Hsin-Hung Yeh

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

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.

Asynchronous Accumulation of Lettuce Infectious Yellows Virus RNAs 1 and 2 and Identification of an RNA 1 trans Enhancer of RNA 2 Accumulation

ABSTRACT Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartiteCrinivirus, Lettuce infectious yellow virus(LIYV; family Closteroviridae). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a trans enhancer for RNA 2 accumulation.

Reassortment and Concerted Evolution in Banana Bunchy Top Virus Genomes

ABSTRACT The nanovirus Banana bunchy top virus (BBTV) has six standard components in its genome and occasionally contains components encoding additional Rep (replication initiation protein) genes. Phylogenetic network analysis of coding sequences of DNA 1 and 3 confirmed the two major groups of BBTV, a Pacific and an Asian group, but show evidence of web-like phylogenies for some genes. Phylogenetic analysis of 102 major common regions (CR-Ms) from all six components showed a possible concerted evolution within the Pacific group, which is likely due to recombination in this region. The CR-M of additional Rep genes is close to that of DNA 1 and 2. Comparison of tree topologies constructed with DNA 1 and DNA 3 coding sequences of 14 BBTV isolates showed distinct phylogenetic histories based on Kishino-Hasegawa and Shimodaira-Hasegawa tests. The results of principal component analysis of amino acid and codon usages indicate that DNA 1 and 3 have a codon bias different from that of all other genes of nanoviruses, including all currently known additional Rep genes of BBTV, which suggests a possible ancient genome reassortment event between distinctive nanoviruses.

Virus-induced gene silencing unravels multiple transcription factors involved in floral growth and development in Phalaenopsis orchids

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.

cis preferential replication of Lettuce infectious yellows virus (LIYV) RNA 1: the initial step in the asynchronous replication of the LIYV genomic RNAs.

A series of Lettuce infectious yellows virus (LIYV) RNA 1 mutants was created to evaluate their ability to replicate in tobacco protoplasts. Mutants DeltaEcoRI, DeltaE-LINK, and Delta1B, having deletions in open reading frames (ORFs) 1A and 1B, did not replicate when individually inoculated to protoplasts or when co-inoculated with wild-type RNA1 as a helper virus. A fragment of the green fluorescent protein (GFP) gene was inserted into the RNA 1 ORF 2 (P34) in order to provide a unique sequence tag. This mutant, P34-GFP TAG, was capable of independent replication in protoplasts. Mutants derived from P34-GFP TAG having frameshift mutations in the ORF 1A or 1B were unable to replicate in protoplasts alone or in trans when co-inoculated with wild-type RNA1 as a helper virus. Taken together, these data strongly suggest that LIYV RNA 1 replication is cis-preferential.

Unusual Events Involved in Banana bunchy top virus Strain Evolution

Banana bunchy top virus (BBTV) can be transmitted by aphids and consists of at least six integral components (DNA-R, -U3, -S, -M, -C, and -N). Several additional replication-competent components (additional Reps) are associated with some BBTV isolates. A collected BBTV strain (TW3) that causes mild symptoms was selected to study the processes in BBTV evolution. Southern blot hybridization, polymerase chain reaction (PCR), and real-time PCR did not detect DNA-N in TW3. Real-time PCR quantification of BBTV components revealed that, except for the copy number of TW3 DNA-U3, each detected integral component of BBTV TW3 was at least two orders lower than that of the severe strains. No infection was observed in plants inoculated with aphids, which were first given acquisition access to the TW3-infected banana leaves. Recombination analysis revealed recombination between the integral component TW3 DNA-U3 and the additional Rep DNA-Y. All BBTV integral components contain a replication initiation region (stem-loop common region) that share high sequence identity. Sequence alignment revealed that TW3 DNA-R, -S, -M, and -C all have a stem-loop common region containing a characteristic 9-nucleotide deletion found only in all reported DNA-N. Our data suggest that the additional Rep DNAs can serve as sources of additional genetic diversity for integral BBTV components.

A High-Throughput Virus-Induced Gene-Silencing Vector for Screening Transcription Factors in Virus-Induced Plant Defense Response in Orchid

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.

An efficient RNA interference screening strategy for gene functional analysis

BackgroundRNA interference (RNAi) is commonly applied in genome-scale gene functional screens. However, a one-on-one RNAi analysis that targets each gene is cost-ineffective and laborious. Previous studies have indicated that siRNAs can also affect RNAs that are near-perfectly complementary, and this phenomenon has been termed an off-target effect. This phenomenon implies that it is possible to silence several genes simultaneously with a carefully designed siRNA.ResultsWe propose a strategy that is combined with a heuristic algorithm to design suitable siRNAs that can target multiple genes and a group testing method that would reduce the number of required RNAi experiments in a large-scale RNAi analysis. To verify the efficacy of our strategy, we used the Orchid expressed sequence tag data as a case study to screen the putative transcription factors that are involved in plant disease responses. According to our computation, 94 qualified siRNAs were sufficient to examine all of the predicated 229 transcription factors. In addition, among the 94 computer-designed siRNAs, an siRNA that targets both TF15 (a previously identified transcription factor that is involved in the plant disease-response pathway) and TF21 was introduced into orchids. The experimental results showed that this siRNA can simultaneously silence TF15 and TF21, and application of our strategy successfully confirmed that TF15 is involved in plant defense responses. Interestingly, our second-round analysis, which used an siRNA specific to TF21, indicated that TF21 is a previously unidentified transcription factor that is related to plant defense responses.ConclusionsOur computational results showed that it is possible to screen all genes with fewer experiments than would be required for the traditional one-on-one RNAi screening. We also verified that our strategy is capable of identifying genes that are involved in a specific phenotype.

Cymbidium mosaic potexvirus isolate-dependent host movement systems reveal two movement control determinants and the coat protein is the dominant.

Abstract Little is known about how plant viruses of a single species exhibit different movement behavior in different host species. Two Cymbidium mosaic potexvirus (CymMV) isolates, M1 and M2, were studied. Both can infect Phalaenopsis orchids, but only M1 can systemically infect Nicotiana benthamiana plants. Protoplast inoculation and whole-mount in situ hybridization revealed that both isolates can replicate in N. benthamiana; however, M2 was restricted to the initially infected cells. Genome shuffling between M1 and M2 revealed that two control modes are involved in CymMV host dependent movement. The M1 coat protein (CP) plays a dominant role in controlling CymMV movement between cells, because all chimeric CymMV viruses containing the M1 CP systemically infected N. benthamiana plants. Without the M1 CP, one chimeric virus containing the combination of the M1 triple gene block proteins (TGBps), the M2 5′ RNA (1–4333), and the M2 CP effectively moved in N. benthamiana plants. Further complementation analysis revealed that M1 TGBp1 and TGBp3 are co-required to complement the movement of the chimeric viruses in N. benthamiana. The amino acids within the CP, TGBp1 and TGBp3 which are required or important for CymMV M2 movement in N. benthamiana plants were mapped. The required amino acids within the CP map to the predicted RNA binding domain. RNA–protein binding assays revealed that M1 CP has higher RNA binding affinity than does M2 CP. Yeast two-hybrid assays to detect all possible interactions of M1 TGBps and CP, and only TGBp1 and CP self-interactions were observed.

A Vicilin-Like Seed Storage Protein, PAP85, Is Involved in Tobacco Mosaic Virus Replication

ABSTRACT One striking feature of viruses with RNA genomes is the modification of the host membrane structure during early infection. This process requires both virus- and host-encoded proteins; however, the host factors involved and their role in this process remain largely unknown. On infection with Tobacco mosaic virus (TMV), a positive-strand RNA virus, the filamentous and tubular endoplasmic reticulum (ER) converts to aggregations at the early stage and returns to filamentous at the late infectious stage, termed the ER transition. Also, membrane- or vesicle-packaged viral replication complexes (VRCs) are induced early during infection. We used microarray assays to screen the Arabidopsis thaliana gene(s) responding to infection with TMV in the initial infection stage and identified an Arabidopsis gene, PAP85 (annotated as a vicilin-like seed storage protein), with upregulated expression during 0.5 to 6 h of TMV infection. TMV accumulation was reduced in pap85-RNA interference (RNAi) Arabidopsis and restored to wild-type levels when PAP85 was overexpressed in pap85-RNAi Arabidopsis. We did not observe the ER transition in TMV-infected PAP85-knockdown Arabidopsis protoplasts. In addition, TMV accumulation was reduced in PAP85-knockdown protoplasts. VRC accumulation was reduced, but not significantly (P = 0.06), in PAP85-knockdown protoplasts. Coexpression of PAP85 and the TMV main replicase (P126), but not their expression alone in Arabidopsis protoplasts, could induce ER aggregations.