Evaluation of Different RNA Extraction Methods from Agropatch Suppressor Assay for Small Quantities of Plant Tissue and Their Application for Analysis of Gene Expression

The agroinfiltration assay provides fast and efficient way to transiently express genes into plant cells by Agrobacterium tumefaciens. Extraction of RNA of high quality and sufficient amounts is prerequisite for gene expression studies such as quantitative Real Time PCR (q-PCR) from infiltrated areas in agropatch suppressor assay with small quantities of plant tissue. To attain prime RNA extraction from small tissues of infiltrated N. benthamiana plants with Potato virus A helper component proteinase viral suppressor protein, the efficiency of three RNA extraction methods (LiCl, TRIzol reagent and commercial kit) was evaluated. The total RNA yield with LiCl method was 2.83 and 33.2-fold greater than that of TRIzol reagent and commercial kit, respectively. Also, total RNA yield using TRIzol reagent was 11.7-fold higher than that with commercial kit. The A260/A280 ratio mean for TRI reagent (1.95) and kit (1.9) extractions were within the optimum range.q-PCR revealed that the cycle threshold values of housekeeping gene, EIF-1α and target genes AGO1 and ATG6 for RNA extracted using LiCl and kit were 1.07 to 1.3 and 1.02 to 1.12 times higher than those evaluated with the TRIzol method. Overall, TRIzol method showed the most effective approach for obtaining RNA from N. benthamiana patches in gene expression studies

Regulatory Network Identification, Promoter and Expression Analysis of Arabidopsis thaliana NPR1 in Defense Responses against Stresses

Salicylic acid (SA) and jasmonic acid (JA) phytohormones have been known for their roles in plant defense behaviour against biotic and abiotic stresses. They regulate defense pathways by antagonistic interaction. NPR1 as a key regulatory factor in the cross-talk between SA and JA, signaling is essential for the inhibition of JA-responsive gene expression by SA. In silico promoter analysis of 1.5 kb promoter regions of NPR1 gene revealed that NPR1 contains 23 MYB and 20 WRKY transcription factor binding sites. Different cis-elements associated with various stress responses were identified in Arabidopsis thaliana NPR1. The most common element was allocated to the defense responses against biotic stresses. Based on gene network analysis, NPR1, TGA2 and TGA3 were predicted to have functional cooperation with each other. Affymetrix microarray data analysis of A. thaliana under SA treatment demonstrated that most genes involved in NPR1 network are up-regulated under SA treatment. Therefore, interaction and cooperation between these factors might serve to fine-tune regulation of defense and immune responses against biotic and abiotic stresses