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Schematic representation of CymMV cDNA infectious clone (A) and derivative vectors (B-H). Rectangles represent open reading frames encoded by CymMV genomic RNA. RNA-dependent RNA polymerase (RdRp), triple gene block 1, 2, and 3, CP, and GFP are indicated. The T3 promoter immediately adjacent to the most 5# end and a poly(A) tail (25 adenosine) at the most 3# end is indicated. The SpeI restriction enzyme digestion site is indicated by an arrow. The numbers below the red lines indicate the selected region of the CP subgenomic promoter; plus or minus corresponds to the upstream and downstream CP translation start codon, respectively. Head-to-head arrows on F indicate that the selected 150 bp of PeMADS6 was cloned as an inverted repeat. The direct arrows on G and H indicate that the PeMADS6 and PDS gene conserved regions were cloned directly. Scale bar, in nucleotides, is shown at the bottom.
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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 reg...
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... selected a mild, symptomless CymMV strain isolated from Phalaenopsis spp. (see ''Materials and Methods'') to construct a cDNA infectious clone. The pCymMV-M1 infectious clone contains a T3 pro- moter immediately adjacent to the 5# end of CymMV and a poly(A) tail (25 adenosines) followed by an SpeI site immediately downstream of the CymMV 3# end (Fig. 1A). No symptoms were observed on pCymMV-inoculated plants even 6 months after inoc- ulation ( Fig. 2A). To compare the infection between the wild-type virus and the derived cDNA infectious clone, sap extracted from plants infected with wild- type virus and transcripts of pCymMV-1 were used to inoculate Phalaenopsis amabilis var. formosa. ...
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... highly transcribe foreign RNA, we duplicated a subgenomic promoter of coat protein (CP) to construct the CymMV vector (Chapman et al., 1992). The dupli- cated promoters were inserted in the CymMV 5,502 nucleotide for CP expression (Fig. 1, B-H). Foreign genes were expressed through subgenomic RNA derived from the original CP subgenomic promoter (Fig. 1, B-H). Two expression vectors with differences in lengths of the duplicated subgenomic promoters were constructed and named pCymMV-pro60 and pCymMV-pro100, respectively ( Fig. 1, B and C). Green fluorescent pro- tein (GFP) was ...
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... highly transcribe foreign RNA, we duplicated a subgenomic promoter of coat protein (CP) to construct the CymMV vector (Chapman et al., 1992). The dupli- cated promoters were inserted in the CymMV 5,502 nucleotide for CP expression (Fig. 1, B-H). Foreign genes were expressed through subgenomic RNA derived from the original CP subgenomic promoter (Fig. 1, B-H). Two expression vectors with differences in lengths of the duplicated subgenomic promoters were constructed and named pCymMV-pro60 and pCymMV-pro100, respectively ( Fig. 1, B and C). Green fluorescent pro- tein (GFP) was introduced to construct the plasmids pCymMV-pro60-GFP and pCymMV-pro100-GFP ( Fig. 1, D and E). To test the ...
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... in the CymMV 5,502 nucleotide for CP expression (Fig. 1, B-H). Foreign genes were expressed through subgenomic RNA derived from the original CP subgenomic promoter (Fig. 1, B-H). Two expression vectors with differences in lengths of the duplicated subgenomic promoters were constructed and named pCymMV-pro60 and pCymMV-pro100, respectively ( Fig. 1, B and C). Green fluorescent pro- tein (GFP) was introduced to construct the plasmids pCymMV-pro60-GFP and pCymMV-pro100-GFP ( Fig. 1, D and E). To test the expression of these vectors, transcripts derived from each vector were inoculated into tobacco protoplasts (a natural host of our selected CymMV isolate). About 20% to 28% of ...
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... the original CP subgenomic promoter (Fig. 1, B-H). Two expression vectors with differences in lengths of the duplicated subgenomic promoters were constructed and named pCymMV-pro60 and pCymMV-pro100, respectively ( Fig. 1, B and C). Green fluorescent pro- tein (GFP) was introduced to construct the plasmids pCymMV-pro60-GFP and pCymMV-pro100-GFP ( Fig. 1, D and E). To test the expression of these vectors, transcripts derived from each vector were inoculated into tobacco protoplasts (a natural host of our selected CymMV isolate). About 20% to 28% of protoplasts emitted fluorescence 14 h postinoculation (Fig. 2, D and F). The same amount of transcripts from pCymMV- M1, pCymMV-pro60-GFP, and ...
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... plants (M1). Genomic RNA (G), triple gene-block subgenomic RNA (TGB sg), and CP subgenomic RNA (CP sg) are indicated. Ribosomal RNA (rRNA) used for loading control is also indicated. CP subgenomic promoter assay (C-F). The subgenomic promoters (see ''Materials and Methods'') were inserted in the CymMV 5,502 nucleotide for CP expression (Fig. 1A). Foreign genes can be expressed through subgenomic RNA derived from the CP subgenomic promoter. GFP was introduced in pCymMV-pro60 to construct the plasmid for pCymMV-pro60-GFP. A total of 5 3 10 5 tobacco protoplasts were inoculated with the transcripts of pCymMV- CP60-GFP. Cells were examined 24 h postinoculation by light (C and E) ...
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... et al., , 2005). To specifically knock down PeMADS6, we amplified a 150-bp fragment at the 3# end of PeMADS6 (Fig. 4). Because inverted repeat sequences can enhance gene silencing ( Smith et al., 2000;Wesley et al., 2001), we cloned the 150-bp fragment into the pCymMV-pro60 as an inverted repeat and named the result pCymMV-pro60-PeMADS6-IR (Fig. ...
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... detected in plants inoculated with both pCymMV-pro60 and pCymMV-pro60-PeMADS6-IR with CymMV CP used as a probe (Fig. 6A, lanes 2 and 3). In contrast, virus-induced siRNA was detected only in plants inoculated with pCymMV-pro60- PeMADS6-IR with PeMADS6 used as a probe ( lane 3). With either probe, siRNA was not detected in mock-inoculated plants (Fig. 6, A and B, lane 1). Similar siRNA findings were detected in P. Sogo Musadium (data not shown). These results indicated that the CymMV VIGS vector induced PeMADS6 siRNA only when the specific sequence of PeMADS6 was inserted in the CymMV vector, which suggests that reduced PeMADS6 expression was indeed medi- ated via the gene-silencing mechanism ...
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... that can easily induce a visible phenotype in orchids during VIGS will be desirable for further research. Therefore, we tried to knock down floral MADS-box genes simultaneously by inserting a 500-bp DNA fragment of PeMADS6 containing a conserved region of the MADS-box genes into pCymMV-pro60l; the resulting plasmid was named pCymMV-pro60- PeMADS6 (Figs. 1G and 4). We expected that several MADS-box genes would be affected, with consequent prominent morphological ...
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... respectively. The two plasmids were digested with NotI and SnaI and then separated on 1% agarose gels. The 3.9-and 5.4-kb fragments derived from pCymMV-1 and pCymMV-2, respectively, were gel purified by use of the gel extraction kit (Qiagen). The purified fragments were ligated with T4 DNA ligase to construct the cDNA infectious clone pCymMV-M1 (Fig. ...
Citations
... Commercial orchid cultivation suffers from several associated breeding barriers such as the large and complex polyploid genome, slow growth, and long life cycle; consequently, it takes a long period to generate new cultivars using cultivation through the traditional breeding system. The low transformation efficiency makes the development of new varieties with desired traits a challenging task [47,48]. For example, the high commercial value orchid Phalaenopsis takes more than 2 years to switch from the vegetative to the reproductive phase [49]. ...
Orchids constitute the largest and most diverse group of flowering plants and are classified in the family Orchidaceae. Exhibiting significance as the most exotic and ubiquitous flowering plant, the cultivation of orchids on a commercial level is gaining momentum worldwide. In addition to its ornamental and aesthetic value, the orchid industry has successfully generated employment for people in developing countries. Recent advances in biotechnological interventions in orchids have substantially contributed to the development of exotic varieties with novel traits, not to forget the inputs of traditional plant breeding methods and tissue culture approaches. In addition, the scientific developments in orchid biology have remarkably bridged the knowledge gaps in areas of orchid classification, phytochemistry, and cultivation strategies. This has facilitated the commercialization of novel varieties, opening new avenues in the orchid industry, and their global marketing as cut flowers and artificially propagated plants. Orchids constitute the first floriculture crops that revolutionized the orchid industry; however, they also hold several challenges in the natural propagation and conservation of several species that are on the verge of extinction. International organizations like CITES have come forward to address challenges associated with illegal global trade and indiscriminate use of orchid varieties, aiming for conservation and legal commercial goals. This thematic review is one-of-a-kind in providing comprehensive insights into the emerging momentum of orchid biology and how its globalization projects to considerably impact the orchid industry in the coming times. However, it is imperative to understand the challenges in the cultivation and conservation of orchid varieties and ensure legislative guidelines both on domestic and global levels to ensure a multipronged approach to the conservation and commercialization of orchids.
... f. was performed (De LC, 2014;Rodrigues et al., 2015). In biotechnology, molecular biology techniques are used, genetic transformation for the development of disease resistance in plants, virus-induced gene silencing (VIGS) techniques for functional validation of genes in orchids, and understanding regulation of flowering in orchid species (Lu et al., 2007) (Table 2). For the efficient propagation and conservation of novel orchid species, different cultivation methods are used, as discussed in Table 2, with key examples. ...
One of the most exotic and ubiquitous plant species, the cultivation of orchids continues to gain momentum worldwide. The growing popularity of orchids and their cultivation worldwide is attributed to substantial progress in approaches like classical breeding, plant tissue culture and biotechnological interventions. In addition to ornamental value, orchids are extensively studied for their pharmacological properties and are widely used as food supplements, flavours, and medicine in different regions of the world. The unprecedented advances in whole-genome sequencing and omics technologies have significantly improved our knowledge of orchid biology, leading to translational success in the production of exotic varieties. With the emerging investigations into the cultivation and global trade popularity of orchids in the present era, this thematic article provides comprehensive insights into the existing and emerging trends in orchid cultivation, diverse ethnomedicinal uses, and multi-faceted applications, and the need for the legalisation of orchids for conservation and trade in the present era. In this way, advances in breeding and molecular approaches aim to significantly improve orchid cultivation and its socio-economic attributes. However, a detailed understanding of challenges in orchid conservation and implementation of domestic/global legislative guidelines are necessary for the protection of endangered species and their legalised trade across the globe.
... However, associated challenges in orchard cultivation include complex genomes, slow growth rates and poor transformation, limiting the conservation of threatened genotypes with novel characteristics [3]. For example, the poplar cultivar has a development stage that lasts more than two years, from vegetative to reproductive [4]. Seventy percent of orchidaceae species are epiphytic, accounting for two-thirds of all epiphytes worldwide [5]. ...
... Commercial orchard cultivation faces several breeding barriers, including a large and complex polyploid genome, slow growth and a long life cycle, making it challenging to generate new cultivars using the traditional breeding system. Furthermore, the low transformation efficiency makes it difficult to develop new varieties with desired traits [4,77]. For example, the high-value orchard Phalaenopsis requires more than two years to transition from the vegetative to reproductive phase [3]. ...
Orchards play a key role in the non-petroleum economy of many developing countries. Developing orchards with good adaptation to abiotic and biotic stresses is the main point for plant researchers to improve and increase fruit yield in many parts all over the world. They have established seed production zones in the best natural stands to provide seed until orchard production can satisfy all needs. Conventional breeding and genetic improvement are time-consuming and don't associate good results in orchards. Because of their great utility, genetic molecular markers will become standard tools for orchard improvement in the future. Biotechnology creates a genetic alteration in orchards comparatively short with slight modifications to other valid characters (tolerance to abiotic and biotic stresses, enhanced post-harvest shelf life of fruit, reduced vegetative propagative phase, and fruit production with higher nutritional values….etc). Thus, it leads to an uprising in orchards sciences, fruit tree breeding, and orchards improvement and management.
... Beside this, when designing VIGS experiments it is also highly desirable to incorporate appropriate positive control VIGS constructs targeting genes whose silencing induces obvious visual phenotypes as well as negative controls carrying heterologous sequences with no known homologous gene targets in the plant species under investigation. [11], Bamboo mosaic virus [12], Cymbidium mosaic virus [13], Cucumber mosaic virus [14], Foxtail mosaic virus [15,16], Chinese wheat mosaic virus [17], and Rice tungro bacilliform virus [18]. Among these, BSMV-based vectors are most widely used for functional genomics study in many cereal crop plants important in agriculture such as wheat and barley [19,20]. ...
Advances made in genome sequencing projects and structural genomics are generating large repertoire of candidate genes in plants associated with specific agronomic traits. Rapid and high-throughput functional genomics approaches are therefore needed to validate the biological function of these genes especially for agronomically important crops beyond the few model plant species. This can be achieved by utilizing available gene knockout or transgenic methodologies, but these can take considerable time and effort particularly in crops with large and complex genomes such as wheat. Therefore, any tool that expedites the validation of gene function is of particular benefit especially in cereal crop plants that are genetically difficult to transform. One such reverse genetics tool is virus-induced gene silencing (VIGS) which relies on the plants’ natural antiviral RNA silencingRNA silencing defence mechanism. VIGS is used to downregulate target gene expression in a transient manner which persists long enough to determine its effect on a specific trait. VIGS based on Barley stripe mosaic virus (BSMV) is rapid, powerful, efficient, and relatively inexpensive tool for the analysis of gene function in cereal species. Here we present detailed protocols for BSMV-mediated VIGS for robust gene silencing in bread wheat and related species.
... At present time, the cultivation of orchids has led to new cultivars and hybrids with multiple ornamental values, contributing to the rapidly growing market and demand for exotic varieties. However, associated challenges in orchid cultivation comprise complex genomes, slow growth rate, and poor transformation projecting limitations in the conservation of threatened varieties with novel attributes (Wang et al., 2017), For instance-the Poplar cultivar comprises a development stage of more than 2 years proceeding from vegetative to the reproductive stage (Lu et al., 2007). Among the family Orchidaceae, 70% of species are epiphytic and constitute two-thirds of global epiphytes . ...
... Commercial orchid cultivation suffers from several associated breeding barriers such as the large and complex polyploid genome, slow growth, and long-life cycle, which takes a long period to generate new cultivars using the cultivation through the traditional breeding system. Also, the low transformation e ciency makes it challenges the development of new varieties having desired traits (Lu et al., 2007;Pan et al., 2012). For example, the high commercial value orchid Phalaenopsis takes more than 2 years to switch from vegetative to reproductive phase (Wang et al., 2017). ...
... The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper. Both genes are highly expressed during the early stages of floral buds and vegetative organs (Chen et al., 2007) P. aphrodite PaAP1-1 and PaAP1-2 ...
Orchids constitute the largest and most diverse group of flowering plants and are classified in Orchidaceae. Exhibiting significance as the most exotic and ubiquitous flowering plants, the cultivation of orchids on a commercial level, is gaining momentum worldwide. In addition to its ornamental and aesthetic value, the orchid industry has been successful in generating employment for people in developing countries. Recent advances in biotechnological interventions in orchids have substantially contributed to the development of exotic varieties with novel traits, not to forget the inputs of traditional plant breeding methods and tissue culture approaches. In addition, the scientific developments in orchid biology have remarkably improved the knowledge in areas of orchid biology, classification, phytochemistry, and cultivation strategies, among other areas. This has facilitated the commercialization of novel varieties, opening new avenues in the orchid industry, witnessed as cultivation of different varieties and globally marketed as a cut flower and artificially propagated plants. The orchids constitute the first floriculture crops, which revolutionized the orchid industry, however, it also withholds several challenges in natural propagation, several species facing extinction. International organizations like CITES have come forward to address challenges associated with illegal global trade and indiscriminate use of orchid varieties, aiming towards conservation and legal commercial goals. This thematic review is one of a kind in providing comprehensive insights on the emerging momentum of orchid biology and how its globalization projects to considerably impact the orchid industry in the coming time. Together with traditional breeding approaches and plant tissue culture, advances in biotechnological interventions continues to make substantial progress in the development of exotic varieties, with multi-faceted attributes. However, it is imperative to understand the challenges in the cultivation and conservation of orchid varieties and ensure legislative guidelines both on domestic and global levels to ensure a multipronged approach for the conservation and commercialization of orchids.
... However, the cultivation of Phalaenopsis orchids suffers from several viral diseases , 2017. We previously developed a cymbidium mosaic virus (CymMV)-induced gene silencing system (VIGS) for gene knockdown assays in Phalaenopsis orchids (Lu et al., 2007, and used this VIGS system for high-throughput screening of genes involved in SA-mediated antiviral immunity in Phalaenopsis orchids . We found that an orchid protein, belonging to the stress-associated protein (SAP) family, Pha13, serves as an important hub in transducing the SA signal for combating virus infection (Chang et al., 2018). ...
... The CymMV isolate used for this study was described previously (Lu et al., 2007. The CymMV isolate used for analysis accumulated more coat protein subunits than other CymMV isolates (Lu et al., 2007), and triggered the expression of the SA marker gene, orchid PR1, in our previous analysis (Chen et al., 2013;Chang et al., 2018). ...
... The CymMV isolate used for this study was described previously (Lu et al., 2007. The CymMV isolate used for analysis accumulated more coat protein subunits than other CymMV isolates (Lu et al., 2007), and triggered the expression of the SA marker gene, orchid PR1, in our previous analysis (Chen et al., 2013;Chang et al., 2018). ...
Viruses cause severe damage on crops, and identification of key gene(s) that can comprehensively activate antiviral immunity will provide insights for designing effective antiviral strategies. Salicylic acid (SA)‐mediated antiviral immunity and RNA interference (RNAi) are two independently discovered antiviral pathways. Previously, we identified the orchid stress‐associated protein (SAP), Pha13, which serves as a hub in SA‐mediated antiviral immunity. As SAPs exist as a protein family, whether duplicated SAPs have redundant or distinctive functions in antiviral immunity remains elusive.
We performed functional assays on orchid Pha21, a homolog of Pha13, using transient and transgenic approaches on orchid, Arabidopsis and Nicotiana benthamiana to overexpress and/or silence Pha21.
The SA treatment induced the expression of both Pha13 and Pha21, whereas Pha21 was found to play a key role in the initiation of the RNAi pathway in Phalaenopsis orchids. We demonstrated that Pha21‐mediated antiviral immunity and enhancement of the RNAi pathway is conserved between dicotyledons and monocotyledons.
We provide new insight that orchid SAPs confer distinctive functions to coordinate both SA‐signaling and RNAi for comprehensive activation of antiviral immunity, and this information will help us develop antiviral strategies on crops.
... Bamboo mosaic virus and its satellite RNA (BaMV) was modified for VIGS in Brachypodium distachyon (Liou et al., 2014); Brome mosaic virus (BMV) in barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and sorghum (Sorghum bicolor; Ding et al., 2006Ding et al., , 2018Zhu et al., 2014;Singh et al., 2018); Barley stripe mosaic virus (BSMV) in many monocot species including Aegilops Tauschii, barley, B. distachyon, ginger (Zingiber officinale), Hedychium coronarium, maize, oat (Avena sativa), rye (Secale cereale), wheat (Triticum aestivum and Haynaldia villosa), and Zingiber zerumbet (Holzberg et al., 2002;Scofield et al., 2005;Renner et al., 2009;Pacak et al., 2010;Wang et al., 2010;Yuan et al., 2011;Mahadevan et al., 2014;Groszyk et al., 2017;Jarugula et al., 2018;Tavakol, 2018;Chen et al., 2019); Chinese wheat mosaic virus (CWMV) in wheat (Yang et al., 2018); Cucumber mosaic virus (CMV) in banana (Musa acuminata), lily (Lilium leichtlinii), and maize (Wang et al., 2016;Tzean et al., 2019;Tasaki et al., 2020); Cymbidium mosaic virus (CymMV) in Phalaenopsis spp. (Lu et al., 2007;Hsieh et al., 2013); Foxtail mosaic virus (FoMV) in barley, foxtail millet (Setaria italica), wheat and maize Mei et al., 2016); Maize rayado fino virus (MRFV) in maize (Mlotshwa et al., 2020); Rice tungro bacilliform virus (RTBV) in rice (Purkayastha et al., 2010); and Tobacco rattle virus (TRV) in wheat and maize (Zhang et al., 2017). These VIGS vectors were all derived from natural monocot hosts except that TRV isolated from dicots (Liu et al., 2002;Zhang et al., 2017). ...
Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 days post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants.
... Relative quantification analysis was carried out using ABI SDS version 1.4 software (Applied Biosystems). Transcript levels were normalized to the endogenous control gene Ubi10 using the comparative cycle threshold method (Lu et al., 2007). The primers used for RT-qPCR are listed in Supplementary Table S1. ...
Petals of the monocot Phalaenopsis aphrodite (Orchidaceae) possess conical epidermal cells on their adaxial surfaces, and a large amount of cuticular wax is deposited on them to serve as a primary barrier against biotic and abiotic stresses. It has been widely reported that subgroup 9A members of the R2R3-MYB gene family, MIXTA and MIXTA-like in eudicots, act to regulate the differentiation of conical epidermal cells. However, the molecular pathways underlying conical epidermal cell development and cuticular wax biosynthesis in monocot petals remain unclear. Here, we characterized two subgroup 9A R2R3-MYB genes, PaMYB9A1 and PaMYB9A2 (PaMYB9A1/2), from P. aphrodite through the transient overexpression of their coding sequences and corresponding chimeric repressors in developing petals. We showed that PaMYB9A1/2 function to coordinate conical epidermal cell development and cuticular wax biosynthesis. In addition, we identified putative targets of PaMYB9A1/2 through comparative transcriptome analyses, revealing that PaMYB9A1/2 act to regulate the expression of cell wall-associated and wax biosynthetic genes. Furthermore, a chemical composition analysis of cuticular wax showed that even-chain n-alkanes and odd-chain primary alcohols are the main chemical constituents of cuticular wax deposited on petals, which is inconsistent with the well-known biosynthetic pathways of cuticular wax, implying a distinct biosynthetic pathway occurring in P. aphrodite flowers. These results reveal that the function of subgroup 9A R2R3-MYB family genes in regulating the differentiation of epidermal cells is largely conserved in monocots and dicots. Furthermore, both PaMYB9A1/2 have evolved additional functions controlling the biosynthesis of cuticular wax.
... Relative quantification analysis was carried out using ABI SDS version 1.4 software (Applied Biosystems). Transcript levels were normalized to the endogenous control gene Ubi10 using the comparative cycle threshold method (Lu et al., 2007). The primers used for RT-qPCR are listed in Supplementary Table S1. ...
Petals of the monocot Phalaenopsis aphrodite (Orchidaceae) possess conical epidermal cells on their adaxial surfaces, and a large amount of cuticular wax is deposited on them to serve as a primary barrier against biotic and abiotic stresses. It has been widely reported that subgroup 9A members of the R2R3-MYB gene family, MIXTA and MIXTA-like in eudicots, act to regulate the differentiation of conical epidermal cells. However, the molecular pathways underlying conical epidermal cell development and cuticular wax biosynthesis in monocot petals remain unclear. Here, we characterized two subgroup 9A R2R3-MYB genes, PaMYB9A1 and PaMYB9A2 (PaMYB9A1/2), from P. aphrodite through the transient overexpression of their coding sequences and corresponding chimeric repressors in developing petals. We showed that PaMYB9A1/2 function to coordinate conical epidermal cell development and cuticular wax biosynthesis. In addition, we identified putative targets of PaMYB9A1/2 through comparative transcriptome analyses, revealing that PaMYB9A1/2 acts to regulate the expression of cell wall-associated and wax biosynthetic genes. Furthermore, a chemical composition analysis of cuticular wax showed that even-chain n-alkanes and odd-chain primary alcohols are the main chemical constituents of cuticular wax deposited on petals, which is inconsistent with the well-known biosynthetic pathways of cuticular wax, implying a distinct biosynthetic pathway occurring in P. aphrodite flowers. These results reveal that the function of subgroup 9A R2R3-MYB family genes in regulating the differentiation of epidermal cells is largely conserved in monocots and dicots. Furthermore, both PaMYB9A1/2 have evolved additional functions controlling the biosynthesis of cuticular wax.
... Clones were confirmed by sequencing. The CymMV construct with a green fluorescent protein (GFP) reporter, pkCy1GFP, was generated by inserting the GFP gene downstream of the duplicated CymMV CP subgenomic promoter (−100 to +22 [40]). A 35S promoterdriven mCherry expression cassette was further amplified by PCR and inserted into the BglII site of pkn vector to obtain pkn::mCherry. ...
Synergistic interactions among viruses, hosts and/or transmission vectors during mixed infection can alter viral titers, symptom severity or host range. Viral suppressors of RNA silencing (VSRs) are considered one of such factors contributing to synergistic responses. Odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV), which are two of the most significant orchid viruses, exhibit synergistic symptom intensification in Phalaenopsis orchids with unilaterally enhanced CymMV movement by ORSV. In order to reveal the underlying mechanisms, we generated infectious cDNA clones of ORSV and CymMV isolated from Phalaenopsis that exerted similar unilateral synergism in both Phalaenopsis orchid and Nicotiana benthamiana. Moreover, we show that the ORSV replicase P126 is a VSR. Mutagenesis analysis revealed that mutation of the methionine in the carboxyl terminus of ORSV P126 abolished ORSV replication even though some P126 mutants preserved VSR activity, indicating that the VSR function of P126 alone is not sufficient for viral replication. Thus, P126 functions in both ORSV replication and as a VSR. Furthermore, P126 expression enhanced cell-to-cell movement and viral titers of CymMV in infected Phalaenopsis flowers and N. benthamiana leaves. Taking together, both the VSR and protein function of P126 might be prerequisites for unilaterally enhancing CymMV cell-to-cell movement by ORSV.
