Volume 5 Issue 2 - July 4, 2008
Strategies for functional validation of genes involved in reproductive stages of orchids
Ming-Hsien Hsieh, and Hong-Hwa Chen*

1Department of Life Sciences, and 2Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan
Email: hhchen@mail.ncku.edu.tw

Plant Physiology 143: 558-569 (2007)

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Orchids have profound diversity of specialized pollination and ecological strategies. Hence, orchid is ideal plant species to study molecular evolution and functional genomics. The sophisticated orchid flower morphology includes two whorls of perianth segments, three sepals and three petals, one of the petals is modified as labellum (or lip). In addition, the male and female reproductive organs are fused to form a gynostemium. The diversity and specialization in orchid floral morphology have fascinated botanists and collectors for centuries.

Phalaenopsis is a member of the Orchidaceae, one of the largest families of flowering plants. Phalaenopsis industry has been developed as an international leading agricultural industry in Taiwan. FloraCulture International (2002) has reported that regarding world trade, the largest orchid plant exporter is Taiwan. Furthermore, Phalaenopsis has been selected by Council of Agriculture as one of the four key representative agricultural products in Taiwan. Thus, there is no need to emphasize how important Phalaenopsis is. The research on Phalaenopsis not only can offer the opportunity to answer the diversity and specialization in orchid biology, but also provide a basis for crop improvement to enhance the competition capability in Phalaenopsis industry worldwide.

Forward and reverse genetics are two methods used to determine gene function. Forward genetics starts with random mutagenesis of an entire genome, after which the trait selection is followed to identify the mutated gene or genes. Reverse genetics disrupts gene expression to search for a phenotype usually beginning with cloned DNA sequences. The problems with forwards genetics include the long life-cycle and lack of high-resolution genetic maps in orchids. The problems with the reverse genetic methods using T-DNA insertion or transposon-tagging include difficulties in plant transformation and long regeneration time in orchids. Here we describe a new approach facilitated by virus-induced gene silencing (VIGS) to accelerate the analysis of orchid gene function.

Virus-induced gene silencing is highly effective at detecting candidate gene function. To carry out VIGS, a fragment of the target gene is cloned into a virus vector. As the virus vector inoculates, the target gene replicates and spreads in the plant. The plant responds with RNA interference as a defense mechanism. The virus vector containing the heterologous insert and their mRNA suffer degradation. This causes a down-regulation of the target gene and often cases a clear phenotype develops. The advantages of VIGS include the short-time required for down-regulation of the target gene and this may give us the ability to study essential genes. VIGS is much easier and more efficient at overcoming functional redundancy by effectively suppressing all or most genes in a given family.

Of all the current viral vectors used in VIGS, potato virus X (PVX) and Tobacco mosaic virus (TMV) are successful VIGS vectors in potato and tobacco respectively. Both PVX and Cymbidium mosaic virus (CymMV) belongs to the same genus, and both TMV and Odontoglossum ringspot virus (ORSV) belongs to the same genus. Thus, CymMV and ORSV are orchid viruses that can be considered as VIGS vectors. After screening, the highly prevalent orchid virus CymMV was chosen as a VIGS vector, which does not induce any symptoms. This vector successfully induced gene silencing by knocking down the RNA levels of Phytoene desaturase ( PDS ) gene in the leaves by 54% in less than 2 months confirming that this virus vector can be used in the study of Phalaenopsis.

Under normal circumstances orchids bloom from March to May, but we need them to bloom through out the year in order to efficiently study their reproductive stage. In order to allow for regular blooming, a grow chamber is required. Commercial orchid growers recommend that orchids be kept at 25℃ during the day and at 20℃ at night with the correct humidity and fertilization that can stimulate stalks for flowering. In order to show that the CymMV vector can induce silencing in all flower organs we use a B-class MADS-box gene, PeMADS6 which are transcribed in all of the flower organs. We selected a stretch of 150 nucleotides (nts) from the less conserved 3’-terminus, and inserted it into the CymMV vector, and named it as pCymMV-pro60-PeMADS6IR. The flowers blossomed six weeks post inoculation of the 6-node emerging stalks of P. amabili var. formosa. Reduced PeMADS6 RNA levels to 63±2%, 33±3%, 23±5% and 33±2% were detected in sepals, petals, lips and columns, respectively in the plants inoculated with pCymMV-pro60-PeMADS6IR as compared to the mock-inoculated plants. To confirm whether the reduced expression of PeMADS6 was due to RNA interference mediated by VIGS, small-molecular-weight RNA was purified from the inoculated plants and then performed northern blot hybridization with probes containing either CymMV Coat protein (CP) gene or PeMADS6 gene. The CymMV CP probe could detect 21-nt small interfering RNA (siRNA) in both the mock-inoculated plant as well as plants inoculated with pCymMV-pro60-PeMADS6IR. However, it could only detect the 21-nt siRNA of PeMADS6 in the plants inoculated with pCymMV-pro60-PeMADS6IR.

In addition, these plants inoculated with pCymMV-pro60-PeMADS6IR showed modified phenotype with several morphological changes. These include streaks or patches of greenish tissue were observed in the sepals, petals and lips. Another interesting change was the initial inability of flower buds to blossom on the lower stalks of inoculated plants. The flower buds were able to blossom on the upper stalks, but did so with streaks or patches of greenish tissue in the sepals, petals and lips. After dissecting the initial flower buds on route to abort, fully formed sepals, petals, lips and columns were found within these plants similar to healthy plants. This indicates that reduced transcript levels of MADS-box family genes still allowed initial flower development to occur normally, but it was insufficient for further development and blossoming to occur.

Interestingly, similar results were obtained when the experiments were conducted in another two orchid varieties, P. amabilis and P. Sogo Musadium. Both varieties are tetrapolid, and in general, loss-of-function assays are not easy to perform in plants with multiple copies. With loss-of-function assays, such as T-DNA insertion or transposon tagging, simultaneously targeting all genes with functional redundancy is difficult. In contrast, VIGS can knock down the RNA level after RNA transcription, regardless of the RNAs transcribed from genes in different genome locations, and thus can silence all genes simultaneously.

These results demonstrate that the newly constructed CymMV-based vector is suitable for analyzing genes involved in the floral morphogenesis of Phalaenopsis spp. As CymMV has a wide host range among various genus of Ochidaceae, including Phalaenopsis, Cymbidium, Cattleya, Dendrobium, Epidendrum, Laelia, laeliocattleya, Oncidium, Zygopetallum, Vanilla, and Vinda, the developed vectors will contribute well to functional genomics studies of these orchids. With the advent of genomics and bioinfomatics, the past few years have seen great strides in the amount of gene information available and development of tools for their analysis of orchids. Thus, the CymMV-based vector can be used for the functional validation of genes in orchids, to overcome their large genome size, low transformation efficiency, long regeneration time, and long life-cycle.
Figure 1. Phenotype on MADS-box gene-silenced plants. Plants of P. amabilis var. Formosa were infected with buffer (A and D), pCumMV-pro60 (B and E), or pCymMV-CP60-PeMADS6 (C and F). The arrows on C indicate greenish streaks of the flower and those on F indicate greenish patches of the lip.
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