The R2R3-MYB transcription factor EVER controls the emission of petunia floral volatiles by regulating epicuticular wax biosynthesis in the petal epidermis.

The epidermal cells of petunia (Petunia × hybrida) flowers are the main site of volatile emission. However, the mechanisms underlying the release of volatiles into the environment are still being explored. Here, using cell-layer-specific transcriptomic analysis, reverse genetics by virus-induced gene silencing and clustered regularly interspaced short palindromic repeat (CRISPR), and metabolomics, we identified EPIDERMIS VOLATILE EMISSION REGULATOR (EVER)-a petal adaxial epidermis-specific MYB activator that affects the emission of volatiles. To generate ever knockout lines, we developed a viral-based CRISPR/Cas9 system for efficient gene editing in plants. These knockout lines, together with transient-suppression assays, revealed EVER's involvement in the repression of low-vapor-pressure volatiles. Internal pools and annotated scent-related genes involved in volatile production and emission were not affected by EVER. RNA-Seq analyses of petals of ever knockout lines and EVER-overexpressing flowers revealed enrichment in wax-related biosynthesis genes. Liquid chromatography/gas chromatography-MS analyses of petal epicuticular waxes revealed substantial reductions in wax loads in ever petals, particularly of monomers of fatty acids and wax esters. These results implicate EVER in the emission of volatiles by fine-tuning the composition of petal epicuticular waxes. We reveal a petunia MYB regulator that interlinks epicuticular wax composition and volatile emission, thus unraveling a regulatory layer in the scent-emission machinery in petunia flowers.

The R2R3-MYB-like regulatory factor EOBI, acting downstream of EOBII, regulates scent production by activating ODO1 and structural scent-related genes in petunia.

Flower scent is a highly dynamic trait, under developmental, spatial, and diurnal regulation. The mechanism governing scent production is only beginning to be unraveled. In petunia (Petunia hybrida), EMISSION OF BENZENOIDS II (EOBII) controls transcription of both the shikimate pathway-regulating MYB factor ODORANT1 (ODO1) and phenylpropanoid scent-related structural genes. A promoter-activation screen identified an R2R3-MYB-like regulatory factor of phenylpropanoid volatile biosynthesis acting downstream of EOBII, designated EOBI. EOBI silencing led to downregulation of ODO1 and numerous structural scent-related genes from both the shikimate and phenylpropanoid pathways. The ability of EOBI to directly activate ODO1, as revealed by electrophoretic mobility shift assay and yeast one-hybrid analysis, place EOBI upstream of ODO1 in regulating substrate availability for volatile biosynthesis. Interestingly, ODO1-silenced transgenic petunia flowers accumulated higher EOBI transcript levels than controls, suggesting a complex feedback loop between these regulatory factors. The accumulation pattern of EOBI transcript relative to EOBII and ODO1, and the effect of up/downregulation of EOBII on transcript levels of EOBI and ODO1, further support these factors' hierarchical relationships. The dependence of scent production on EOBI expression and its direct interaction with both regulatory and structural genes provide evidence for EOBI's wide-ranging involvement in the production of floral volatiles.

EOBII, a gene encoding a flower-specific regulator of phenylpropanoid volatiles' biosynthesis in petunia.

Floral scent, which is determined by a complex mixture of low molecular weight volatile molecules, plays a major role in the plant's life cycle. Phenylpropanoid volatiles are the main determinants of floral scent in petunia (Petunia hybrida). A screen using virus-induced gene silencing for regulators of scent production in petunia flowers yielded a novel R2R3-MYB-like regulatory factor of phenylpropanoid volatile biosynthesis, EMISSION OF BENZENOIDS II (EOBII). This factor was localized to the nucleus and its expression was found to be flower specific and temporally and spatially associated with scent production/emission. Suppression of EOBII expression led to significant reduction in the levels of volatiles accumulating in and emitted by flowers, such as benzaldehyde, phenylethyl alcohol, benzylbenzoate, and isoeugenol. Up/downregulation of EOBII affected transcript levels of several biosynthetic floral scent-related genes encoding enzymes from the phenylpropanoid pathway that are directly involved in the production of these volatiles and enzymes from the shikimate pathway that determine substrate availability. Due to its coordinated wide-ranging effect on the production of floral volatiles, and its lack of effect on anthocyanin production, a central regulatory role is proposed for EOBII in the biosynthesis of phenylpropanoid volatiles.

A T7-driven silencing system in transgenic plants expressing T7 RNA polymerase is a nuclear process.

We previously demonstrated a case of silencing in transgenic plants expressing T7 RNA polymerase in which expression of a reporter gene placed under the control of the T7 promoter was silenced. Here we demonstrate that endogenous genes can be silenced by the same system. The T7-driven silencing system does not conform to several aspects characteristic of post-transcriptional RNA silencing in plants, and this prompted an investigation into the mechanisms underlying this type of silencing. The present paper demonstrates that T7-driven silencing is a post-transcriptional process that is restricted to the nucleus. Nuclear run-on assays indicated the presence of silenced gene transcripts in both orientations. SiRNA corresponding to the silenced gene could not be traced in the cytoplasm but was found in nuclei. The silenced gene was hypermethylated. We present evidence that a tobacco RNA-dependent RNA polymerase (RdRP) is not involved in T7-mediated silencing, but indicate the involvement of a nuclear RdRP in this type of silencing.

Isolation and characterization of Israeli acute paralysis virus, a dicistrovirus affecting honeybees in Israel: evidence for diversity due to intra- and inter-species recombination.

We report the isolation, purification, genome-sequencing and characterization of a picorna-like virus from dead bees in Israel. Sequence analysis indicated that IAPV (Israeli acute paralysis virus) is a distinct dicistrovirus. It is most homologous to Kashmir bee virus and acute bee paralysis virus. The virus carries a 9487 nt RNA genome in positive orientation, with two open reading frames separated by an intergenic region, and its coat comprises four major proteins, the sizes of which suggest alternate processing of the polyprotein. IAPV virions also carry shorter, defective-interfering (DI)-like RNAs. Some of these RNAs are recombinants of different segments of IAPV RNA, some are recombinants of IAPV RNA and RNA from another dicistrovirus, and yet others are recombinants of IAPV and non-viral RNAs. In several of the DI-like RNAs, a sense-oriented fragment has recombined with its complement, forming hairpins and stem-loop structures. In previous reports, we have shown that potyviral and IAPV sequences are integrated into the genome of their respective hosts. The dynamics of information exchange between virus and host and the possible resistance-engendering mechanisms are discussed.

Reverse genetics of floral scent: application of tobacco rattle virus-based gene silencing in Petunia.

Floral fragrance is responsible for attracting pollinators as well as repelling pathogens and pests. As such, it is of immense biological importance. Molecular dissection of the mechanisms underlying scent production would benefit from the use of model plant systems with big floral organs that generate an array of volatiles and that are amenable to methods of forward and reverse genetics. One candidate is petunia (Petunia hybrida), which has emerged as a convenient model system, and both RNAi and overexpression approaches using transgenes have been harnessed for the study of floral volatiles. Virus-induced gene silencing (VIGS) is characterized by a simple inoculation procedure and rapid results relative to transgenesis. Here, we demonstrate the applicability of the tobacco rattle virus-based VIGS system to studies of floral scent. Suppression of the anthocyanin pathway via chalcone synthase silencing was used as a reporter, allowing easy visual identification of anthocyaninless silenced flowers/tissues with no effect on the level of volatile emissions. Use of tobacco rattle virus constructs containing target genes involved in phenylpropanoid volatile production, fused to the chalcone synthase reporter, allowed simple identification of flowers with suppressed activity of the target genes. The applicability of VIGS was exemplified with genes encoding S-adenosyl-l-methionine:benzoic acid/salicylic acid carboxyl methyltransferase, phenylacetaldehyde synthase, and the myb transcription factor ODORANT1. Because this high-throughput reverse-genetics approach was applicable to both structural and regulatory genes responsible for volatile production, it is expected to be highly instrumental for large-scale scanning and functional characterization of novel scent genes.

Reproductive development and phenotypic differences in garlic are associated with expression and splicing of LEAFY homologue gaLFY.

Modern garlic (Allium sativum L.) cultivars are sterile and propagated only vegetatively. The recent discovery of fertile genotypes in Central Asia and the restoration of flowering and fertility by environmental manipulations open the way for in-depth florogenetic, genetic, and molecular research in garlic. In the present work, two bolting garlic accessions were employed: #3026, developing normal flowers and seeds, and #2509, in which flowers abort at the early stages of development. Morphological studies showed transition of the apical meristems from the vegetative to the reproductive stage and inflorescence initiation in both genotypes. Low temperatures promote transition of the apex and stem elongation, but have no effect on the phenotypic expression of the inflorescence development. The initial stages of reproductive development in non-flowering #2509 plants were followed by abortion of floral primordia at the differentiation stage. A search for genes involved in the control of flowering in garlic resulted in identification of the garlic LEAFY/FLO homologue, gaLFY. Further comparative analyses of gene expression revealed two gaLFY transcripts, differing in 64 nucleotides, with clear splicing borders. The short variant transcript was identified in both genotypes throughout all development stages, whereas the long variant appears in the flowering genotype #3026 only during reproductive development. The phenotypic differences in garlic, with regard to flowering, may be associated with the efficacy of the splicing process.

Tobacco mosaic virus regulates the expression of its own resistance gene N.

The N gene of tobacco (Nicotiana tabacum) is a typical resistance (R) gene engendering localization of tobacco mosaic virus (TMV) infection and the elicitation of a hypersensitive necrotic response. The consensus model for R gene-derived resistance is at the level of protein:protein interactions, in which proteins of the pathogen interact with already present receptor-like proteins produced by the plant's R genes. This article demonstrates, by quantitative real-time reverse transcription-PCR analysis, that in tobacco carrying the dominant allele N, a basal level of transcription indeed occurs in noninfected plants. However, accumulation of N-mRNA in infected plants indicates that transcription is stimulated by TMV infection (up to 38-fold in locally infected leaves and up to 165-fold in upper, noninoculated leaves). Potato virus Y infection did not result in accumulation of N-mRNA, indicating a specific TMV-related phenomenon. The possible uncoupling of viral restriction from necrosis is discussed.

Identification and characterization of phytoplasmal genes, employing a novel method of isolating phytoplasmal genomic DNA.

Phytoplasmas are unculturable, insect-transmissible plant pathogens belonging to the class Mollicutes. To be transmitted, the phytoplasmas replicate in the insect body and are delivered to the insect's salivary glands, from where they are injected into the recipient plant. Because phytoplasmas cannot be cultured, any attempt to recover phytoplasmal DNA from infected plants or insects has resulted in preparations with a large background of host DNA. Thus, studies of the phytoplasmal genome have been greatly hampered, and aside from the rRNA genes, only a few genes have hitherto been isolated and characterized. We developed a unique method to obtain host-free phytoplasmal genomic DNA from the insect vector's saliva, and we demonstrated the feasibility of this method by isolating and characterizing 78 new putative phytoplasmal open reading frames and their deduced proteins. Based on the newly accumulated information on phytoplasmal genes, preliminary characteristics of the phytoplasmal genome are discussed.

Vaccination with E. coli recombinant empty viral particles of infectious bursal disease virus (IBDV) confer protection.

The A genome segment of the highly virulent Infectious bursal disease virus (IBDV) was amplified using long and accurate-RT-PCR (LA-RT-PCR). The entire sequence region encoding VP2, VP4, and VP3 in that order was cloned and sequenced. Following subcloning into the Escherichia coli expression vector pET21a under the T7 promoter, viral proteins were expressed and processed as demonstrated by Western blot analysis. Virus-like particles could be visualized by immuno-electron microscopy in IPTG-induced cells suggesting that viral assembly can take place in E. coli. Induction of anti-IBDV antibodies was detected in chickens immunized with purified recombinant IBDV by intra muscular (i.m.) injection. Furthermore, the vaccinated chickens were protected when challenged with the Gep 5 isolate of IBDV.