Journal: Life

Article Title: Transient Overexpression of the Pepper WRKY2 Gene in Nicotiana benthamiana Markedly Delays the Systemic Necrosis Caused by Tobacco Mosaic Virus

doi: 10.3390/life15040669

Figure Lengend Snippet: Sequences of primer pairs in 5′ to 3′ directions used for semi-quantitative PCR and qPCR investigations. Gene identification numbers are shown in parenthesis (databases: NCBI and Solanaceae Genomics Network).

Article Snippet: The PCR program consisted of using a temperature of 94 °C for 2 min, and then 25 cycles as follows: 94 °C for 0:45 min, 52 °C for 0:45 min, 72 °C for 2:30 min, and finally, incubation at 72 °C for 10 min. From the positive E. coli TOP10 colonies, the entry vector plasmids carrying the CaWRKY2 gene were purified by MiniPrep Express Matrix (MP Biomedicals, Irvine, CA, USA).

Techniques:

Journal: Life

Article Title: Transient Overexpression of the Pepper WRKY2 Gene in Nicotiana benthamiana Markedly Delays the Systemic Necrosis Caused by Tobacco Mosaic Virus

doi: 10.3390/life15040669

Figure Lengend Snippet: The effects of Obuda pepper virus (ObPV), pepper mild mottle virus (PMMoV), and mock-inoculations on the transcript abundance of the CaWRKY2 gene in inoculated pepper leaves at various time points following inoculation as detected by RT-qPCR with the specific primer pair CaWRKY2 . The expression of a ubiquitin gene ( UBI-3 ) was examined as a control housekeeping gene. The PCR conditions and GenBank accession numbers of all genes are shown in . The open, gray, and black columns represent mock-, ObPV-, and PMMoV-inoculated leaves, respectively. The mean values of three independent experiments ± SD are shown. The symbols *, **, and *** show significant differences between mock- and virus-inoculated plants at p < 5%, < 1%, and < 0.1%, respectively.

Article Snippet: The PCR program consisted of using a temperature of 94 °C for 2 min, and then 25 cycles as follows: 94 °C for 0:45 min, 52 °C for 0:45 min, 72 °C for 2:30 min, and finally, incubation at 72 °C for 10 min. From the positive E. coli TOP10 colonies, the entry vector plasmids carrying the CaWRKY2 gene were purified by MiniPrep Express Matrix (MP Biomedicals, Irvine, CA, USA).

Techniques: Virus, Quantitative RT-PCR, Expressing, Control

Journal: Life

Article Title: Transient Overexpression of the Pepper WRKY2 Gene in Nicotiana benthamiana Markedly Delays the Systemic Necrosis Caused by Tobacco Mosaic Virus

doi: 10.3390/life15040669

Figure Lengend Snippet: Expression of CaWRKY2 gene in transiently transformed N. benthamiana leaves. CaWRKY2 expression detected by RT-qPCR 3–10 days after agroinfiltration with specific primer pair CaWRKY2rt . Means of three independent parallel experiments ± SD are shown.

Article Snippet: The PCR program consisted of using a temperature of 94 °C for 2 min, and then 25 cycles as follows: 94 °C for 0:45 min, 52 °C for 0:45 min, 72 °C for 2:30 min, and finally, incubation at 72 °C for 10 min. From the positive E. coli TOP10 colonies, the entry vector plasmids carrying the CaWRKY2 gene were purified by MiniPrep Express Matrix (MP Biomedicals, Irvine, CA, USA).

Techniques: Expressing, Transformation Assay, Quantitative RT-PCR

Journal: Life

Article Title: Transient Overexpression of the Pepper WRKY2 Gene in Nicotiana benthamiana Markedly Delays the Systemic Necrosis Caused by Tobacco Mosaic Virus

doi: 10.3390/life15040669

Figure Lengend Snippet: The monitoring of TMV replication in N. benthamiana leaves overexpressing CaWRKY2 and in control leaves carrying an empty expression vector at various time points after TMV inoculation. The expression of the coat protein gene ( CP ) of TMV was detected by RT-PCR with the specific primer pair TMV-CP . The expression of an actin was also detected as a constitutive control gene. The representative results of three independent parallel experiments are shown.

Article Snippet: The PCR program consisted of using a temperature of 94 °C for 2 min, and then 25 cycles as follows: 94 °C for 0:45 min, 52 °C for 0:45 min, 72 °C for 2:30 min, and finally, incubation at 72 °C for 10 min. From the positive E. coli TOP10 colonies, the entry vector plasmids carrying the CaWRKY2 gene were purified by MiniPrep Express Matrix (MP Biomedicals, Irvine, CA, USA).

Techniques: Control, Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Life

Article Title: Transient Overexpression of the Pepper WRKY2 Gene in Nicotiana benthamiana Markedly Delays the Systemic Necrosis Caused by Tobacco Mosaic Virus

doi: 10.3390/life15040669

Figure Lengend Snippet: The effect of TMV inoculation on the transcript abundance of PR-1b and LOX1 genes in N. benthamiana leaves overexpressing CaWRKY2 and in empty vector control leaves at various time points after TMV inoculation, as detected by RT-qPCR. The expression values were normalized to those of the constitutive control actin gene and then related to empty vector leaf samples at 0 dpi. The mean values of three independent experiments ± SD are shown. The symbols * and ** show significant differences between CaWRKY2 -overexpressing and empty vector control plants at p < 5% and <1%, respectively.

Article Snippet: The PCR program consisted of using a temperature of 94 °C for 2 min, and then 25 cycles as follows: 94 °C for 0:45 min, 52 °C for 0:45 min, 72 °C for 2:30 min, and finally, incubation at 72 °C for 10 min. From the positive E. coli TOP10 colonies, the entry vector plasmids carrying the CaWRKY2 gene were purified by MiniPrep Express Matrix (MP Biomedicals, Irvine, CA, USA).

Techniques: Plasmid Preparation, Control, Quantitative RT-PCR, Expressing

Journal: Life

Article Title: Transient Overexpression of the Pepper WRKY2 Gene in Nicotiana benthamiana Markedly Delays the Systemic Necrosis Caused by Tobacco Mosaic Virus

doi: 10.3390/life15040669

Figure Lengend Snippet: Systemic necrosis caused by TMV inoculation in N. benthamiana plants transformed with CaWRKY2 gene or with empty expression vector at 7, 8, and 9 days after TMV inoculation (dpi). TMV inoculation was performed on three previously agroinfiltrated leaves. Experiment was performed in six replicates with same results.

Article Snippet: The PCR program consisted of using a temperature of 94 °C for 2 min, and then 25 cycles as follows: 94 °C for 0:45 min, 52 °C for 0:45 min, 72 °C for 2:30 min, and finally, incubation at 72 °C for 10 min. From the positive E. coli TOP10 colonies, the entry vector plasmids carrying the CaWRKY2 gene were purified by MiniPrep Express Matrix (MP Biomedicals, Irvine, CA, USA).

Techniques: Transformation Assay, Expressing, Plasmid Preparation