Application of VIGS to floral gene function studies of the orchid, a nonmodel plant

Orchidaceae is one of the largest and most diverse families of flowering plants. It occupies a wide range of ecological habitats and exhibits highly specialized morphological, structural, and physiological characteristics. Thus, orchid reproductive biology is of special interest in terms of floral color, morphology, size, and number of flowers. Virus-induced gene silencing (VIGS) is a reverse genetic approach used for functional analysis of plant genes, especially those with long life cycles and having few genetic resources, such as orchids. In this study, the modified Cymbidium mosaic virus was used as a VIGS-based vector. To explore the applicability and improve the viral silencing efficiency for demarcated functional genes in Phalaenopsis, this VIGS vector was used for silencing PeUFGT3, PeMADS5, and PeMADS6, and induced significant phenotypes with a silencing efficiency of up to 95.8% reduction in target gene expressions. In addition, the size of target gene fragments for inserting into VIGS vectors can be as small as 79-85 bp and can still reach 61.5%-95.8% reduction. The silencing efficiency of PeMADS6 has been increased significantly when the inflorescences of the Phalaenopsis plants contained eight internodes with one visible floral bud used as experimental materials. Furthermore, leaf injection saves time and causes less damage to inflorescences for agroinfiltration, and there is no difference between leaf and inflorescence injection for efficiencies of virus infection and silenced PeMADS6. To understand individual transcription factors (TFs) that affect orchid floral morphogenesis and development, we used high-throughput VIGS to silence the expression of 126 floral ESTs that encode TFs and examined their functions during floral morphogenesis in Phalaenopsis. Five TFs were associated with obvious phenotypes. Silencing PeMADS7, PeHB, or PebZIP led to abortion of the first three floral buds, and silencing PeMADS1 or PebHLH affected floral morphogenesis and reduced floral size. Silencing PeMADS1 caused a homeotic transformation of the column into a petallike column, whereas silencing PebHLH affected the development of lateral lobes of the lip. Cosilencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, produced flowers with the combined characteristics of the silencing of each gene, indicating that the functions of the two MADS-box genes ensured the normal morphogenesis and epidermal cellular types within the lip and column. The cosilencing technique will facilitate studies for unraveling the interactions among different classes of MADS-box genes and elucidating the regulatory mechanisms of floral morphogenesis in orchids.