Translation initiation landscape profiling reveals hidden open-reading frames required for the pathogenesis of tomato yellow leaf curl Thailand virus.

Plant viruses with densely packed genomes employ noncanonical translational strategies to increase the coding capacity for viral function. However, the diverse translational strategies used make it challenging to define the full set of viral genes. Here, using tomato yellow leaf curl Thailand virus (TYLCTHV, genus Begomovirus) as a model system, we identified genes beyond the annotated gene sets by experimentally profiling in vivo translation initiation sites (TISs). We found that unanticipated AUG TISs were prevalent and determined that their usage involves alternative transcriptional and/or translational start sites and is associated with flanking mRNA sequences. Specifically, two downstream in-frame TISs were identified in the viral gene AV2. These TISs were conserved in the begomovirus lineage and led to the translation of different protein isoforms localized to cytoplasmic puncta and at the cell periphery, respectively. In addition, we found translational evidence of an unexplored gene, BV2. BV2 is conserved among TYLCTHV isolates and localizes to the endoplasmic reticulum and plasmodesmata. Mutations of AV2 isoforms and BV2 significantly attenuated disease symptoms in tomato (Solanum lycopersicum). In conclusion, our study pinpointing in vivo TISs untangles the coding complexity of a plant viral genome and, more importantly, illustrates the biological significance of the hidden open-reading frames encoding viral factors for pathogenicity.

PbABCG1 and PbABCG2 transporters are required for the emission of floral monoterpenes in Phalaenopsis bellina.

Terrestrial plants emit volatiles into the atmosphere to attract both pollinators and the enemies of herbivores, for defense. Phalaenopsis bellina is a scented orchid species in which the main scent components are monoterpenes, including linalool and geraniol, and their derivatives. Here, we investigated whether ABC transporters are involved in floral scent emission. We carried out whole-genome identification of ABC transporter-related genes using four floral transcriptomics libraries of P. bellina. We identified 86 ABC subfamily G genes related to terpenoid transport. After comparing the gene expression patterns of P. bellina with that of Phalaenopsis aphrodite subsp. formosana, a scentless species, followed by gene-to-gene correlation analysis, PbABCG1 and PbABCG2 were selected. The temporal expression of both PbABCG1 and PbABCG2 was highly correlated with that of the key enzyme PbGDPS and the major transcription factor PbbHLH4 in monoterpene biosynthesis, with optimal expression on day 5 post-anthesis. Spatial gene expression analysis showed that PbABCG1 was highly expressed in sepals, whereas PbABCG2 was expressed in the lip. Subcellular localization with a GFP fusion protein revealed that both PbABCG1 and PbABCG2 are cytoplasmic membrane proteins. Co-downregulation of PbABCG1 and PbABCG2 using both double-strand RNA interference and tobacco rattle virus-based gene silencing led to a significant decrease in monoterpene emission, accompanied by an increase in the internal monoterpene pools. Furthermore, ectopic expression of PbABCG1 and PbABCG2 in an ABC16- mutant yeast strain rescued its tolerance to geraniol. Altogether, our results indicate that PbABCG1 and PbABCG2 play substantial roles in monoterpene transport/emission in P. bellina floral scent.

Correction: Plant A20/AN1 protein serves as the important hub to mediate antiviral immunity.

[This corrects the article DOI: 10.1371/journal.ppat.1007288.].

Stress associated proteins coordinate the activation of comprehensive antiviral immunity in Phalaenopsis orchids.

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.

ICTV Virus Taxonomy Profile: Nanoviridae.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus) or eight (genus Nanovirus) genomic DNAs is 0.9-1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0-1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae, which is available at ictv.global/report/nanoviridae.

Identification of MaWRKY40 and MaDLO1 as Effective Marker Genes for Tracking the Salicylic Acid-Mediated Immune Response in Bananas.

Bananas are among the world's most important cash and staple crops but are threatened by various devastating pathogens. The phytohormone salicylic acid (SA) plays a key role in the regulation of plant immune response. Tracking the expression of SA-responsive marker genes under pathogen infection is important in pathogenesis elucidation. However, the common SA-responsive marker genes are not consistently induced in different banana cultivars or different organs. Here, we conducted transcriptome analysis for SA response of a banana cultivar, 'Pei-Chiao' (Cavendish, AAA genome), and identified three genes, MaWRKY40, MaWRKY70, and Downy Mildew Resistant 6 (DMR6)-Like Oxygenase 1 (MaDLO1) that are robustly induced upon SA treatment in both the leaves and roots. Consistent induction of these three genes by SA treatment was also detected in both the leaves and roots of bananas belonging to different genome types such as 'Tai-Chiao No. 7' (Cavendish, AAA genome), 'Pisang Awak' (ABB genome), and 'Lady Finger' (AA genome). Furthermore, the biotrophic pathogen cucumber mosaic virus elicited the expression of MaWRKY40 and MaDLO1 in infected leaves of susceptible cultivars. The hemibiotrophic fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) also consistently induced the expression of MaWRKY40 and MaDLO1 in the infected roots of the F. oxysporum f. sp. cubense TR4-resistant cultivar. These results indicate that MaWRKY40 and MaDLO1 can be used as reliable SA-responsive marker genes for the study of plant immunity in banana. Revealing SA-responsive marker genes provides a stepping stone for further studies in banana resistance to pathogens.

Engineering Plant Resistance to Tomato Yellow Leaf Curl Thailand Virus Using a Phloem-Specific Promoter Expressing Hairpin RNA.

Transgenic approaches employing RNA interference (RNAi) strategies have been successfully applied to generate desired traits in plants; however, variations between RNAi transgenic siblings and the ability to quickly apply RNAi resistance to diverse cultivars remain challenging. In this study, we assessed the promoter activity of a cauliflower mosaic virus 35S promoter (35S) and a phloem-specific promoter derived from rice tungro bacilliform virus (RTBV) and their efficacy to drive RNAi against the endogenous glutamate-1-semialdehyde aminotransferase gene (GSA) that acts as a RNAi marker, through chlorophyll synthesis inhibition, and against tomato yellow leaf curl Thailand virus (TYLCTHV), a begomovirus (family Geminiviridae) reported to be the prevalent cause of tomato yellow leaf curl disease (TYLCD) in Taiwan. Transgenic Nicotiana benthamiana expressing hairpin RNA of GSA driven by either the 35S or RTBV promoter revealed that RTBV::hpGSA induced stronger silencing along the vein and more uniformed silencing phenotype among its siblings than 35S::hpGSA. Analysis of transgenic N. benthamiana, 35S::hpTYLCTHV, and RTBV::hpTYLCTHV revealed that, although 35S::hpTYLCTHV generated a higher abundance of small RNA than RTBV::hpTYLCTHV, RTBV::hpTYLCTHV transgenic plants conferred better TYLCTHV resistance than 35S::hpTYLCTHV. Grafting of wild-type (WT) scions to TYLCTHV RNAi rootstocks allowed transferable TYLCTHV resistance to the scion. A TYLCTHV-inoculation assay showed that noninfected WT scions were only observed when grafted to RTBV::hpTYLCTHV rootstocks but not 35S::hpTYLCTHV nor WT rootstocks. Together, our findings demonstrate an approach that may be widely applied to efficiently confer TYLCD resistance.

Plant A20/AN1 protein serves as the important hub to mediate antiviral immunity.

Salicylic acid (SA) is a key phytohormone that mediates a broad spectrum of resistance against a diverse range of viruses; however, the downstream pathway of SA governed antiviral immune response remains largely to be explored. Here, we identified an orchid protein containing A20 and AN1 zinc finger domains, designated Pha13. Pha13 is up-regulated upon virus infection, and the transgenic monocot orchid and dicot Arabidopsis overexpressing orchid Pha13 conferred greater resistance to different viruses. In addition, our data showed that Arabidopsis homolog of Pha13, AtSAP5, is also involved in virus resistance. Pha13 and AtSAP5 are early induced by exogenous SA treatment, and participate in the expression of SA-mediated immune responsive genes, including the master regulator gene of plant immunity, NPR1, as well as NPR1-independent virus defense genes. SA also induced the proteasome degradation of Pha13. Functional domain analysis revealed that AN1 domain of Pha13 is involved in expression of orchid NPR1 through its AN1 domain, whereas dual A20/AN1 domains orchestrated the overall virus resistance. Subcellular localization analysis suggested that Pha13 can be found localized in the nucleus. Self-ubiquitination assay revealed that Pha13 confer E3 ligase activity, and the main E3 ligase activity was mapped to the A20 domain. Identification of Pha13 interacting proteins and substrate by yeast two-hybrid screening revealed mainly ubiquitin proteins. Further detailed biochemical analysis revealed that A20 domain of Pha13 binds to various polyubiquitin chains, suggesting that Pha13 may interact with multiple ubiquitinated proteins. Our findings revealed that Pha13 serves as an important regulatory hub in plant antiviral immunity, and uncover a delicate mode of immune regulation through the coordination of A20 and/or AN1 domains, as well as through the modulation of E3 ligase and ubiquitin chain binding activity of Pha13.

A Polysaccharide Derived from a Trichosporon sp. Culture Strongly Primes Plant Resistance to Viruses.

Plant viruses cause devastating diseases in plants, yet no effective viricide is available for agricultural application. We screened cultured filtrates derived from various soil microorganisms cultured in vegetable broth that enhanced plant viral resistance. A cultured filtrate, designated F8 culture filtrate, derived from a fungus belonging to the genus Trichosporon, induced strong resistance to various viruses on different plants. Our inoculation assay found the infection rate of Tobacco mosaic virus (TMV)-inoculated Nicotiana benthamiana with F8 culture filtrate pretreatment may decrease to 0%, whereas salicylic acid (SA)-pretreated N. benthamiana attenuated TMV-caused symptoms but remained 100% infected. Tracking Tobacco mosaic virus tagged with green fluorescence protein in plants revealed pretreatment with F8 culture filtrate affected the initial establishment of the virus infection. From F8 culture filtrate, we identified a previously unknown polysaccharide composed of D-mannose, D-galactose, and D-glucose in the ratio 1.0:1.2:10.0 with a α-D-1,4-glucan linkage to be responsible for the induction of plant resistance against viruses through priming of SA-governed immune-responsive genes. Notably, F8 culture filtrate only triggered local defense but was much more effective than conventional SA-mediated systematic acquired resistance. Our finding revealed that microbial cultured metabolites provided a rich source for identification of potent elicitors in plant defense.

Multiplex detection, distribution, and genetic diversity of Hop stunt viroid and Citrus exocortis viroid infecting citrus in Taiwan.

BACKGROUND: Two citrus viroids, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), have been reported and become potential threats to the citrus industry in Taiwan. The distributions and infection rates of two viroids have not been investigated since the two diseases were presented decades ago. The genetic diversities and evolutionary relationships of two viroids also remain unclear in the mix citrus planted region. METHODS: Multiplex RT-PCR was used to detect the two viroids for the first time in seven main cultivars of citrus. Multiplex real-time RT-PCR quantified the distributions of two viroids in four citrus tissues. Sequence alignment and phylogenetic analysis were performed using the ClustalW and MEGA6 (neighbor-joining with p-distance model), respectively. RESULTS: HSVd was found more prevalent than CEVd (32.2% vs. 30.4%). Both CEVd and HSVd were commonly found simultaneously in the different citrus cultivars (up to 55%). Results of the multiplex quantitative analysis suggested that uneven distributions of both viroids with twig bark as the most appropriate material for studies involving viroid sampling such as quarantine inspection. Sequence alignment against Taiwanese isolates, along with analysis of secondary structure, revealed the existence of 10 and 5 major mutation sites in CEVd and HSVd, respectively. The mutation sites in CEVd were located at both ends of terminal and variability domains, whereas those in HSVd were situated in left terminal and pathogenicity domains. A phylogenetic analysis incorporating worldwide viroid isolates indicated three and two clusters for the Taiwanese isolates of CEVd and HSVd, respectively. CONCLUSIONS: Moderately high infection and co-infection rates of two viroids in certain citrus cultivars suggest that different citrus cultivars may play important roles in viroid infection and evolution. These data also demonstrate that two multiplex molecular detection methods developed in the present study provide powerful tools to understand the genetic diversities among viroid isolates and quantify viroids in citrus host. Our field survey can help clarify citrus-viroid relationships as well as develop proper prevention strategies.

A vicilin-like seed storage protein, PAP85, is involved in tobacco mosaic virus replication.

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.

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.

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.

BACKGROUND: RNA 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. RESULTS: We 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. CONCLUSIONS: Our 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.

Characterization of the early response of the orchid, Phalaenopsis amabilis, to Erwinia chrysanthemi infection using expression profiling.

Erwinia chrysanthemi is a devastating bacterial pathogen in Phalaenopsis amabilis and causes soft-rotting disease by secretion of cell wall-degrading enzymes. However, the molecular mechanisms underlying the interaction of P. amabilis with E. chrysanthemi remain elusive. In this study, early molecular events of the plant in response to the pathogen attack were investigated. The alteration in reactive oxygen species accumulation and peroxidase activity occurred at the site of infection. Subsequently, a systematic sequencing of expressed sequence tags (ESTs) using suppression subtractive hybridization (SSH) was performed to obtain the first global picture of the assembly of genes involved in the pathogenesis. The majority of the SSH clones showed a high identity with genes coding for proteins that have known roles in redox homeostasis, responses to pathogens and metabolism. A notable number of the SSH clones were those encoding WRKY, MYB and basic leucine zipper transcription factors, indicating the stimulation of intracellular signal transduction. An orchid gene encoding trans-2-enoyl-CoA reductase (ECR) was the most abundant transcripts in the EST library. ECR is an enzyme catalyzing the very long chain fatty acids (VLCFAs) biosynthesis, and the full-length cDNA of the ECR gene (PaECR1) was obtained. Functional analysis of PaECR1 was conducted by virus-induced gene silencing to knock down the gene expression in P. amabilis. The PaECR1-silenced plants were more susceptible to E. chrysanthemi infection, implying potential roles for VLCFAs in the pathogenesis. In summary, the pathogen-responsive gene expression profiles facilitated a more comprehensive view of the molecular events that underlie this economically important plant-pathogen interaction.

Fungal F8-Culture Filtrate Induces Tomato Resistance against Tomato Yellow Leaf Curl Thailand Virus.

Tomato (Solanum lycopersicum) is an important economic crop worldwide. However, tomato production is jeopardized by the devastating tomato yellow leaf curl disease caused by whitefly-transmitted begomoviruses (WTBs). In this study, we evaluated the efficacy of our previously developed plant antiviral immunity inducer, fungal F8-culture filtrate, on tomato to combat tomato yellow leaf curl Thailand virus (TYLCTHV), the predominant WTB in Taiwan. Our results indicated that F8-culture filtrate treatment induced strong resistance, did not reduce the growth of tomato, and induced prominent resistance against TYLCTHV both in the greenhouse and in the field. Among TYLCTHV-inoculated Yu-Nu tomato grown in the greenhouse, a greater percentage of plants treated with F8-culture filtrate (43-100%) were healthy-looking compared to the H2O control (0-14%). We found that TYLCTHV cannot move systemically only on the F8-culture filtrate pretreated healthy-looking plants. Tracking the expression of phytohormone-mediated immune maker genes revealed that F8-culture filtrate mainly induced salicylic acid-mediated plant immunity. Furthermore, callose depositions and the expression of the pathogen-induced callose synthase gene, POWDERY MILDEW RESISTANT 4 were only strongly induced by TYLCTHV on tomato pretreated with F8-culture filtrate. This study provides an effective way to induce tomato resistance against TYLCTHV.

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.

Cucumber mosaic virus-induced gene silencing in banana.

Banana (Musa spp.) is one of the world's most important staple and cash crops. Despite accumulating genetic and transcriptomic data, low transformation efficiency in agronomically important Musa spp. render translational researches in banana difficult by using conventional knockout approaches. To develop tools for translational research in bananas, we developed a virus induced-gene silencing (VIGS) system based on a banana-infecting cucumber mosaic virus (CMV) isolate, CMV 20. CMV 20 genomic RNA 1, 2, and 3, were separately cloned in Agrobacterium pJL89 binary vectors, and a cloning site was introduced on RNA 2 immediately after the 2a open reading frame to insert the gene targeted for silencing. An efficient Agrobacterium inoculation method was developed for banana, which enabled the CMV 20 VIGS vector infection rate to reach 95% in our experiments. CMV 20-based silencing of Musa acuminata cv. Cavendish (AAA group) glutamate 1-semialdehyde aminotransferase (MaGSA) produced a typical chlorotic phenotype and silencing of M. acuminata phytoene desaturase (MaPDS) produced a photobleachnig phenotype. We show this approach efficiently reduced GSA and PDS transcripts to 10% and 18% of the control, respectively. The high infection rate and extended silencing of this VIGS system will provide an invaluable tool to accelerate functional genomic studies in banana.

Optimizing virus-induced gene silencing efficiency with Cymbidium mosaic virus in Phalaenopsis flower.

Virus-induced gene silencing (VIGS) is a good way to study floral gene functions of orchids, especially those with a long life cycle. To explore the applicability and improve viral silencing efficiency for application of Cymbidium mosaic virus (CymMV)-induced gene silencing, we examined several variables, including the optimal length of the DNA fragment, the effect of developmental maturation status of inflorescence, and suitable inoculation sites. A CymMV-based VIGS system can be used with orchids to silence genes including PeUFGT3, PeMADS5 and PeMADS6 and induce prominent phenotypes with silencing efficiency up to 95.8% reduction. The DNA fragment size used for silencing can be as small as 78-85 bp and still reach 61.5-95.8% reduction. The effect of cDNA location as a target in VIGS varies among genes because of non-target gene influence when using the 5' terminus of the coding region of both PeMADS5 and PeMADS6. Use of VIGS to knock down a B-class MADS-box gene (PeMADS6) in orchids with different maturation status of inflorescence allowed for observing discernable knockdown phenotypes in flowers. Furthermore, silencing effects with Agro-infiltration did not differ with both leaf and inflorescence injections, but injection in the leaf saved time and produced less damage to plants. We propose an optimized approach for VIGS using CymMV as a silencing vector for floral functional genomics in Phalaenopsis orchid with Agro-infiltration: (1) DNA fragment length about 80 bp, (2) a more mature status of inflorescence and (3) leaf injection.