A High-Throughput Virus-Induced Gene-Silencing Vector for Screening Transcription Factors in Virus-Induced Plant Defense Response in Orchid

Abstract

The large number of species and worldwide spread of species of Orchidaceae indicates their successful adaptation to environmental stresses. Thus, orchids provide rich resources to study how plants have evolved to cope with stresses. This report describes our improvement of our previously reported orchid virus-induced gene silencing vector, pCymMV-pro60, with a modified Gateway cloning system which requires only one recombination and can be inoculated by agroinfiltration. We cloned 1,700 DNA fragments, including 187 predicted transcription factors derived from an established expression sequence tag library of orchid, into pCymMV-Gateway. Phalaenopsis aphrodite was inoculated with these vectors that contained DNA fragments of the 187 predicted transcription factors. The viral vector initially triggered the expression of the salicylic acid (SA)-related plant defense responses and later induced silencing of the endogenous target transcription factor genes. By monitoring the expression of the SA-related plant defense marker PhaPR1 (homolog of PR1), we identified a gene, PhaTF15, involved in the expression of PhaPR1. Knockdown of PhaTF15 by virus-induced gene silencing and by transient delivery of double-stranded RNA (dsRNA) reduced expression of the orchid homolog of the conserved positive defense regulator NPR1, PhaNPR1. Cymbidium mosaic virus also accumulated to high levels with knockdown of PhaTF15 by transient delivery of dsRNA. We demonstrated efficient cloning and screening strategies for high-throughput analysis of orchid and identify a gene, PhaTF15, involved in regulation of SA-related plant defense.