Over-expression of GbRac1 gene in transgenic tobacco enhances disease resistance to Alternaria alternata in vitro

2005 ◽  
Vol 2 (2) ◽  
pp. 73-77 ◽  
Author(s):  
Li Wei-Min ◽  
Wang Zhi-Xing ◽  
Jia Shi-Rong
Keyword(s):  
Disease Resistance ◽  
Transgenic Plants ◽  
Alternaria Alternata ◽  
Northern Blot Analysis ◽  
Challenge Test ◽  
Constitutive Expression ◽  
Degenerate Primers ◽  
Over Expression ◽  
Detached Leaves

AbstractGbRac1 gene was cloned from Gossypium barbadense with degenerate primers and 3′-RACE. Northern blot analysis indicated that GbRac1 mRNA was expressed abundantly in G. barbadense seedlings inoculated with Verticillium dehliae compared with mock-inoculated plants. A plant constitutive expression vector pRac harbouring GbRac1 gene was constructed and leaf discs of tobacco (Nicotiana tabacum L. cv. NC89) were transformed with pRac by Agrobacterium-mediated transformation. Disease challenge test of detached leaves of the transgenic plants by inoculation with Alternaria alternata showed that resistance was enhanced dramatically compared with the non-transgenic plants. Results suggest that GbRac1 gene might have potential application in the genetic engineering of plants with enhanced disease resistance.

scholarly journals Constitutive expression of transcription factor SirZ blocks pathogenicity in Leptosphaeria maculans independently of sirodesmin production

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252333
Author(s):  
Andrew S. Urquhart ◽  
Candace E. Elliott ◽  
Wei Zeng ◽  
Alexander Idnurm
Keyword(s):  
Transcription Factor ◽  
Pathogenic Fungus ◽  
Leptosphaeria Maculans ◽  
Constitutive Expression ◽  
Blackleg Disease ◽  
Gene Encoding ◽  
Over Expression ◽  
Expression Construct ◽  
New Strategies

Sirodesmin, the major secondary metabolite produced by the plant pathogenic fungus Leptosphaeria maculans in vitro, has been linked to disease on Brassica species since the 1970s, and yet its role has remained ambiguous. Re-examination of gene expression data revealed that all previously described genes and two newly identified genes within the sir gene cluster in the genome are down-regulated during the crucial early establishment stages of blackleg disease on Brassica napus. To test if this is a strategy employed by the fungus to avoid damage to and then detection by the host plant during the L. maculans asymptomatic biotrophic phase, sirodesmin was produced constitutively by overexpressing the sirZ gene encoding the transcription factor that coordinates the regulation of the other genes in the sir cluster. The sirZ over-expression strains had a major reduction in pathogenicity. Mutation of the over-expression construct restored pathogenicity. However, mutation of two genes, sirP and sirG, required for specific steps in the sirodesmin biosynthesis pathway, in the sirZ over-expression background resulted in strains that were unable to synthesize sirodesmin, yet were still non-pathogenic. Elucidating the basis for this pathogenicity defect or finding ways to overexpress sirZ during disease may provide new strategies for the control of blackleg disease.

scholarly journals In Vitro Mutagenesis followed by Polymorphism Detection Using Start Codon Targeted Markers to Engineer Brown Spot Resistance in Kalanchoe

2019 ◽  
Vol 144 (3) ◽  
pp. 193-200
Author(s):  
Rui Li ◽  
Lu Fan ◽  
Jingdong Lin ◽  
Mingyang Li ◽  
Daofeng Liu ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Brown Spot ◽  
Start Codon ◽  
Chemical Mutagenesis ◽  
In Vitro Mutagenesis ◽  
Aesthetic Value ◽  
Disease Resistant ◽  
Detached Leaves ◽  
Mutant Lines

Kalanchoe (Kalanchoe blossfeldiana) is a common potted flower that is popular throughout the world. Brown spot (caused by Stemphylium lycopersici) is one of the common foliage diseases in kalanchoe. This disease tends to infect leaves of kalanchoe plants in hot and humid environments, reducing their aesthetic value. The current investigation aimed to generate mutations resistant to brown spot in ‘Mary’ kalanchoe through chemical mutagenesis followed by molecular marker identification. Putative mutants were developed by treating embryogenic calluses with ethyl methanesulfonate (EMS) at median lethal doses (LD50)–either a 0.8% concentration for 2 hours or a 1.0% concentration for 0.5 hours. Brown spot crude toxin solution was used as the selection agent to identify disease-resistant calluses during tissue culture. The optimal crude concentration (60%) was determined by soaking calluses with different concentrations of crude pathogen: 0%, 20%, 40%, 60%, and 80% (v/v). A total of 32 anti-brown spot lines were regenerated and tested for disease resistance with detached leaves. Three regenerated EMS mutant lines showed no obvious brown spot lesions on their leaves after the disease resistance assay and were subjected to polymorphism identification by start codon targeted (SCoT) molecular markers. Three (SCoT40, SCoT71, and SCoT72) of 45 selected primers were chosen to identify the mutants. This work may lay the foundation for further development of new disease-resistant cultivars of kalanchoe.

Cloning of a Na+-driven Cl/HCO3 exchanger from squid giant fiber lobe

2003 ◽  
Vol 285 (4) ◽  
pp. C771-C780 ◽  
Author(s):  
Leila V. Virkki ◽  
Inyeong Choi ◽  
Bruce A. Davis ◽  
Walter F. Boron
Keyword(s):  
Northern Blot Analysis ◽  
Stellate Ganglion ◽  
Disulfonic Acid ◽  
Giant Fiber ◽  
Slow Recovery ◽  
Degenerate Primers ◽  
Bicarbonate Transporter ◽  
Loligo Pealei ◽  
Full Length Clone

We extracted RNA from the giant fiber lobe (GFL) of the squid Loligo pealei and performed PCR with degenerate primers that were based on highly conserved regions of Na+-coupled HCO3- transporters. This approach yielded a novel, 290-bp sequence related to the bicarbonate transporter superfamily. Using an L. opalescens library, we extended the initial fragment in the 3′ and 5′ directions by a combination of library screening and PCR and obtained the full-length clone (1,198 amino acids) by PCR from L. pealei GFL. The amino acid sequence is 46% identical to mammalian electrogenic and electroneutral Na-HCO3 cotransporters and 33% identical to the anion exchanger AE1. Northern blot analysis showed strong signals in L. pealei GFL, optic lobe, and heart and weaker signals in gill and stellate ganglion. To assess function, we injected in vitro-transcribed cRNA into Xenopus oocytes and subsequently used microelectrodes to monitor intracellular pH (pHi) and membrane voltage ( Vm). Superfusing these oocytes with 5% CO2-33 mM HCO3- caused a CO2-induced fall in pHi, followed by a slow recovery. The absence of a rapid HCO3--induced hyperpolarization indicates that the pHi recovery mechanism is electroneutral. Ion substitutions showed that Na+ and Cl- are required on opposite sides of the membrane. Transport was blocked by 50 μM 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The characteristics of our novel clone fit those of a Na+-driven Cl/HCO3 exchanger (NDCBE).

scholarly journals Constitutive Expression of Pea Defense Gene DRR206 Confers Resistance to Blackleg (Leptosphaeria maculans) Disease in Transgenic Canola (Brassica napus)

1999 ◽  
Vol 12 (5) ◽  
pp. 410-418 ◽  
Author(s):  
Yaping Wang ◽  
Goska Nowak ◽  
David Culley ◽  
Lee A. Hadwiger ◽  
Brian Fristensky
Keyword(s):  
Brassica Napus ◽  
Transgenic Plants ◽  
Leptosphaeria Maculans ◽  
Constitutive Expression ◽  
Single Copy ◽  
Cauliflower Mosaic Virus ◽  
Adult Plant ◽  
35S Promoter ◽  
Transgenic Lines

To identify genes effective against the blackleg fungus Leptosphaeria maculans (Phoma lingam), we have transformed canola (Brassica napus) with four pea (Pisum sativum) genes under constitutive control by the cauliflower mosaic virus 35S promoter: PR10.1, chitinase, DRR206, and defensin. Transgenic lines containing single-copy T-DNA insertions for each gene were screened for both seedling (cotyledonary) and adult plant resistance. Lines for which pea DRR206 mRNA was expressed showed decreased disease scores, compared with non-expressing transgenic lines. Transgenic plants expressing pea defensin showed a slight enhancement of resistance, while for PR10 and chitinase transgenics there was little or no enhancement of resistance. Resistance to L. maculans cosegregated with DRR206 transgenes. Extracts from DRR206 and defensin transgenic plants inhibited fungal germination in vitro. DRR206 transgenic plants also demonstrated decreased hyphal growth at inoculation sites. While the precise function of DRR206 remains to be determined, these results suggest that it does play an important role in defense against fungi.

scholarly journals Rapid Screening of Musa Species for Resistance to Black Leaf Streak Using In Vitro Plantlets in Tubes and Detached Leaves

Plant Disease ◽  
2007 ◽  
Vol 91 (3) ◽  
pp. 308-314 ◽  
Author(s):  
M. Twizeyimana ◽  
P. S. Ojiambo ◽  
A. Tenkouano ◽  
T. Ikotun ◽  
R. Bandyopadhyay
Keyword(s):  
Disease Resistance ◽  
Leaf Area ◽  
Incubation Time ◽  
T Test ◽  
Infected Leaf ◽  
Strongly Correlated ◽  
In Vitro Plantlets ◽  
Detached Leaves ◽  
Black Leaf Streak

This study investigated the utility of inoculation of in vitro plantlets in tubes and detached leaves as reliable and rapid assays for screening Musa genotypes against Mycosphaerella fijiensis, the causal agent of black leaf streak. In the first part of the study, three types of inocula were evaluated to determine suitability for in vitro inoculation. Inoculation of in vitro plantlets with mycelial fragments resulted in significantly (P < 0.05) higher levels of disease severity and faster rates of disease progress compared with inoculations using conidial suspensions. In the detached leaf assay, amending agar medium with plant hormones significantly (P < 0.0001) aided retention of green leaf color. Leaf pieces on medium containing gibberellic acid at 5 mg/liter had about 5% chlorosis at 52 days after plating. When in vitro plantlets in tubes and detached leaves of 10 Musa genotypes with different levels of disease resistance were inoculated with M. fijiensis, there were significant (P < 0.05) differences among genotypes in leaf area infected, incubation time, and symptom evolution time. For incubation time and leaf area infected, cultivars responded depending on their level of disease resistance, with resistant genotypes Calcutta-4 and PITA-17 having significantly (P = 0.001) longer incubation times and lower infected leaf areas compared with the susceptible cultivar Agbagba and moderately resistant cultivar FHIA-23. A similar pattern in cultivar response was observed for symptom evolution time. Leaf area infected was not significantly (P = 0.2817 for two-tailed t test) different when assessed using the two assays, and infected leaf areas in both assays were strongly correlated (r = 0.88, n = 48, P < 0.0001). Although incubation times were significantly (P = 0.0062 for two-tailed t test) different between the two assays, values from the two assays were strongly correlated (r = 0.69, n = 48, P < 0.0001). These results show that these two assays are rapid and space-effective, and can reliably be used for screening Musa genotypes for resistance to black leaf streak.

scholarly journals Over-Expression of Endogenous SUGARWIN Genes Exalted Tolerance against Colletotrichum Infection in Sugarcane

Plants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 869
Author(s):  
Aqsa Parvaiz ◽  
Ghulam Mustafa ◽  
Muhammad Sarwar Khan ◽  
Muhammad Amjad Ali
Keyword(s):  
Transgenic Plants ◽  
Fungal Pathogens ◽  
Red Rot ◽  
Colletotrichum Falcatum ◽  
Molecular Tools ◽  
Embryogenic Calli ◽  
Over Expression ◽  
Potential Source ◽  
First Time

Sugarcane being the major contributor of sugar and potential source of biofuel around the globe, occupies significant commercial importance. Red rot is the most devastating disease of sugarcane, severely affecting its quality as well as yield. Here we report the overexpression of SUGARWIN1 and SUGARWIN2 genes in any field crop for the first time. For this purpose, SUGAWIN1 and SUGARWIN2 were cloned downstream of maize ubiquitin (Ubi-1) promoter to construct two independent expression cassettes. The bar gene conferring resistance against phosphinothricin was used as selectable marker. Embryogenic calli of sugarcane were bombarded with both expression cassettes and selected on regeneration medium supplemented with phosphinothricin. The phosphinothricin-resistant shoots were rooted and then, analyzed using molecular tools at the genomic as well as transcriptomic levels. The transcriptomic analysis, using real time qPCR, showed that expression of SUGARWIN1 (SWO) and SUGARWIN2 (SWT) was higher in transgenic plants as compared to untransformed plants. Our results further demonstrated that over expression of these genes under maize ubiquitin (Ubi-1) promoter causes significant restriction in proliferation of red rot causal agent, Colletotrichum falcatum in sugarcane transgenic plants, under in vitro conditions. This report may open up exciting possibilities to extend this technology to other monocots for the development of crops with better ability to withstand fungal pathogens.

1442: CXCL12 Over-Expression and Secretion by Aging Fibroblasts Stimulates Human Prostate Epithelial Proliferation in Vitro

2006 ◽  
Vol 175 (4S) ◽  
pp. 466-466
Author(s):  
Jill A. Macoska ◽  
Lesa Begley ◽  
Christine Monteleon ◽  
James W. MacDonald ◽  
Rajal B. Shah
Keyword(s):  
Human Prostate ◽  
Epithelial Proliferation ◽  
Over Expression ◽  
Proliferation In Vitro

Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon

2020 ◽  
Vol 139 ◽  
pp. 153-160
Author(s):  
S Peeralil ◽  
TC Joseph ◽  
V Murugadas ◽  
PG Akhilnath ◽  
VN Sreejith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virulence Genes ◽  
Vibrio Harveyi ◽  
Penaeus Monodon ◽  
Challenge Test ◽  
Virulence Gene ◽  
Economic Losses ◽  
Virulence Gene Expression

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

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