Strategies for Functional Validation of Genes Involved in Reproductive Stages of Orchids

Abstract

Plants in the largest family of angiosperms, Orchidaceae, are diverse in both specialized pollination and ecological strategies and provide a rich source for investigating evolutionary relationships and developmental biology. However, studies in orchids have been hindered by several challenges that include low transformation efficiency and long regeneration time. To overcome such obstacles, we selected a symptomless cymbidium mosaic virus (CymMV) isolate for constructing virus-induced gene-silencing vectors. The feasibility of the virus vectors was first assessed with use of an orchid phytoene desaturase gene. The vector was able to induce gene silencing in orchids; however, because of the slow growth of orchids, the commonly used phytoene desaturase gene was not a good visual marker in orchids. We inserted a 150-nucleotide unique region of a B-class MADS-box family gene, PeMADS6, into pCymMV-pro60. The transcription level of PeMADS6 in inoculated Phalaenopsis plants was reduced by up to 73%, but no effect was observed for other MADS-box family genes. In contrast, in Phalaenopsis plants inoculated with CymMV transcripts containing 500 nucleotides of PeMADS6, a conserved region among MADS-box genes, the transcription level of PeMADS6 and the B-and C-class MADS-box genes was reduced by up to 97.8% as compared with plants inoculated with the vector alone. Flower morphology was affected in the MADS-box family gene-silenced plants as well. This in vivo experiment demonstrates an efficient way to study genes involved in the reproductive stage of plants with a long life cycle.Functional genomics studies of nonmodel organisms to reveal how changes in regulatory networks contribute to diversity have been a big challenge in biology (Pennisi, 2004;Costa et al., 2005;Gewin, 2005). Studies of nonmodel organisms may be hindered by their large genome size, low transformation efficiency and long regeneration time, and long life cycle. Advances in sequencing technology have alleviated the difficulties in obtaining genetic information from organisms with large genome size. For example, construction of an expressed sequence tag (EST) database can reveal a massive amount of genetic information reflecting an important biological state in a short period. However, this information needs further analysis to define its biological significance. Most gene function validation approaches require extensive genetic screening and plant transformation. Likewise, these approaches are suitable for model plants, but are not practical for plants with low transformation efficiency, long regeneration time, and long life cycle. In particular, a formidable obstacle rests in functional genomics studies of genes involved in the final stage of the life cycle, the reproductive stage, in plants with long life cycles. For such plants, years may be required to obtain data by conventional approaches to study genes involved in the reproductive stage. Unfortunately, the life cycles of most plants are longer than 1 year.

PaPTP1, a Gene Encoding Protein Tyrosine Phosphatase from Orchid, Phalaenopsis amabilis, is Regulated During Floral Development and Induced by Wounding

“…The expression of PaPTP1 gene is mainly localized in floral organs and in response to wounding. Generation of loss-of-function orchid plants by virus-induced gene silencing has been described and achievable (Lu et al 2007). Further functional analysis using PaPTP1-silenced orchid plants should provide novel information on the role of reversible tyrosine phosphorylation in developmental biology and stress responses.…”

The expression of two DEFICIENS-like genes was reduced in the sepaloid tepals of viridiflora tulips

“…Until now, concerning GLO-like genes except higher eudicots, there have also been reports using virus-induced gene silencing. For example, AqvPI in Aquilegia (Kramer et al 2007) and PeMADS6 in Phalaenopsis (Lu et al 2007), which showed that petaloid organs changed to sepaloid organs upon down-regulation of these class B genes. With regard to the DEF-like genes, it has been reported that in the superwoman mutant of rice and the silky1 mutant of maize, lodicules, which are considered to correspond to petals, change to palea or lemma-like organs and stamens change to carpels (Nagasawa et al 2003, Ambrose et al 2000.…”

A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize

“…Six different viruses have been developed into viral vectors for VIGS applications in monocots to date: Barley stripe mosaic virus (Scofield et al, 2005), Brome mosaic virus (BMV; Ding et al, 2006), Cymbidium mosaic virus (Lu et al, 2007), Rice tungro bacilliform virus (Purkayastha et al, 2010), Bamboo mosaic virus together with its associated satellite RNA (Liou et al, 2014), and, most recently, Cucumber mosaic virus (CMV; Wang et al, 2016). Among these, the BMV and CMV vectors have the ability to infect and induce gene silencing in maize (Zea mays; Ding et al, 2006;Lee et al, 2015;Wang et al, 2016).…”