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Dianthus chinensis is a widely used landscaping plant with high commercial value. Understanding the molecular mechanisms responsible for floral organ formation can help to improve the traits (e.g.,, petal size, floral organ shape, etc.) of D. chinensis. However, there has no comprehensive study of ABCE MADS-box genes in D. chinensis. In this study, we identified 15 ABCE MADS-box genes of D. chinensis. The expression and interactions patterns among ABCE MADS-box genes were analysis by in situ, qRT-PCT, Yeast two‐hybrid and bimolecular fluorescent complimentary experiments. In different stages of petal development, the expression of DcNAC56 changes with DcPI2. The overexpression of DcPI2 in D. chinensis resulted in high expression levels of DcNAC56. 35S: DcPI2 transgenic plants had a larger petal size with petals expansion. Furthermore, we observed that the DcPI2 protein can bind to the DcNAC56 promoter in D. chinensis. On the basis of these results, we propose that spatiotemporal expression of class ABCE genes and different combinations between class ABCE proteins might regulate floral organ specification. And DcPI2 can regulate the expansion of petals by binding to DcNAC56 involved in cell proliferation and differentiation. These results provide new insights into the improving cultivars of Dianthus by change floral traits.

International audience; Dianthus chinensis is widely used as a landscaping plant in rock gardens and has high commercial value. However, few studies of this plant's regeneration and genetic transformation have been reported. In this study, cotyledons of D. chinensis were used as explants for regeneration and infection with Agrobacterium. The results indicated that the efficiency of regeneration was markedly improved by using 1-naphthylcetic acid (NAA). The cotyledons were cocultured with Agrobacterium tumefaciens EHA105 containing pK7GWIWG2-GFP plasmids for 15-20 min. Transgenic regenerated plants were obtained by kanamycin (80 mg.L-1) screening. qRT-PCR results confirmed the expression of GFP and the DcIPT1 (which is related to cell division) gene in the transgenic DcIPT1 plants of D. chinensis. The results show that the proliferation ability of transgenic DcIPT1 plants is enhanced. Overall, we report the successful establishment of a reliable and efficient method for cotyledon transformation and plant regeneration in D. chinensis based on the combination of infection with Agrobacterium and plant regeneration. This work will contribute to functional gene research and genetic improvement of Dianthus species plants.

The rose is the queen of flowers and is widely used as a garden plant and for the cut flower market. Roses are also used for the production of essential oil for the cosmetic and perfume industries. A lot of botanical roses are scented and use their volatiles to attract pollinators. Fragrances in garden roses are very diverse and scent has always been an important character in the selection process. Breeders have recently tried to introduce new fragrances, for instance reminiscent of fruit or spice odors. But despite their efforts, some roses on the market are not very fragrant, especially the ones selected for the cut flower market. The cause of this lack of scent is not known. In modern roses or Hybrid Tea roses, scent is mainly produced by petals, although stamens can also contribute to the emission of volatiles. Scent compounds are concentrated in rose petal epidermis, probably in small lipid droplets. In spite of numerous chemical studies, the biosynthetic pathways of many rose scent compounds are unknown. We are studying several genes involved in the biosynthesis of scent in rose. For example, we recently characterized the enzymes responsible for the so-called “tea scent” emitted by Chinese roses. Apart from “tea scent”, terpenoids, especially monoterpenes, are also major constituents of rose flowers, mostly responsible for the “typical rose scent”. Generally, terpenoid biosynthesis in plants is achieved by various terpene synthases. However, with a combination of transcriptomic and genetic approaches, our research group recently discovered a terpene synthase-independent pathway. A key enzyme of this pathway is RhNUDX1, belonging to the Nudix protein family. It has geranyl diphosphate diphosphohydrolase activity in vitro. A positive correlation was found between the expression levels of RhNUDX1 gene and the production of the monoterpene geraniol, indicating its essential role in scent production in roses.

An SNP mutation in the miR172 target site of the DcAP2L gene is associated with a double flower phenotype in Dianthus chinensis., The double flower is a highly important breeding trait that affects the ornamental value in many flowering plants. To get a better understanding of the genetic mechanism of double flower formation in Dianthus chinensis, we have constructed a high-density genetic map using 140 F2 progenies derived from a cross between a single flower genotype and a double flower genotype. The linkage map was constructed using double-digest restriction site-associated DNA sequencing (ddRAD-seq) with 2353 single nucleotide polymorphisms (SNPs). Quantitative trait locus (QTL) mapping analysis was conducted for 12 horticultural traits, and major QTLs were identified for nine of the 12 traits. Among them, two major QTLs accounted for 20.7% and 78.1% of the total petal number variation, respectively. Bulked segregant RNA-seq (BSR-seq) was performed to search accurately for candidate genes associated with the double flower trait. Integrative analysis of QTL mapping and BSR-seq analysis using the reference genome of Dianthus caryophyllus suggested that an SNP mutation in the miR172 cleavage site of the A-class flower organ identity gene APETALA2 (DcAP2L) is responsible for double flower formation in Dianthus through regulating the expression of DcAG genes.

The Translationally Controlled Tumor Protein (TCTP) is a conserved protein which expression was associated with several biochemical and cellular functions. Loss-of-function mutants are lethal both in animals and in plants, making the identification of its exact role difficult. Recent data using the model plant Arabidopsis thaliana provided the first viable adult knockout for TCTP and helped addressing the biological role of TCTP during organ development and the functional conservation between plants and animals. This chapter summarizes our up to date knowledge about the role of TCTP in plants and discuss about conserved functions and mechanisms between plants and animals.

International audience; Roses hold high symbolic value and great cultural importance in different societies throughout human history. They are widely used as garden ornamental plants, as cut flowers, and for the production of essential oils for the perfume and cosmetic industries. Domestication of roses has a long and complex history, and the rose species have been hybridized across vast geographic areas such as Europe, Asia, and the Middle East. The domestication processes selected several flower characters affecting floral quality, such as recurrent flowering, double flowers, petal colours, and fragrance. The molecular and genetic events that determine some of these flower characters cannot be studied using model species such as Arabidopsis thaliana, or at least only in a limited manner. In this review, we comment on the recent development of genetic, genomic, and transcriptomic tools for roses, and then focus on recent advances that have helped unravel the molecular mechanisms underlying several rose floral traits.

International audience; Phyllody is a flower abnormality in which leaf-like structures replace flower organs in all whorls. Here, we investigated the origin and the molecular mechanism of phyllody phenotype in Rosa chinensis cv. Viridiflora, an ancient naturally occurring Chinese mutant cultivar. Reciprocal grafting experiments and microscopy analyses, demonstrated that the phyllody phenotype in Viridiflora is not associated with phytoplasmas infection. Transcriptome comparisons by the mean of RNA-Seq identified 672 up regulated and 666 down-regulated genes in Viridiflora compared to its closely related genotype R. chinensis cv. Old Blush. A fraction of these genes are putative homologs of genes known to be involved in flower initiation and development. We show that in flower whorl 2 of Viridiflora, a down-regulation of the floral organ identity genes RcPISTILLATA (RcPI), RcAPETALA3 (RcAP3) and RcSEPALLATA3 (RcSEP3), together with an up -regulation of the putative homolog of the gene SUPPRESSOR of OVEREXPRESSION of CONSTANS1 (RcSOC1) are likely at the origin of the loss of petal identity and leaf -like structures formation. In whorl 3 of Viridiflora, ectopic expression of RcAPETALA2 (RcAP2) along with the down regulation of RcPI, RcAP3, and RcSEP3 is associated with loss of stamens identity and leaf -like structures formation. In whorl 4, the ectopic expression of RcAP2 associated with a down -regulation of RcSEP3 and of the C-class gene RcAGAMOUS correlate with loss of pistil identity. The latter also suggested the antagonist effect between the A and C class genes in the rose. Together, these data suggest that modified expression of the ABCE flower organ identity genes is associated with the phyllody phenotype in the rose Viridiflora and that these genes are important for normal flower organs development.

*† ‡ SUMMARY In Arabidopsis, four homeotic gene classes, A, B, C and E, are required for the patterning of floral organs. However, very little is known about how the activity of these master genes is translated into regulatory processes leading to specific growth patterns and the formation of organs with specific shapes and sizes. Previously we showed that the transcript variant BPEp encodes a bHLH transcription factor that is involved in limiting petal size by controlling post-mitotic cell expansion. Here we show that the phytohormone jasmonate is required for control of BPEp expression. Expression of BPEp was negatively regulated in opr3 mutant flowers that are deficient in jasmonate synthesis. Moreover, the expression of BPEp was restored in opr3 flowers following exogenous jasmonate treatments. Expression of the second transcript variant BPEub, which originates from the same gene as BPEp via an alternative splicing event, was not affected, indicating that BPEp accumulation triggered by jasmonate occurs at the post-transcriptional level. Consistent with these data, opr3 exhibited an increase in petal size as a result of increased cell size, as well as a modified vein pattern, phenotypes that are similar to those of thebpe-1 mutant. Furthermore, exogenous treatments with jasmonate rescued petal phenotypes associated with loss of function of OPR3. Our data demonstrate that jasmonate signaling downstream of OPR3 is involved in the control of cell expansion and in limiting petal size, and that BPEp is a downstream target that functions as a component mediating jasmonate signaling during petal growth.

Stop to smell the roses Some roses smell beautiful, yet others only look beautiful. Magnard et al. leveraged this distinction to study the biosynthesis of geraniol, a monoterpene alcohol in rose scent (see the Perspective by Tholl and Gershenzon). Enzymes known for geraniol synthesis in other plants, such as basil, did not seem to provide that function for roses. Instead, a diphosphohydrolase, which functions in the cytoplasm of cells in rose petals, generates the geraniol emitted by fragrant roses. Identification of the enzyme and its gene enables marker-assisted breeding to put the perfume back into beauty. Science , this issue p. 81 ; see also p. 28

International audience; Rosa chinensis ‘Pallida’ (Rosa L.) is one of the most important ancient rose cultivars originating from China. It contributed the ‘tea scent’ trait to modern roses. However, little information is available on the gene regulatory networks involved in scent biosynthesis and metabolism in Rosa. In this study, the transcriptome of R. chinensis ‘Pallida’ petals at different developmental stages, from flower buds to senescent flowers, was investigated using Illumina sequencing technology. De novo assembly generated 89,614 clusters with an average length of 428 bp. Based on sequence similarity search with known proteins, 62.9% of total clusters were annotated. Out of these annotated transcripts, 25,705 and 37,159 sequences were assigned to gene ontology and clusters of orthologous groups, respectively. The dataset provides information on transcripts putatively associated with known scent metabolic pathways. Digital gene expression (DGE) was obtained using RNA samples from flower bud, open flower and senescent flower stages. Comparative DGE and quantitative real time PCR permitted the identification of five transcripts encoding proteins putatively associated with scent biosynthesis in roses. The study provides a foundation for scent-related gene discovery in roses.

The link between gene regulation/function and organ shape (morphogenesis) is poorly understood and remains one of the major issues in developmental biology. Petals are attractive model organs for studying organogenesis mainly because they have a simple laminar structure with a small number of cell types. Moreover, because petals are dispensable for plant growth and reproduction, one can experimentally manipulate petal development and dissect the genetic mechanisms behind the changes without serious effects on plant viability. Here, we describe the methods used to study petal development at the molecular, cytological, and genetic level, and more specifically mitotic and post-mitotic growth control during petal morphogenesis.

The biophysical properties of the tobacco mosaic tobamovirus (TMV) coat protein (CP) make it possible to display foreign peptides on the surface of TMV. The immunogenic epitopes G5-24 from the rabies virus (RV) glycoprotein, and 5B19 from murine hepatitis virus (MHV) S-glycoprotein were successfully displayed on the surface of TMV, and viruses accumulated to high levels in infected leaves of Nicotiana tabacum Xanthi-nn. The peptide RB19, which contains an arginine residue plus the 5B19 epitope fused to the CP (TMV-RB19), resulted in the induction of necrotic local lesions on inoculated leaves of N. tabacum Xanthi-nn and cell death of infected BY2 protoplasts. RNA dot blot assays confirmed that expression of the acidic and basic pathogenesis-related PR2 genes were induced in infected Xanthi-nn leaf tissue. TMV that carried epitope 31D from the RV nucleoprotein did not accumulate in inoculated tobacco leaves. Analysis of hybrid CPs predicted that the isoelectric points (pI):charge value was 5.31:-2 for wild-type CP, 5.64:-1 for CP-RB19, and 9.14:+2 for CP-31D. When acidic amino acids were inserted in CP-RB19 and CP-31D to bring their pI:charge to near that of wild-type CP, the resulting viruses TMV-RB19E and TMV-4D:31D infected N. tabacum Xanthi-nn plants and BY2 protoplasts without causing cell death. These data show the importance of the pI of the epitope and its effects on the hybrid CP pI:charge value for successful epitope display as well as the lack of tolerance to positively charged epitopes on the surface of TMV.

To monitor infection of Nicotiana benthamiana by tobacco mosaic virus (TMV), leaves were inoculated with viral constructs expressing the green fluorescent protein (GFP) from jellyfish (Aequorea victoria) fused to the movement protein (MP) of TMV (MP:GFP) or as a free GFP in place of the coat protein (CP). Infection sites produced by TMV expressing the MP:GFP appeared as fluorescent rings larger in diameter and less fluorescent than fluorescent disks induced by constructs encoding free GFP. These results suggest that protein expression driven by the MP subgenomic promoter (sgp) initiates and ends earlier and is at lower level than that observed for proteins driven by the CP sgp. Similarly, analyses of cross sections through the infection sites revealed that in different cell types the accumulation of MP:GFP was regulated differently than the accumulation of free GFP. Immunocytochemistry and electron microscopy showed that near the leading edge of the fluorescent ring the MP:GFP and the viral 126 kDa and 183 kDa replicase proteins accumulated in paired cytoplasmic bodies that formed often on opposite sides of adjacent cell walls containing plasmodesmata. In the dimly fluorescent center of the rings the 126 kDa and 183 kDa proteins, but not the MP:GFP, were localized in unpaired cytoplasmic bodies containing ropelike, fibrillar structures. The paired bodies were similar to previously described viroplasms, while the unpaired bodies were similar to X-bodies. These data indicate that the accumulation of proteins expressed from different sgps of TMV has a specific spatial and temporal pattern in planta. In addition, the cytoplasmic bodies containing the 126 kDa and 183 kDa proteins are dynamic entities whose protein content and subcellular location change during infection.

Coat protein-mediated resistance (CP-MR) has been widely used to protect transgenic plants against virus diseases. To characterize the mechanisms of CP-MR to tobacco mosaic tobamovirus (TMV) we developed mutants of the coat protein that affected subunit-subunit interactions. Mutant CPs were expressed during TMV replication as well as in transgenic Nicotiana tabacum plants. The mutation T42-->W increased protein aggregation and T28-->W abolished aggregation and assembly, while the mutations T28-->W plus T42-->W and T89-->W altered normal CP subunit-subunit interactions. The mutant T28W was unable to assemble virus-like particles (VLPs) during infection and in transgenic plants failed to aggregate; this protein conferred no protection against challenge of transgenic plants by TMV. The mutant T42W had strong CP subunit-subunit interactions and formed VLPs but not infectious virions. Transgenic lines with this protein exhibited stronger protection against TMV infection than transgenic plants that contained the wild-type (wt) CP. It is proposed that increased resistance conferred by the T42W mutant results from strong interaction between transgenic CP subunits and challenge virus CP subunits. CP carrying the mutation T89-->W formed flexuous and unstable VLPs whereas the double mutant T28W:T42W formed open helical structures that accumulated as paracrystalline arrays. In transgenic plants, T89W and the double mutant CPs showed reduced ability to aggregate and provided lower protection against TMV infection than wt CP. A strong correlation between normal CP subunit-subunit interactions and CP-MR is observed, and a model for CP-MR involving interactions between the transgenic CP and the CP of the challenge virus as well as interference with virus movement is discussed.

One of the most severe diseases of cultivated tomato worldwide is caused by tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci. Here we describe the application of antisense RNAs to interfere with the disease caused by TYLCV. The target of the antisense RNA is the rare messenger RNA of the Rep protein, encoded by the C1 gene. Transgenic Nicotiana benthamiana plants expressing C1 antisense RNA were obtained and shown to resist infection by TYLCV. Some of the resistant lines are symptomless, and the replication of challenge TYLCV almost completely suppressed. The transgenes mediating resistance were shown to be effective through at least two generations of progeny.

International audience; Cultivated for centuries, the varieties of rose have been selected based on a number of flower traits. Understanding the genetic and molecular basis that contributes to these traits will impact on future improvements for this economically important ornamental plant. In this study, we used scanning electron microscopy and sections of meristems and flowers to establish a precise morphological calendar from early rose flower development stages to senescing flowers. Global gene expression was investigated from floral meristem initiation up to flower senescence in three rose genotypes exhibiting contrasted floral traits including continuous versus once flowering and simple versus double flower architecture, using a newly developed Affymetrix microarray (Rosa1_Affyarray) tool containing sequences representing 4765 unigenes expressed during flower development. Data analyses permitted the identification of genes associated with floral transition, floral organs initiation up to flower senescence. Quantitative real time PCR analyses validated the mRNA accumulation changes observed in microarray hybridizations for a selection of 24 genes expressed at either high or low levels. Our data describe the early flower development stages in Rosa sp, the production of a rose microarray and demonstrate its usefulness and reliability to study gene expression during extensive development phases, from the vegetative meristem to the senescent flower.

Plant organ growth and final size are determined by coordinated cell proliferation and expansion. The BIGPETALp (BPEp) basic helix-loop-helix (bHLH) transcription factor was shown to limit Arabidopsis thaliana petal growth by influencing cell expansion. We demonstrate here that BPEp interacts with AUXIN RESPONSE FACTOR8 (ARF8) to affect petal growth. This interaction is mediated through the BPEp C-terminal domain (SDBPEp) and the C-terminal domain of ARF8. Site-directed mutagenesis identified an amino acid consensus motif in SDBPEp that is critical for mediating BPEp-ARF8 interaction. This motif shares sequence similarity with motif III of ARF and AUXIN/INDOLE-3-ACETIC ACID proteins. Petals of arf8 mutants are significantly larger than those of the wild type due to increased cell number and increased cell expansion. bpe arf8 double mutant analyses show that during early petal development stages, ARF8 and BPEp work synergistically to limit mitotic growth. During late stages, ARF8 and BPEp interact to limit cell expansion. The alterations in cell division and cell expansion observed in arf8 and/or bpe mutants are associated with a change in expression of early auxin-responsive genes. The data provide evidence of an interaction between an ARF and a bHLH transcription factor and of its biological significance in regulating petal growth, with local auxin levels likely influencing such a biological function.

Background Roses have been cultivated for centuries and a number of varieties have been selected based on flower traits such as petal form, color, and number. Wild-type roses have five petals (simple flowers), whereas high numbers of petals (double flowers) are typical attributes of most of the cultivated roses. Here, we investigated the molecular mechanisms that could have been selected to control petal number in roses. Methodology/Principal Findings We have analyzed the expression of several candidate genes known to be involved in floral organ identity determination in roses from similar genetic backgrounds but exhibiting contrasting petal numbers per flower. We show that the rose ortholog of AGAMOUS (RhAG) is differentially expressed in double flowers as compared to simple flowers. In situ hybridization experiments confirm the differential expression of RhAG and demonstrate that in the double-flower roses, the expression domain of RhAG is restricted toward the center of the flower. Conversely, in simple-flower roses, RhAG expression domain is wider. We further show that the border of RhAG expression domain is labile, which allows the selection of rose flowers with increased petal number. Double-flower roses were selected independently in the two major regions for domestication, China and the peri-Mediterranean areas. Comparison of RhAG expression in the wild-type ancestors of cultivated roses and their descendants both in the European and Chinese lineages corroborates the correlation between the degree of restriction of RhAG expression domain and the number of petals. Our data suggests that a restriction of RhAG expression domain is the basis for selection of double flowers in both the Chinese and peri-Mediterranean centers of domestication. Conclusions/Significance We demonstrate that a shift in RhAG expression domain boundary occurred in rose hybrids, causing double-flower phenotype. This molecular event was selected independently during rose domestication in Europe/Middle East and in China.

The growth of an organism and its size determination require the tight regulation of cell proliferation and cell growth. However, the mechanisms and regulatory networks that control and integrate these processes remain poorly understood. Here, we address the biological role of Arabidopsis translationally controlled tumor protein (AtTCTP) and test its shared functions in animals and plants. The data support a role of plant AtTCTP as a positive regulator of mitotic growth by specifically controlling the duration of the cell cycle. We show that, in contrast to animal TCTP , plant AtTCTP is not implicated in regulating postmitotic growth. Consistent with this finding, plant AtTCTP can fully rescue cell proliferation defects in Drosophila loss of function for dTCTP. Furthermore, Drosophila dTCTP is able to fully rescue cell proliferation defects in Arabidopsis tctp knockouts. Our data provide evidence that TCTP function in regulating cell division is part of a conserved growth regulatory pathway shared between plants and animals. The study also suggests that, although the cell division machinery is shared in all multicellular organisms to control growth, cell expansion can be uncoupled from cell division in plants but not in animals.

International audience; Somatic embryogenesis was induced from in vitro-derived leaf explants of Rosa chinensis cultivar (cv) Old Blush. Calli producing embryos with expanded cotyledons (RcOBType1 embryos) were obtained. Further refinements of the callus maintenance medium generated a more typical rose embryogenic callus (RcOBType2) displaying high levels of secondary embryogenesis and embryos with limited cotyledon expansion Agrobacterium tumefaciens-mediated transformation assays using β-glucuronidase (GUS) reporter gene showed that both types of embryos were competent for transformation. Under selection conditions, transformed RcOBType1 explants produced non chimaeric transformed embryos, from which shoots could be adventitiously regenerated. In contrast to RcOBType1, transformed RcOBType2 embryos directly yielded transformed shoots when repeatedly cultured in selective regeneration conditions. Transformation efficiency ranged between three to nine percent and shoots suitable for rooting were obtained within 6-8 months. Transgenic plants were transferred into the greenhouse and molecularly confirmed. The availability of transformation methods in a diploid rose, R. chinensis cv. Old Blush, will be useful for gene functional studies.

The genome of an isolate of tomato yellow leaf curl virus from Sardinia, Italy (TYLCV-S), a geminivirus transmitted by the whitefly Bemisia tabaci, has been cloned and sequenced. The single circular DNA molecule comprises 2770 nucleotides. Genome organisation closely resembles that of the DNA A component of the whitefly-transmitted geminiviruses with a bipartite genome. A 1.8 mer of the TYLCV-S genome in a binary vector of Agrobacterium tumefaciens is infectious upon agroinoculation of tomato plants. Typical tomato yellow leaf curl disease symptoms developed about three weeks after inoculation. The disease was transmitted by the natural vector B.tabaci from agroinfected plants to test plants, reproducing in this way the full biological cycle and proving that the genome of TYLCV-S consists of only one circular single-stranded DNA molecule. Contrary to the other whitefly-transmitted geminiviruses described so far, there is no evidence for the existence nor the necessity of a second component (B DNA) in the TYLCV-S genome.

In Arabidopsis, APETALA1, PISTILLATA, APETALA3 and SEPALLATA interact to form multimeric protein complexes required to specify petal identity. However, the downstream events that lead to petal specific shape and size remain largely unknown. Organ final size can be influenced by cell number or cell expansion or both. To date, no gene that specifically limits petal size by controlling postmitotic cell expansion has been identified. Here we have identified a novel petal-expressed, basic helix-loop-helix encoding gene (BIGPETAL, BPE) that is involved in the control of petal size. BPE is expressed via two mRNAs derived from an alternative splicing event. The BPEub transcript is expressed ubiquitously, whereas the BPEp transcript is preferentially expressed in petals. We demonstrate that BPEp is positively regulated downstream of APETALA3, PISTILLATA, APETALA1 and PISTILLATA3 and is negatively regulated downstream of AGAMOUS. Plants that lack the petal-expressed variant BPEp have larger petals as a result of increased cell size, showing that BPEp interferes with postmitotic cell expansion. BPEp is therefore a part of the network that links the patterning genes to final morphogenesis.

Expression of tobacco mosaic virus (TMV) coat protein (CP) in plants confers resistance to infection by TMV and related tobamoviruses. Certain mutants of the CP (CP T42W ) provide much greater levels of resistance than wild-type (wt) CP. In the present work, infection induced by RNA transcripts of TMV clones that contain wt CP or mutant CP T42W fused to the green fluorescent protein (GFP) (TMV-CP:GFP, TMV-CP T42W :GFP) and clones harboring TMV movement protein (MP):GFP were followed in nontransgenic and transgenic tobacco BY-2 protoplasts and Nicotiana tabaccum Xanthi-nn plants that express wt CP or CP T42W . On nontransgenic and wt CP transgenic plants, TMV-CP:GFP produced expanding, highly fluorescent disk-shaped areas. On plants expressing CP T42W , infection by TMV-CP:GFP or TMV-MP:GFP-CP produced infection sites of smaller size that were characterized by low fluorescence, reflecting reduced levels of virus spread and reduced accumulation of both CP:GFP and MP:GFP. TMV-CP T42W :GFP failed to produce visible infection sites on nontransgenic plants, yet produced normal infection sites on MP-transgenic plants that produce MP. TMV infection of transgenic BY-CP T42W protoplasts resulted in very low levels of MP accumulation, whereas on BY-CP protoplasts (containing wt CP), infection produced higher levels of MP than in nontransgenic BY-2 cells. The results suggest that wt CP has a positive effect on the production of MP, whereas the CP T42W has a negative effect on MP accumulation and/or function. This effect results in very high levels of resistance to TMV infection in plants containing CP T42W . This report shows that the CP of a plant virus regulates production of the MP, and that a mutant CP interferes with MP accumulation and cell-to-cell movement of infection.

International audience; Single-pass sequences were obtained from the 5P P-ends of a total of 1794 rose petal cDNA clones. Cluster analysis identified 242 groups of sequences and 635 singletons indicating that the database represents a total of 877 genes. Putative functions could be assigned to 1151 of the transcripts. Expression analysis indicated that transcripts of several of the genes identified accumulated specifically in petals and stamens. The cDNA library and expressed sequence tag database described here represent a valuable resource for future research aimed at improving economically important rose characteristics such as flower form, longevity and scent. ß

A chimeric tobacco mosaic virus (TMV) was constructed by inserting sequences representing peptide 9-14mer (TDAYNQKLSERRAN) of outer membrane (OM) protein F of Pseudomonas aeruginosa between amino acids Ser154 and Gly155 of the TMV coat protein (CP). This is the first example of TMV being used to construct a chimera containing a bacterial epitope. Mice immunized with TMV-9-14 produced anti-peptide-9-14mer-specific antibodies that reacted in whole-cell ELISA with all seven Fisher-Devlin (FD) immunotype strains of P. aeruginosa, reacted specifically by Western blotting with OM protein F extracted from all seven FD immunotypes, and were opsonic in opsonophagocytic assays. The chimeric TMV-9-14 vaccine afforded immunoprotection against challenge with wild-type P. aeruginosa in a mouse model of chronic pulmonary infection. TMV-9-14 is an excellent candidate for further development as a vaccine for possible use in humans to protect against P. aeruginosa infections.

Hybrids of tobacco mosaic virus (TMV) were constructed with the use of fusion to the coat protein peptides of 10 or 15 amino acids, containing the 5B19 epitope from the spike protein of murine hepatitis virus (MHV) and giving rise to TMV-5B19 and TMV-5B19L, respectively. The TMV hybrids were propagated in tobacco plants, and the virus particles were purified. Immunogold labeling, with the use of the monoclonal MAb5B19 antibody, showed specific decoration of hybrid TMV particles, confirming the expression and display of the MHV epitope on the surface of the TMV. Mice were immunized with purified hybrid viruses after several regimens of immunization. Mice that received TMV-5B19L intranasally developed serum IgG and IgA specific for the 5B19 epitope and for the TMV coat protein. Hybrid TMV-5B19, administered by subcutaneous injections, elicited high titers of serum IgG that was specific for the 5B19 epitope and for coat protein, but IgA that was specific against 5B19 was not observed. Mice that were immunized with hybrid virus by subcutaneous or intranasal routes of administration survived challenge with a lethal dose (10 × LD 50 ) of MHV strain JHM, whereas mice administered wild-type TMV died 10 d post challenge. Furthermore, there was a positive correlation between the dose of administered immunogen and protection against MHV infection. These studies show that TMV can be an effective vaccine delivery vehicle for parenteral and mucosal immunization and for protection from challenge with viral infection.