No abstract

Orchidaceae, one of the largest angiosperm families, has significant commercial value. Isolation of genes involved in orchid floral development and morphogenesis, scent production, and colouration will advance knowledge of orchid flower formation and facilitate breeding new varieties to increase the commercial value. With high-throughput virus-induced gene silencing (VIGS), this study identified five transcription factors involved in various aspects of flower morphogenesis in the orchid Phalaenopsis equestris. These genes are PeMADS1, PeMADS7, PeHB, PebHLH, and PeZIP. Silencing PeMADS1 and PebHLH resulted in reduced flower size together with a pelaloid column containing petal-like epidermal cells and alterations of epidermal cell arrangement in lip lateral lobes, respectively. Silencing PeMADS7, PeHB, and PeZIP alone resulted in abortion of the first three fully developed flower buds of an inflorescence, which indicates the roles of the genes in late flower development. Furthermore, double silencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, respectively, produced a combinatorial phenotype with two genes cloned in separate vectors. Both PeMADS1 and PeMADS6 are required to ensure the normal development of the lip and column as well as the cuticle formation on the floral epidermal cell surface. Thus, VIGS allows for unravelling the interaction between two classes of MADS transcription factors for dictating orchid floral morphogenesis.

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.