  
Root Surface ◽  
Histochemical Staining ◽  
Gus Activity ◽  
Vascular Bundles ◽  
Interaction Sites ◽  
Inoculation Density ◽  
Tomato Roots ◽  
Cell Layers

Histochemical staining, β-glucuronidase (GUS) activity, or placing roots on agar were methods used to characterize interactions between the pathogenic fungus, Fusarium oxysporum f. sp. lycopersici, and the nonpathogenic biocontrol F. oxysporum strain 70T01 with respect to colonization behaviors, interaction sites, and population densities on tomato roots. Mycelia of strain 70T01, a genetic transformant expressing stable GUS activity, hygromycin B resistance, and effective disease control, were localized in epidermal and cortex cell layers of tomato roots in a discontinuous and uneven pattern. In contrast, mycelia of F. oxysporum f. sp. lycopersici were found in the vascular bundles. Thus, direct interactions between the two fungi likely happen in the root surface cell layers. Colonization density of strain 70T01 was related to the inoculation density but decreased with distance from the inoculation site. Host defense reactions, including increased cell wall thickness or papilla deposits, were adjacent to 70T01 hyphae. Experiments done in soil showed that strain 70T01 densities in roots were highest at inoculation zones and barely detectable for root segments more than 2 cm away from the inoculation sites. F. oxysporum f. sp. lycopersici densities were lowest at 70T01 inoculation zones and highest (>10 times) where strain 70T01 was not directly applied. Newly elongating roots where strain 70T01 did not reach were available for infection by the pathogen. The higher strain 70T01 density was always found when the plants were simultaneously infected by F. oxysporum f. sp. lycopersici, suggesting that F. oxysporum f. sp. lycopersici has as much influence in predisposing the plant to colonization by strain 70T01 as strain 70T01 has on providing disease protection against the pathogen.

scholarly journals Functional Analysis of the Metallothionein Gene cgMT1 Isolated from the Actinorhizal Tree Casuarina glauca

2007 ◽  
Vol 20 (10) ◽  
pp. 1231-1240 ◽  
Author(s):  
Mariana Obertello ◽  
Luis Wall ◽  
Laurent Laplaze ◽  
Michel Nicole ◽  
Florence Auguy ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Xanthomonas Campestris ◽  
Virulent Strain ◽  
Lateral Roots ◽  
Bacterial Pathogen ◽  
Gus Expression ◽  
35S Promoter ◽  
Nitrogen Fixing ◽  
Symbiotic Interaction ◽  
Casuarina Glauca

cgMT1 is a metallothionein (MT)-like gene that was isolated from a cDNA library of young nitrogen-fixing nodules resulting from the symbiotic interaction between Frankia spp. and the actinorhizal tree Casuarina glauca. cgMT1 is highly transcribed in the lateral roots and nitrogen-fixing cells of actinorhizal nodules; it encodes a class I type 1 MT. To obtain insight into the function of cgMT1, we studied factors regulating the expression of the MT promoter region (PcgMT1) using a β-glucuronidase (gus) fusion approach in transgenic plants of Arabidopsis thaliana. We found that copper, zinc, and cadmium ions had no significant effect on the regulation of PcgMT1-gus expression whereas wounding and H2O2 treatments led to an increase in reporter gene activity in transgenic leaves. Strong PcgMT1-gus expression also was observed when transgenic plants were inoculated with a virulent strain of the bacterial pathogen Xanthomonas campestris pv. campestris. Transgenic Arabidopsis plants expressing cgMT1 under the control of the constitutive 35S promoter were characterized by reduced accumulation of H2O2 when leaves were wounded and by increased susceptibility to the bacterial pathogen X. campestris. These results suggest that cgMT1 could play a role during the oxidative response linked to biotic and abiotic stresses.

scholarly journals A flexible target-specific anti-infection therapeutic platform that can be applied to different microbial species

2020 ◽  
Author(s):  
Makoto Mitsunaga ◽  
Kimihiro Ito ◽  
Takashi Nishimura ◽  
Hironori Miyata ◽  
Yoshimitsu Mizunoe ◽  
...  
Keyword(s):  
Binding Sites ◽  
Fungal Pathogen ◽  
Animal Experiments ◽  
Microbial Pathogens ◽  
Drug Resistant ◽  
Viral Particles ◽  
New Type ◽  
Resistant Strains ◽  
Therapeutic Platform ◽  
Drug Resistant Strains

AbstractThe emergence of new microbial pathogens, including drug-resistant strains, complicates treatment, thereby threatening global health. We demonstrated a photoimmuno-antimicrobial strategy (PIAS) that eliminated antibody-targets using a photo-activated anti-pathogen antibody generating mechanical stress that damaged the target’s binding sites. PIAS is effective against many pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), the fungal pathogen Candida albicans, and viral particles irrespective of their species or drug-resistance status. Animal experiments demonstrated that PIAS saved mice from fatal infections; microbiome and histochemical analyses indicated no apparent effect on normal host microflora and tissues in PIAS-treated mice. Resistance to PIAS was not observed during the eight years of this study. As a new type of anti-infection therapy, PIAS may contribute to advances in anti-infection strategies.

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