No Evidence for Seed Transmission of Tomato Yellow Leaf Curl Sardinia Virus in Tomato.

Seed transmission is an important factor in the epidemiology of plant pathogens. Geminiviruses are serious pests spread in tropical and subtropical regions. They are transmitted by hemipteran insects, but a few cases of transmission through seeds were recently reported. Here, we investigated the tomato seed transmissibility of the begomovirus tomato yellow leaf curl Sardinia virus (TYLCSV), one of the agents inducing the tomato yellow leaf curl disease, heavily affecting tomato crops in the Mediterranean area. None of the 180 seedlings originating from TYLCSV-infected plants showed any phenotypic alteration typical of virus infection. Moreover, whole viral genomic molecules could not be detected in their cotyledons and true leaves, neither by membrane hybridization nor by rolling-circle amplification followed by PCR, indicating that TYLCSV is not a seed-transmissible pathogen for tomato. Examining the localization of TYLCSV DNA in progenitor plants, we detected the virus genome by PCR in all vegetative and reproductive tissues, but viral genomic and replicative forms were found only in leaves, flowers and fruit flesh, not in seeds and embryos. Closer investigations allowed us to discover for the first time that these embryos were superficially contaminated by TYLCSV DNA but whole genomic molecules were not detectable. Therefore, the inability of TYLCSV genomic molecules to colonize tomato embryos during infection justifies the lack of seed transmissibility observed in this host.

A 2-kb Mycovirus Converts a Pathogenic Fungus into a Beneficial Endophyte for Brassica Protection and Yield Enhancement.

Mycoviruses are viruses that infect fungi, and hypovirulence-associated mycoviruses have the potential to control fungal diseases. However, it is unclear how mycovirus-mediated hypovirulent strains live and survive in the field, and no mycovirus has been applied for field crop protection. In this study, we found that a previously identified small DNA mycovirus (SsHADV-1) can convert its host, Sclerotinia sclerotiorum, from a typical necrotrophic pathogen to a beneficial endophytic fungus. SsHADV-1 downregulates the expression of key pathogenicity factor genes in S. sclerotiorum during infection. When growing in rapeseed, the SsHADV-1-infected strain DT-8 significantly regulates the expression of rapeseed genes involved in defense, hormone signaling, and circadian rhythm pathways. As a result, plant growth is promoted and disease resistance is enhanced. Field experiments showed that spraying DT-8 at the early flowering stage can reduce the disease severity of rapeseed stem rot by 67.6% and improve yield by 14.9%. Moreover, we discovered that SsHADV-1 could also infect other S. sclerotiorum strains on DT-8-inoculated plants and that DT-8 could be recovered from dead plants. These findings suggest that the mycoviruses may have the ability to shape the origin of endophytism. Our discoveries suggest that mycoviruses may influence the origin of endophytism and may also offer a novel strategy for disease control in which mycovirus-infected strains are used to improve crop health and release mycoviruses into the field.

Evolutionary dynamics of Tomato spotted wilt virus within and between alternate plant hosts and thrips.

Tomato spotted wilt virus (TSWV) is a generalist pathogen with one of the broadest known host ranges among RNA viruses. To understand how TSWV adapts to different hosts, we experimentally passaged viral populations between two alternate hosts, Emilia sochifolia and Datura stramonium, and an obligate vector in which it also replicates, western flower thrips (Frankliniella occidentalis). Deep sequencing viral populations at multiple time points allowed us to track the evolutionary dynamics of viral populations within and between hosts. High levels of viral genetic diversity were maintained in both plants and thrips between transmission events. Rapid fluctuations in the frequency of amino acid variants indicated strong host-specific selection pressures on proteins involved in viral movement (NSm) and replication (RdRp). While several genetic variants showed opposing fitness effects in different hosts, fitness effects were generally positively correlated between hosts indicating that positive rather than antagonistic pleiotropy is pervasive. These results suggest that high levels of genetic diversity together with the positive pleiotropic effects of mutations have allowed TSWV to rapidly adapt to new hosts and expand its host range.

Identification and full genomic sequence of nerine yellow stripe virus.

This study reports the first complete genome sequence of nerine yellow stripe virus (NeYSV, GenBank MT396083). The genome consists of 10,165 nucleotides, excluding the 3'-terminal poly(A) tail. A single open reading frame encodes a large polyprotein of 3294 amino acids with typical potyvirus features. The nuclear inclusion b and coat protein region shares 95% identity with a previously reported partial NeYSV sequence (NC_043153.1). Phylogenetic analysis of the polyprotein amino acid sequence showed that NeYSV clustered with hippeastrum mosaic virus (HiMV YP_006382256.1).

Association between flower stalk elongation, an Arabidopsis developmental trait, and the subcellular location and movement dynamics of the nonstructural protein P3 of Turnip mosaic virus.

Virus infections affect plant developmental traits but this aspect of the interaction has not been extensively studied so far. Two strains of Turnip mosaic virus differentially affect Arabidopsis development, especially flower stalk elongation, which allowed phenotypical, cellular, and molecular characterization of the viral determinant, the P3 protein. Transiently expressed wild-type green fluorescent protein-tagged P3 proteins of both strains and selected mutants of them revealed important differences in their behaviour as endoplasmic reticulum (ER)-associated peripheral proteins flowing along the reticulum, forming punctate accumulations. Three-dimensional (3D) model structures of all expressed P3 proteins were computationally constructed through I-TASSER protein structure predictions, which were used to compute protein surfaces and map electrostatic potentials to characterize the effect of amino acid changes on features related to protein interactions and to phenotypical and subcellular results. The amino acid at position 279 was the main determinant affecting stalk development. It also determined the speed of ER-flow of the expressed proteins and their final location. A marked change in the protein surface electrostatic potential correlated with changes in subcellular location. One single amino acid in the P3 viral protein determines all the analysed differential characteristics between strains differentially affecting flower stalk development. A model proposing a role of the protein in the intracellular movement of the viral replication complex, in association with the viral 6K2 protein, is proposed. The type of association between both viral proteins could differ between the strains.

Virus-Induced Gene Silencing in Rose Flowers.

Virus-induced gene silencing (VIGS) is a favorable method to study gene function by posttranscriptional gene silencing in plants. Here we describe a methodology of graft-accelerated VIGS in rose aimed at obtaining posttranscriptional gene silencing in the flower. The resulting phenotype can be observed within 5-6 weeks post infiltration. By using this method, we successfully silenced the expression of several genes involved in processes such as scent production, petal coloration, or flower architecture. We showed that graft-accelerated VIGS was faster, more efficient, and more convenient than conventional methods previously developed in rose such as agroinfiltration of young plantlets and in vitro cultured tissues or seeds.

Complete genome sequence of begonia flower breaking virus, a novel member of the genus Potyvirus.

We determined the complete genomic sequence of begonia flower breaking virus (BFBV), a novel putative member of the genus Potyvirus isolated from Begonia bowerae cv. 'Tiger' plants grown in Kunming. The genomic RNA comprises 9540 nucleotides (nt), excluding the 3'-terminal poly(A) tail, and contains a typical open reading frame (ORF) of potyviruses. The ORF consists of 9219 nucleotides and encodes a 3073-amino-acid polyprotein that is predicted to be proteolytically cleaved into 10 mature peptides. Sequence comparison indicated that BFBV shares 43.9-55.12% amino acid sequence identity with known potyviruses and that BFBV shares the highest amino acid sequence identity (55.12%) with beet mosaic virus. The results from the complete genomic sequence analysis further suggest that BFBV is a member of a novel species in the genus Potyvirus.

Disturbance of floral colour pattern by activation of an endogenous pararetrovirus, petunia vein clearing virus, in aged petunia plants.

White areas of star-type bicolour petals of petunia (Petunia hybrida) are caused by post-transcriptional gene silencing (PTGS) of the key enzyme of anthocyanin biosynthesis. We observed blotched flowers and a vein-clearing symptom in aged petunia plants. To determine the cause of blotched flowers, we focused on an endogenous pararetrovirus, petunia vein clearing virus (PVCV), because this virus may have a suppressor of PTGS (VSR). Transcripts and episomal DNAs derived from proviral PVCVs accumulated in aged plants, indicating that PVCV was activated as the host plant aged. Furthermore, DNA methylation of CG and CHG sites in the promoter region of proviral PVCV decreased in aged plants, suggesting that poor maintenance of DNA methylation activates PVCV. In parallel, de novo DNA methylation of CHH sites in its promoter region was also detected. Therefore, both activation and inactivation of PVCV occurred in aged plants. The accumulation of PVCV transcripts and episomal DNAs in blotched regions and the detection of VSR activity support a mechanism in which suppression of PTGS by PVCV causes blotched flowers.

A thrips vector of tomato spotted wilt virus responds to tomato acylsugar chemical diversity with reduced oviposition and virus inoculation.

There is increasing evidence that acylsugars deter insect pests and plant virus vectors, including the western flower thrips (WFT), Frankliniella occidentalis (Pergande), vector of tomato spotted wilt virus (TSWV). Acylsugars are sugar-polyesters composed of saturated, un-saturated, and variously branched short and long chain fatty acids (FAs) esterified to a glucose (acylglucose) or sucrose (acylsucrose) moiety. We sought to understand how acylsucrose amount and composition of associated FA profiles interacted to mediate resistance to WFT oviposition and TSWV inoculation on tomato leaves. Towards this goal, we examined WFT oviposition and TSWV inoculation behavior on tomato lines bred to exude varying amounts of acylsucrose in association with diverse FA profiles. Our data show that as acylsucrose amounts increased, WFT egg-laying (oviposition) decreased and TSWV inoculation was suppressed. Western flower thrips also responded to FA profiles that included iC4, iC11, nC12 and nC10 FA. These findings support improving acylsugar-mediated resistance against WFT by breeding tomatoes exuding greater amounts of acylsucrose associated with specific FA profiles. We show that increasing acylsucrose amount output by type IV trichomes and selecting for particular FA profiles through advanced breeding profoundly affects WFT behavior in ways that benefit management of WFT as direct pests and as TSWV vectors.

Flowers as viral hot spots: Honey bees (Apis mellifera) unevenly deposit viruses across plant species.

RNA viruses, once considered specific to honey bees, are suspected of spilling over from managed bees into wild pollinators; however, transmission routes are largely unknown. A widely accepted yet untested hypothesis states that flowers serve as bridges in the transmission of viruses between bees. Here, using a series of controlled experiments with captive bee colonies, we examined the role of flowers in bee virus transmission. We first examined if honey bees deposit viruses on flowers and whether bumble bees become infected after visiting contaminated flowers. We then examined whether plant species differ in their propensity to harbor viruses and if bee visitation rates increase the likelihood of virus deposition on flowers. Our experiment demonstrated, for the first time, that honey bees deposit viruses on flowers. However, the two viruses we examined, black queen cell virus (BQCV) and deformed wing virus (DWV), were not equally distributed across plant species, suggesting that differences in floral traits, virus ecology and/or foraging behavior may mediate the likelihood of deposition. Bumble bees did not become infected after visiting flowers previously visited by honey bees suggesting that transmission via flowers may be a rare occurrence and contingent on multiplicative factors and probabilities such as infectivity of virus strain across bee species, immunocompetence, virus virulence, virus load, and the probability a bumble bee will contact a virus particle on a flower. Our study is among the first to experimentally examine the role of flowers in bee virus transmission and uncovers promising avenues for future research.

A virome from ornamental flowers in an Australian rural town.

Samples of leaves exhibiting symptoms resembling those caused by virus infection were collected from ornamental street flowers in a rural town in Western Australia. Thirty-seven leaf samples were collected from plants of iris, tulip, lily, daffodil, stock and grape hyacinth. Shotgun sequencing of cDNA derived from leaf samples was done, and analysis showed that about 6% of the sequences obtained were of viral origin. Assembly of virus-like sequences revealed complete or partial genome sequences of 13 virus isolates representing 11 virus species. Eight of the isolates were of potyviruses, one was of a macluravirus, three were of potexviruses, and one was of a bunya-like virus. The complete genome of an isolate originally classified as ornithogalum mosaic virus was genetically divergent and differed in polyprotein cleavage motifs, and we propose that this isolate represents a distinct species. The implications of importing to Australia live plant propagules infected with viruses are discussed.

Insights into the maternal pathway for Cucumber green mottle mosaic virus infection of cucurbit seeds.

Cucumber green mottle mosaic virus (CGMMV), genus Tobamovirus, is a major pathogen of cucurbits that primarily affects cucumber, melon, and watermelon crops. The aim of this study was to reveal the contribution of CGMMV-infected female flowers to disease spread. Using a fluorescent in situ hybridization (FISH) technique, we show that ovaries and ovules of CGMMV-infected cucumber and melon plants showed a CGMMV-specific fluorescence signal prior to and following anthesis. The fluorescence signal was prominent but sporadic. Ripe fruits of infected melon plants showed strong signals in the funiculus, the seed stalk, which connects the developing seed to the interior ovary wall. Importantly, in seeds, a strong fluorescence signal was observed in the perisperm-endosperm (PE) envelope, which underlies the seed coat and surrounds the embryo. Interestingly, the fluorescence signal was not uniformly distributed in the PE envelope but was localized to a specific envelope layer. These results have important epidemiological implications for CGMMV management and commercial seed production, particularly regarding the improvement of seed disinfection methods that will contribute to limit the global distribution of the virus.

Development of the VIGS System in the Dioecious Plant Silene latifolia.

(1) Background: Silene latifolia is a dioecious plant, whose sex is determined by XY-type sex chromosomes. Microbotryum lychnidis-dioicae is a smut fungus that infects S. latifolia plants and causes masculinization in female flowers, as if Microbotryum were acting as a sex-determining gene. Recent large-scale sequencing efforts have promised to provide candidate genes that are involved in the sex determination machinery in plants. These candidate genes are to be analyzed for functional characterization. A virus vector can be a tool for functional gene analyses; (2) Methods: To develop a viral vector system in S. latifolia plants, we selected Apple latent spherical virus (ALSV) as an appropriate virus vector that has a wide host range; (3) Results: Following the optimization of the ALSV inoculation method, S. latifolia plants were infected with ALSV at high rates in the upper leaves. In situ hybridization analysis revealed that ALSV can migrate into the flower meristems in S. latifolia plants. Successful VIGS (virus-induced gene silencing) in S. latifolia plants was demonstrated with knockdown of the phytoene desaturase gene. Finally, the developed method was applied to floral organ genes to evaluate its usability in flowers; (4) Conclusion: The developed system enables functional gene analyses in S. latifolia plants, which can unveil gene functions and networks of S. latifolia plants, such as the mechanisms of sex determination and fungal-induced masculinization.

Vertical and Horizontal Transmission of Pospiviroids.

Viroids are highly structured, single-stranded, non-protein-coding circular RNA pathogens. Some viroids are vertically transmitted through both viroid-infected ovule and pollen. For example, potato spindle tuber viroid, a species that belongs to Pospiviroidae family, is delivered to the embryo through the ovule or pollen during the development of reproductive tissues before embryogenesis. In addition, some of Pospiviroidae are also horizontally transmitted by pollen. Tomato planta macho viroid in pollen infects to the ovary from pollen tube during pollen tube elongation and eventually causes systemic infection, resulting in the establishment of horizontal transmission. Furthermore, fertilization is not required to accomplish the horizontal transmission. In this review, we will overview the recent research progress in vertical and horizontal transmission of viroids, mainly by focusing on histopathological studies, and also discuss the impact of seed transmission on viroid dissemination and seed health.

Biological and Genomic Characterization of a Novel Tobamovirus Infecting Hoya spp.

Foliar symptoms suggestive of virus infection were observed on the ornamental plant hoya (Hoya spp.; commonly known as waxflower) in Florida. An agent that reacted with commercially available tobamovirus detection reagents was mechanically transmitted to Chenopodium quinoa and Nicotiana benthamiana. Rod-shaped particles ∼300 nm in length and typical of tobamoviruses were observed in partially purified virion preparations by electron microscopy. An experimental host range was determined by mechanical inoculation with virions, and systemic infections were observed in plants in the Asclepiadaceae, Apocynaceae, and Solanaceae families. Some species in the Solanaceae and Chenopodiaceae families allowed virus replication only in inoculated leaves, and were thus only local hosts for the virus. Tested plants in the Amaranthaceae, Apiaceae, Brassicaceae, Cucurbitaceae, Fabaceae, and Malvaceae did not support either local or systemic virus infection. The complete genome for the virus was sequenced and shown to have a typical tobamovirus organization. Comparisons of genome nucleotide sequence and individual gene deduced amino acid sequences indicate that it is a novel tobamovirus sharing the highest level of sequence identity with Streptocarpus flower break virus and members of the Brassicaceae-infecting subgroup of tobamoviruses. The virus, for which the name Hoya chlorotic spot virus (HoCSV) is proposed, was detected in multiple hoya plants from different locations in Florida.

Insights into carotenoid accumulation using VIGS to block different steps of carotenoid biosynthesis in petals of California poppy.

KEY MESSAGE: Viral-induced gene silencing of selected biosynthetic genes decreased overall carotenoid accumulation in California poppy. Regulation of carotenogenesis was linked with pigment sequestration, not changes in biosynthetic gene expression. Genes of carotenogenesis are well described, but understanding how they affect carotenoid accumulation has proven difficult because of plant lethality when the pigments are lacking. Here, we used a Tobacco Rattle Virus-based virus-induced-gene-silencing (VIGS) approach in California poppy (Eschscholzia californica) to investigate how silencing of the carotenoid biosynthetic pathway genes affects carotenoid metabolite accumulation and RNA transcript abundance of the carotenoid biosynthetic pathway genes. VIGS of upstream (PDS and ZDS) and downstream (ßOH and ZEP) genes reduced transcript abundance of the targeted genes in the poppy petals while having no effect on abundance of the other carotenogenesis genes. Silencing of PDS, ZDS, ßOH and ZEP genes reduced total pigment concentration by 75-90% and altered petal colour. HPLC and LC-MS measurements suggested that petal colour changes were caused by substantially altered pigment profiles and quantity. Carotenoid metabolites were different to those normally detected in wild-type petals accumulated but overall carotenoid concentration was less, suggesting the chemical form of carotenoid was important for whether it could be stored at high amounts. In poppy petals, eschscholtzxanthin and retro-carotene-triol were the predominant carotenoids, present mainly as esters. Specific esterification enzymes for specific carotenoids and/or fatty acids appear key for enabling petal carotenoids to accumulate to high amounts. Our findings argue against a direct role for carotenoid metabolites regulating carotenogenesis genes in the petals of California poppy as transcript abundance of carotenogenesis genes studied was unchanged, while the petal carotenoid metabolite profile changed substantially.

An efficient and improved method for virus-induced gene silencing in sorghum.

BACKGROUND: Although the draft genome of sorghum is available, the understanding of gene function is limited due to the lack of extensive mutant resources. Virus-induced gene silencing (VIGS) is an alternative to mutant resources to study gene function. This study reports an improved and efficient method for Brome mosaic virus (BMV)-based VIGS in sorghum. METHODS: Sorghum plants were rub-inoculated with sap prepared by grinding 2 g of infected Nicotiana benthamiana leaf in 1 ml 10 mM potassium phosphate buffer (pH 6.8) and 100 mg of carborundum abrasive. The sap was rubbed on two to three top leaves of sorghum. Inoculated plants were covered with a dome to maintain high humidity and kept in the dark for two days at 18 °C. Inoculated plants were then transferred to 18 °C growth chamber with 12 h/12 h light/dark cycle. RESULTS: This study shows that BMV infection rate can be significantly increased in sorghum by incubating plants at 18 °C. A substantial variation in BMV infection rate in sorghum genotypes/varieties was observed and BTx623 was the most susceptible. Ubiquitin (Ubiq) silencing is a better visual marker for VIGS in sorghum compared to other markers such as Magnesium Chelatase subunit H (ChlH) and Phytoene desaturase (PDS). The use of antisense strand of a gene in BMV was found to significantly increase the efficiency and extent of VIGS in sorghum. In situ hybridization experiments showed that the non-uniform silencing in sorghum is due to the uneven spread of the virus. This study further demonstrates that genes could also be silenced in the inflorescence of sorghum. CONCLUSION: In general, sorghum plants are difficult to infect with BMV and therefore recalcitrant to VIGS studies. However, by using BMV as a vector, a BMV susceptible sorghum variety, 18 °C for incubating plants, and antisense strand of the target gene fragment, efficient VIGS can still be achieved in sorghum.

Investigation of Petal Senescence by TRV-Mediated Virus-Induced Gene Silencing in Rose.

The classic reverse genetic screening, such as EMS-induced or T-DNA-mediated mutation, is a powerful tool to identify senescence-related genes in many model plants. For most non-model plants, however, this strategy is hard to achieve. Even for model plants, construction of a mutant library is usually labor and time-consuming. Virus-induced gene silencing (VIGS) provides an alternative to characterize gene function in a wide spectrum of plants through transient gene expression. To date, more than a dozen of VIGS vector systems have been developed from different RNA and DNA viruses, while Tobacco rattle virus (TRV) system might be one of the most used due to its wide host range and ease of use. Here, we describe a modified TRV vector, TRV-GFP, in which a green fluorescent protein (GFP) is fused to 3'-end of the coat protein (CP) gene in the TRV2 vector. Since the GFP-tagged CP protein could be traced under UV light in planta, identification of TRV-GFP-infected plants is easy. Application of this system in identifying genes regulating petal senescence in rose is described.

Identification, transmission and genomic characterization of a new member of the family Caulimoviridae causing a flower distortion disease of Rudbeckia hirta.

A disease of Rudbeckia hirta (Black-eyed Susan), characterized by severe flower deformation, was observed in Minnesota during 2010-2016. A previously undescribed virus species, named Rudbeckia flower distortion virus (RuFDV, family Caulimoviridae, genus unassigned), was determined to be the causal agent of the disease. Symptoms induced by RuFDV infection resemble those characteristic of phytoplasma-induced diseases, but no phytoplasmas were detected in RuFDV-infected R. hirta. The virus, and the disease were transmitted readily by mechanical inoculation and by the aphid Myzus persicae, but only to R. hirta. Virions of RuFDV are icosahedral, 42-45nm in diameter, and contain a circular 8222bp dsDNA genome containing seven open reading frames (ORFs). The ORFs 2-6 have 28-52% amino acid sequence identity to the movement protein (MP), coat protein (CP), aspartic protease (AP), reverse transcriptase (RT) and RNase H, and translational transactivator (TA) domains of known caulimoviruses. The two remaining ORFs (1 and 7) have no significant amino acid sequence similarity to known viruses. Although the RuFDV ORF 6 is significantly truncated relative to those of other known caulimoviruses, neither this nor the concomitant absence of characteristic virus-encoded cytoplasmic inclusion bodies appears to adversely affect aphid transmission of this virus. Phylogenetic analysis based on the sequence of the RT region revealed no close relationship to known members of the family Caulimoviridae. Based on sequence similarity, genome organization and phylogenetic relatedness, RuFDV appears to be distinct from any currently recognized taxonomic grouping in the family Caulimoviridae.

A Virus-Induced Assay for Functional Dissection and Analysis of Monocot and Dicot Flowering Time Genes.

Virus-induced flowering (VIF) uses virus vectors to express Flowering Locus T (FT) to induce flowering in plants. This approach has recently attracted wide interest for its practical applications in accelerating breeding in crops and woody fruit trees. However, the insight into VIF and its potential as a powerful tool for dissecting florigenic proteins remained to be elucidated. Here, we describe the mechanism and further applications of Potato virus X (PVX)-based VIF in the short-day Nicotiana tabacum cultivar Maryland Mammoth. Ectopic delivery of Arabidopsis (Arabidopsis thaliana) AtFT by PVX/AtFT did not induce the expression of the endogenous FT ortholog NtFT4; however, it was sufficient to trigger flowering in Maryland Mammoth plants grown under noninductive long-day conditions. Infected tobacco plants developed no systemic symptoms, and the PVX-based VIF did not cause transgenerational flowering. We showed that the PVX-based VIF is a much more rapid method to examine the impacts of single amino acid mutations on AtFT for floral induction than making individual transgenic Arabidopsis lines for each mutation. We also used the PVX-based VIF to demonstrate that adding a His- or FLAG-tag to the N or C terminus of AtFT could affect its florigenic activity and that this system can be applied to assay the function of FT genes from heterologous species, including tomato (Solanum lycopersicum) SFT and rice (Oryza sativa) Hd3a Thus, the PVX-based VIF represents a simple and efficient system to identify individual amino acids that are essential for FT-mediated floral induction and to test the ability of mono- and dicotyledonous FT genes and FT fusion proteins to induce flowering.