Computational protocol: An Asparagine-Rich Protein Nbnrp1 Modulate Verticillium dahliae Protein PevD1-Induced Cell Death and Disease Resistance in Nicotiana benthamiana

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Protocol publication

[…] To investigate the transcription of defense-related genes, Nbnrp1-overexpressing lines, Nbnrp1-silence lines and wild type plant leaves were infiltrated with 10 μM PevD1. The samples were collected from the upper leaves at the indicated times and then rapidly frozen in liquid nitrogen. Total RNA was extracted with the RNA prep pure Plant Kit (TIANGEN Biotech). Residual genomic DNA was eliminated by treatment with a gDNA Eraser. First-strand cDNA was synthesized from 100 ng of total RNA using reverse transcriptase (TIANGEN Biotech) according to the supplier’s protocol. Quantitative Real-time quantitative PCR (qPCR) was performed to determine the relative expression levels of several defense-related genes using SYBR Green PCR Master Mix (TIANGEN Biotech). Specific genes primers were designed according to the coding sequences of each gene using Beacon Designer 8. PCR mixture was processed on a Bio-Rad CFX Manager (Bio-Rad). Three technical replicates were amplified for each sample, including negative controls. EF-1a was used as an internal standard. Quantification of the relative changes in gene transcript levels was performed using the 2-ΔΔCT method (; ). […]

Pipeline specifications

Software tools Beacon Designer, CFX Manager
Application qPCR
Organisms Fungi, Verticillium dahliae, Gossypium hirsutum, Nicotiana tabacum, Arabidopsis thaliana, Tobacco mosaic virus, Bacteria, Pseudomonas syringae, Nicotiana benthamiana
Diseases Drug Hypersensitivity
Chemicals Amino Acids