Three Strains of Tobacco etch virus Distinctly Alter the Transcriptome of Apical Stem Tissue in Capsicum annuum during Infection.

Tobacco etch virus (TEV; genus Potyvirus) is flexuous rod shaped with a single molecule of single-stranded RNA and causes serious yield losses in species in the Solanaceae. Three TEV strains (HAT, Mex21, and N) are genetically distinct and cause different disease symptoms in plants. Here, a transcriptomic RNA sequencing approach was taken for each TEV strain to evaluate gene expression of the apical stem segment of pepper plants during two stages of disease development. Distinct profiles of Differentially Expressed Genes (DEGs) were identified for each TEV strain. DEG numbers increased with degree of symptom severity: 24 from HAT, 1190 from Mex21, and 4010 from N. At 7 days post-inoculation (dpi), when systemic symptoms were similar, there were few DEGs for HAT- and Mex21-infected plants, whereas N-infected plants had 2516 DEGs. DEG patterns from 7 to 14 dpi corresponded to severity of disease symptoms: milder disease with smaller DEG changes for HAT and Mex21 and severe disease with larger DEG changes for N. Strikingly, in each of these comparisons, there are very few overlapping DEGs among the TEV strains, including no overlapping DEGs between all three strains at 7 or 14 dpi.

Identifying optimal reference genes for the normalization of microRNA expression in cucumber under viral stress.

Cucumber green mottle mosaic virus (CGMMV) is an economically important pathogen and causes significant reduction of both yield and quality of cucumber (Cucumis sativus). Currently, there were no satisfied strategies for controlling the disease. A better understanding of microRNA (miRNA) expression related to the regulation of plant-virus interactions and virus resistance would be of great assistance when developing control strategies for CGMMV. However, accurate expression analysis is highly dependent on robust and reliable reference gene used as an internal control for normalization of miRNA expression. Most commonly used reference genes involved in CGMMV-infected cucumber are not universally expressed depending on tissue types and stages of plant development. It is therefore crucial to identify suitable reference genes in investigating the role of miRNA expression. In this study, seven reference genes, including Actin, Tubulin, EF-1α, 18S rRNA, Ubiquitin, GAPDH and Cyclophilin, were evaluated for the most accurate results in analyses using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene expression was assayed on cucumber leaves, stems and roots that were collected at different days post inoculation with CGMMV. The expression data were analyzed using algorithms including delta-Ct, geNorm, NormFinder, and BestKeeper as well as the comparative tool RefFinder. The reference genes were subsequently validated using miR159. The results showed that EF-1α and GAPDH were the most reliable reference genes for normalizing miRNA expression in leaf, root and stem samples, while Ubiquitin and EF-1α were the most suitable combination overall.

Occurrence and genetic diversity analysis of apple stem pitting virus isolated from apples in China.

Two primer pairs were used to detect apple stem pitting virus (ASPV) using a reverse transcription (RT)-PCR test. 82 out of the 141 randomly collected samples, from ten orchards in five provinces and regions of China, tested positive. In the positive samples forty-five (55%) were infected by ASPV and two other viruses. The full coat protein (CP) and the triple gene block (TGB) gene 1, 2 and 3 of partial ASPV isolates were subsequently cloned. The nucleotide and amino acid identities of 39 CP sequence variants from 31 Chinese apple samples were compared with that of previously reported ASPV isolates and were 67.4-96.0% and 68.4-97.7%, respectively. All ASPV sequence variants from Chinese apples separated into two clades with CP- and TGB-based phylogenetic trees, whilst the grouping of TGB2 and TGB3 trees was the same. Three recombinants (FS06-2, X5-2, and XLF-C-2) for CP and six (TH2-5, X8-2, FS05-2, X6-2 and XLF-A-1) recombinants for TGB were identified from the Chinese apple isolates. Two recombinants were found in the TGB sequence of isolate XLF-A-1. The results presented here may assist in the development of a more comprehensive screening tool for apple viruses.

Distribution of Tomato spotted wilt virus in dahlia plants.

Tomato spotted wilt virus (TSWV) causes significant losses in the production of the ornamental plant Dahlia variabilis in Japan. The purpose of this study was to examine the distribution of TSWV in dahlia plants and identify plant parts that can be used in the selection of TSWV-free plants. The distribution of TSWV was investigated using reverse transcriptional polymerase chain reaction (RT-PCR) and tissue blot immunoassay. The detection rate of TSWV in latent infected compound leaves was the highest in the petiole, and it decreased from the veins and rachis to the lamina. The tissue blot immunoassays of the leaflets showed an uneven distribution of TSWV, especially along the edge of the leaf blade. In stems, the detection rate of TSWV was high partway up the stem compared to that in the upper and the lower parts of the stem during the vegetative growth stage. A highly uneven distribution was observed in the bulb. Our results indicated that middle parts of the stem as well as the petioles, rachis, and veins of compound leaves are suitable for detection of TSWV in dahlias. This study is the first to report uneven distribution of TSWV in dahlia plants. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the distribution of Tomato spotted wilt virus (TSWV) in various parts of dahlia plants was investigated for the first time. The distribution of TSWV was uneven in compound leaves, leaflets, stems, and bulbs. The middle parts of the stem or the petiole and leaf veins should be sampled to detect TSWV when selecting healthy plants.

A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets.

Rose rosette disease caused by Rose rosette virus (RRV; genus Emaravirus) is the most economically relevant disease of Knock Out® series roses in the U.S. As there are no effective chemical control options for the disease, the most critical disease management strategies include the use of virus free clean plants for propagation and early detection and destruction of infected plants. The current diagnostic techniques for RRV including end-point reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RT-qPCR) are highly sensitive, but limited to diagnostic labs with the equipment and expertise; and is time consuming. To address this limitation, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay based on multiple gene targets for specific detection of RRV was developed. The assay is highly specific and did not cross react with other viruses belonging to the inclusive and exclusive genus. Dilution assays using the in vitro transcripts showed that the primer sets designed (RPA-267, RPA-131, and RPA-321) are highly sensitive, consistently detecting RRV with a detection limit of 1fg/µL. Testing of the infected plants using the primer sets indicated that the virus could be detected from leaves, stems and petals of roses. The primer pair RPA-267 produced 100% positive detection of the virus from infected leaf tissues, while primer set RPA-131 produced 100% detection from stems and petals. The primer set RPA-321 produced 83%, 87.5% and 75% positive detection from leaves, petals and stem tissues, respectively. In addition, the assay has been efficiently used in the detection of RRV infecting Knock Out® roses, collected from different states in the U.S. The assay can be completed in 20min as compared to the end-point RT-PCR assay (3-4h) and RT-qPCR (1.5h). The RT-RPA assay is reliable, rapid, highly sensitive, and can be easily used in diagnostic laboratories for detection of RRV with no need for any special equipment.

Characterization of a soybean mosaic virus variant causing different diseases in Glycine max and Nicotiana benthamiana.

We discovered a soybean mosaic virus (SMV) variant (4278-1) that caused systemic infections in Nicotiana benthamiana plants, resulting in stem stunting and leaf shriveling. The virus had a particle morphology and incubation period similar to those of other SMV isolates but differed from them in the leaf symptoms it caused when infecting soybean and N. benthamiana. The genome of this variant consisted of a 9994-nt single-stranded RNA, which was different from most of the other known SMV isolates (approximately 9600 nt). Interestingly, we found evidence that two recombination events (nt 1-476 and nt 1145-1349) had occurred between 4278-1 and a watermelon mosaic virus analogue (WMV analogue), in the 5' untranslated region and the P1 cistron.

Cassava brown streak virus has a rapidly evolving genome: implications for virus speciation, variability, diagnosis and host resistance.

Cassava is a major staple food for about 800 million people in the tropics and sub-tropical regions of the world. Production of cassava is significantly hampered by cassava brown streak disease (CBSD), caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). The disease is suppressing cassava yields in eastern Africa at an alarming rate. Previous studies have documented that CBSV is more devastating than UCBSV because it more readily infects both susceptible and tolerant cassava cultivars, resulting in greater yield losses. Using whole genome sequences from NGS data, we produced the first coalescent-based species tree estimate for CBSV and UCBSV. This species framework led to the finding that CBSV has a faster rate of evolution when compared with UCBSV. Furthermore, we have discovered that in CBSV, nonsynonymous substitutions are more predominant than synonymous substitution and occur across the entire genome. All comparative analyses between CBSV and UCBSV presented here suggest that CBSV may be outsmarting the cassava immune system, thus making it more devastating and harder to control.

Occurrence of Viruses and Associated Grain Yields of Paired Symptomatic and Nonsymptomatic Tillers in Kansas Winter Wheat Fields.

Vector-borne virus diseases of wheat are recurrent in nature and pose significant threats to crop production worldwide. In the spring of 2011 and 2012, a state-wide sampling survey of multiple commercial field sites and university-managed Kansas Agricultural Experiment Station variety performance trial locations spanning all nine crop-reporting regions of the state was conducted to determine the occurrence of Barley yellow dwarf virus-PAV (BYDV-PAV), Cereal yellow dwarf virus-RPV, Wheat streak mosaic virus (WSMV), High plains virus, Soilborne wheat mosaic virus, and Wheat spindle streak mosaic virus using enzyme-linked immunosorbent assays (ELISA). As a means of directly coupling tiller infection status with tiller grain yield, multiple pairs of symptomatic and nonsymptomatic plants were selected and individual tillers were tagged for virus species and grain yield determination at the variety performance trial locations. BYDV-PAV and WSMV were the two most prevalent species across the state, often co-occurring within location. Of those BYDV-PAV- or WSMV-positive tillers, 22% and 19%, respectively, were nonsymptomatic, a finding that underscores the importance of sampling criteria to more accurately assess virus occurrence in winter wheat fields. Symptomatic tillers that tested positive for BYDV-PAV produced significantly lower grain yields compared with ELISA-negative tillers in both seasons, as did WSMV-positive tillers in 2012. Nonsymptomatic tillers that tested positive for either of the two viruses in 2011 produced significantly lower grain yields than tillers from nonsymptomatic, ELISA-negative plants, an indication that these tillers were physiologically compromised in the absence of virus-associated symptoms. Overall, the virus survey and tagged paired-tiller sampling strategy revealed effects of virus infection on grain yield of individual tillers of plants grown under field conditions and may provide a complementary approach toward future estimates of the impact of virus incidence on crop health in Kansas.

Detection of a New Luteovirus in Imported Nectarine Trees: A Case Study to Propose Adoption of Metagenomics in Post-Entry Quarantine.

In spring 2013, 5-year-old nectarine (Prunus persica) trees, grafted on peach rootstock Nemaguard, were found stunted in a propagation block in California. These trees had been propagated from budwood of three nectarine cultivars imported from France and cleared through the post-entry quarantine procedure. Examination of the canopy failed to reveal any obvious symptoms. However, examination of the trunks, after stripping the bark, revealed extensive pitting on the woody cylinder. To investigate the etiological agent, double-stranded RNA was extracted from bark scrapings from the scion and rootstock portions, and a cDNA library was prepared and sequenced using the Illumina platform. BLAST analysis of the contigs generated by the de novo assembly of sequence reads indicated the presence of a novel luteovirus. Complete sequence of the viral genome was determined by sequencing of three overlapping cDNA clones generated by reverse transcription-polymerase chain reaction (RT-PCR) and by rapid amplification of the 5'- and 3'-termini. The virus genome was comprised of 4,991 nucleotides with a gene organization similar to members of the genus Luteovirus (family Luteoviridae). The presence of the virus, tentatively named Nectarine stem pitting-associated virus, was confirmed in symptomatic trees by RT-PCR. Discovery of a new virus in nectarine trees after post-entry quarantine indicates the importance of including (i) metagenomic analysis by next-generation sequencing approach as an essential tool to assess the plant health status, and (ii) examination of the woody cylinders as part of the indexing process.

Turnip mosaic virus moves systemically through both phloem and xylem as membrane-associated complexes.

Plant viruses move systemically in plants through the phloem. They move as virions or as ribonucleic protein complexes, although it is not clear what these complexes are made of. The approximately 10-kb RNA genome of Turnip mosaic virus (TuMV) encodes a membrane protein, known as 6K2, that induces endomembrane rearrangements for the formation of viral replication factories. These factories take the form of vesicles that contain viral RNA (vRNA) and viral replication proteins. In this study, we report the presence of 6K2-tagged vesicles containing vRNA and the vRNA-dependent RNA polymerase in phloem sieve elements and in xylem vessels. Transmission electron microscopy observations showed the presence in the xylem vessels of vRNA-containing vesicles that were associated with viral particles. Stem-girdling experiments, which leave xylem vessels intact but destroy the surrounding tissues, confirmed that TuMV could establish a systemic infection of the plant by going through xylem vessels. Phloem sieve elements and xylem vessels from Potato virus X-infected plants also contained lipid-associated nonencapsidated vRNA, indicating that the presence of membrane-associated ribonucleic protein complexes in the phloem and xylem may not be limited to TuMV. Collectively, these studies indicate that viral replication factories could end up in the phloem and the xylem.

Differential disease symptoms and full-length genome sequence analysis for three strains of Tobacco etch virus.

Tobacco etch virus (TEV) strains HAT, Mex21, and N have been the focus of numerous studies to dissect a host resistance mechanism in Capsicum spp. Little is known, however, about their general pathogenicity and genomic sequence data are not available on the TEV strains Mex21 and N. Four Nicotiana spp. were evaluated after inoculation with each TEV strain. Nicotiana tabacum 'Kentucky 14' and N. clevelandii plants expressed varied systemic symptoms dependent on the TEV strain; however, disease severity increased from HAT (mild mosaic symptoms) to Mex21 (more severe mosaic symptoms with stunting) to N (severe chlorosis and stunting). Nicotiana tabacum 'Samsun' plants developed relatively milder symptoms and N. glutinosa plants remained symptomless, although they were systemically infected. The genome of each TEV strain was sequenced and shown to consist of 9,495 nucleotides and a polyprotein of 3,054 amino acids. Comparison of their nucleotide sequences relative to the original HAT sequence (GenBank Accession No. M11458) revealed 95, 92, and 92 % identity for HAT-AU (from Auburn University), Mex21, and N, respectively. HAT-AU had 91 % sequence identity with Mex21 and N, while Mex21 and N were more closely related with 98 % nucleotide sequence identity. Similarly, the amino acid sequence identities for the full-length polyprotein ranged from 95 % for HAT-AU when compared with N to a high of 98 % identity between Mex21 and N.

Sweet pepper confirmed as a reservoir host for tomato yellow leaf curl virus by both agro-inoculation and whitefly-mediated inoculation.

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, has a single-stranded DNA genome. TYLCV can induce severe disease symptoms on tomato plants, but other hosts plants such as cucurbits and peppers are asymptomatic. A full-length DNA clone of a Korean TYLCV isolate was constructed by rolling-circle amplification from TYLCV-infected tomatoes in Korea. To assess relative susceptibility of sweet pepper varieties to TYLCV, 19 cultivars were inoculated with cloned TYLCV by agro-inoculation. All TYLCV-infected sweet peppers were asymptomatic, even though Southern hybridization and polymerase chain reaction analysis showed TYLCV genomic DNA accumulation in roots, stems, and newly produced shoots. Southern hybridization indicated that TYLCV replicated and moved systemically from agro-inoculated apical shoot tips to roots or newly produced shoots of sweet peppers. Whitefly-mediated inoculation experiments showed that TYLCV can be transmitted to tomatoes from TYLCV-infected sweet peppers. Taken together, these results indicate that sweet pepper can be a reservoir for TYLCV in nature.

Dynamics of Cryphonectria hypovirus infection in chestnut blight cankers.

Virulent strains of the chestnut blight fungus Cryphonectria parasitica cause lethal bark cankers on chestnut trees. Infection of C. parasitica with Cryphonectria hypovirus 1 in Europe biologically controls this disease, leading to nonlethal and inactive cankers. Unexpectedly, virus-free C. parasitica strains have been isolated from inactive cankers. In this study, we compared the virulence of virus-infected and virus-free C. parasitica strains isolated from either inactive or active cankers on chestnut seedlings and sprouts. In the seedling experiment, we assessed canker growth and seedling mortality. In the sprout experiment, we also assessed canker growth and made fungal reisolations to determine virus infection and immigration of foreign vegetative compatibility (vc) types over a period of 13 years in a coppice forest. Overall, the virulence of virus-free C. parasitica strains isolated from inactive versus active cankers did not differ. Significant differences were only attributed to virus infection. Virus infection and fungal strain composition in cankers changed over time. Foreign vc types immigrated into cankers and virus-free cankers became virus-infected within a few years. Most of the cankers were callused over time and became inactive. However, we observed that the virus did not always persist in these cankers. This study demonstrates that virus spread occurs effectively in European chestnut forests and that this biocontrol system is highly dynamic.

TRV-GFP: a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function.

Virus-induced gene silencing (VIGS) is a useful tool for functional characterization of genes in plants. Unfortunately, the efficiency of infection by Tobacco rattle virus (TRV) is relatively low for some non-Solanaceae plants, which are economically important, such as rose (Rosa sp.). Here, to generate an easy traceable TRV vector, a green fluorescent protein (GFP) gene was tagged to the 3' terminus of the coat protein gene in the original TRV2 vector, and the silencing efficiency of the modified TRV-GFP vector was tested in several plants, including Nicotiana benthamiana, Arabidopsis thaliana, rose, strawberry (Fragaria ananassa), and chrysanthemum (Dendranthema grandiflorum). The results showed that the efficiency of infection by TRV-GFP was equal to that of the original TRV vector in each tested plant. Spread of the modified TRV virus was easy to monitor by using fluorescent microscopy and a hand-held UV lamp. When TRV-GFP was used to silence the endogenous phytoene desaturase (PDS) gene in rose cuttings and seedlings, the typical photobleached phenotype was observed in 75-80% plants which were identified as GFP positive by UV lamp. In addition, the abundance of GFP protein, which represented the concentration of TRV virus, was proved to correlate negatively with the level of the PDS gene, suggesting that GFP could be used as an indicator of the degree of silencing of a target gene. Taken together, this work provides a visualizable and efficient tool to predict positive gene silencing plants, which is valuable for research into gene function in plants, especially for non-Solanaceae plants.

Efficient transmission of cassava brown streak disease viral pathogens by chip bud grafting.

BACKGROUND: Techniques to study plant viral diseases under controlled growth conditions are required to fully understand their biology and investigate host resistance. Cassava brown streak disease (CBSD) presents a major threat to cassava production in East Africa. No infectious clones of the causal viruses, Cassava brown streak virus (CBSV) or Ugandan cassava brown streak virus (UCBSV) are available, and mechanical transmission to cassava is not effective. An improved method for transmission of the viruses, both singly and as co-infections has been developed using bud grafts. FINDINGS: Axillary buds from CBSD symptomatic plants infected with virulent isolates of CBSV and UCBSV were excised and grafted onto 6-8 week old greenhouse-grown, disease-free cassava plants of cultivars Ebwanateraka, TME204 and 60444. Plants were assessed visually for development of CBSD symptoms and by RT-PCR for presence of the viruses in leaf and storage root tissues. Across replicated experiments, 70-100% of plants inoculated with CBSV developed CBSD leaf and stem symptoms 2-6 weeks after bud grafting. Infected plants showed typical, severe necrotic lesions in storage roots at harvest 12-14 weeks after graft inoculation. Sequential grafting of buds from plants infected with UCBSV followed 10-14 days later by buds carrying CBSV, onto the same test plant, resulted in 100% of the rootstocks becoming co-infected with both pathogens. This dual transmission rate was greater than that achieved by simultaneous grafting with UCBSV and CBSV (67%), or when grafting first with CBSV followed by UCBSV (17%). CONCLUSIONS: The bud grafting method described presents an improved tool for screening cassava germplasm for resistance to CBSD causal viruses, and for studying pathogenicity of this important disease. Bud grafting provides new opportunities compared to previously reported top and side grafting systems. Test plants can be inoculated as young, uniform plants of a size easily handled in a small greenhouse or large growth chamber and can be inoculated in a controlled manner with CBSV and UCBSV, either singly or together. Disease symptoms develop rapidly, allowing better studies of interactions between these viral pathogens, their movement within shoot and root systems, and how they induce their destructive disease symptoms.

Recruitment of the host plant heat shock protein 70 by Tomato yellow leaf curl virus coat protein is required for virus infection.

A functional capsid protein (CP) is essential for host plant infection and insect transmission of Tomato yellow leaf curl virus (TYLCV) and other monopartite begomoviruses. We have previously shown that TYLCV CP specifically interacts with the heat shock protein 70 (HSP70) of the virus insect vector, Bemisia tabaci. Here we demonstrate that during the development of tomato plant infection with TYLCV, a significant amount of HSP70 shifts from a soluble form into insoluble aggregates. CP and HSP70 co-localize in these aggregates, first in the cytoplasm, then in the nucleus of cells associated with the vascular system. CP-HSP70 interaction was demonstrated by co-immunopreciptation in cytoplasmic - but not in nuclear extracts from leaf and stem. Inhibition of HSP70 expression by quercetin caused a decrease in the amount of nuclear CP aggregates and a re-localization of a GFP-CP fusion protein from the nucleus to the cytoplasm. HSP70 inactivation resulted in a decrease of TYLCV DNA levels, demonstrating the role of HSP70 in TYLCV multiplication in planta. The current study reveals for the first time the involvement of plant HSP70 in TYLCV CP intracellular movement. As described earlier, nuclear aggregates contained TYLCV DNA-CP complexes and infectious virions. Showing that HSP70 localizes in these large nuclear aggregates infers that these structures operate as nuclear virus factories.

Altered invertase activities of symptomatic tissues on Beet severe curly top virus (BSCTV) infected Arabidopsis thaliana.

Arabidopsis thaliana infected with Beet severe curly top virus (BSCTV) exhibits systemic symptoms such as stunting of plant growth, callus induction on shoot tips, and curling of leaves and shoot tips. The regulation of sucrose metabolism is essential for obtaining the energy required for viral replication and the development of symptoms in BSCTV-infected A. thaliana. We evaluated the changed transcript level and enzyme activity of invertases in the inflorescence stems of BSCTV-infected A. thaliana. These results were consistent with the increased pattern of ribulose-1,5-bisphosphate carboxylase/oxygenase activity and photosynthetic pigment concentration in virus-infected plants to supply more energy for BSCTV multiplication. The altered gene expression of invertases during symptom development was functionally correlated with the differential expression patterns of D-type cyclins, E2F isoforms, and invertase-related genes. Taken together, our results indicate that sucrose sensing by BSCTV infection may regulate the expression of sucrose metabolism and result in the subsequent development of viral symptoms in relation with activation of cell cycle regulation.

Imbalance in expression of hop (Humulus lupulus) chalcone synthase H1 and its regulators during hop stunt viroid pathogenesis.

Viroid-derived small RNAs generated during hop stunt viroid (HSVd) pathogenesis may induce the symptoms found in the hop cultivar "Admiral", including observed shifts in phenylpropanoid metabolites and changes in petiole coloration. Using quantitative RT-PCR, we examined hop lupulin gland-specific genes that have been shown to be involved in phenylpropanoid metabolism, for altered expression in response to infection with two HSVd isolates, HSVd-g and CPFVd. Most notably, the expression of a gene encoding a key enzyme for phenylpropanoid synthesis, naringenin-chalcone synthase H1 (chs_H1), decreased up to 40-fold in infected samples. In addition, a marked decrease in the expression of HlbHLH2 and an increase in the expression of HlMyb3 were observed. These two genes encode transcription factors that form a ternary complex with HlWDR1 for chs_H1 promoter activation. In a transient expression assay, a decrease in the ternary complex potential to activate the chs_H1 promoter was observed upon the decrease of HlbHLH2 expression. In addition, targeting of the chs_H1 transcript by vd-sRNAs may contribute to these expression changes. Our data show that HSVd infection causes a significant imbalance in the expression of phenylpropanoid metabolite-affecting genes via a complex mechanism, possibly involving regulatory disorders and direct targeting by vd-sRNA.

Phloem-mobile signals affecting flowers: applications for crop breeding.

Transport of endogenous macromolecules within and between tissues serves as a signaling pathway to regulate numerous aspects of plant growth. The florigenic FT gene product moves via the phloem from leaves to apical tissues and induces the flowering program in meristems. Similarly, short interfering RNA (siRNA) signals produced in source or sink tissues move cell-to-cell and long distance via the phloem to apical tissues. Recent advances in identifying these mobile signals regulating flowering or the epigenetic status of targeted tissues can be applicable to crop-breeding programs. In this review, we address the identity of florigen, the mechanism of allocation, and how virus-induced flowering and grafting of transgenes producing siRNA signals affecting meiosis can produce transgene-free progenies useful for agriculture.

Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus.

Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.