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.

Seed Transmission of Beet Curly Top Virus and Beet Curly Top Iran Virus in a Local Cultivar of Petunia in Iran.

Beet curly top virus (BCTV) and beet curly top Iran virus (BCTIV) are known as the causal agents of curly top disease in beet and several other dicotyledonous plants in Iran. These viruses are transmitted by Circulifer species, and until now, there has been no confirmed report of their seed transmission. A percentage (38.2-78.0%) of the seedlings developed from the seeds of a petunia local cultivar under insect-free conditions showed stunting, interveinal chlorosis, leaf curling, and vein swelling symptoms, and were infected by BCTV when tested by PCR. Presence of BCTV in seed extracts of petunia local cultivar was confirmed by PCR and IC-PCR, followed by sequencing. Agroinoculation of curly top free petunia plants with a BCTV infectious clone resulted in BCTV infection of plants and their developed seeds. These results show the seed infection and transmission of BCTV in a local cultivar of petunia. Similar experiments performed with BCTIV showed that this virus is also seed transmissible in the same cultivar of petunia, although with a lower rate (8.8-18.5%). Seed transmission of curly top viruses may have significant implications in the epidemiology of these viruses.

Integrated transcriptome catalogue and organ-specific profiling of gene expression in fertile garlic (Allium sativum L.).

BACKGROUND: Garlic is cultivated and consumed worldwide as a popular condiment and green vegetable with medicinal and neutraceutical properties. Garlic cultivars do not produce seeds, and therefore, this plant has not been the subject of either classical breeding or genetic studies. However, recent achievements in fertility restoration in a number of genotypes have led to flowering and seed production, thus enabling genetic studies and breeding in garlic. RESULTS: A transcriptome catalogue of fertile garlic was produced from multiplexed gene libraries, using RNA collected from various plant organs, including inflorescences and flowers. Over 32 million 250-bp paired-end reads were assembled into an extensive transcriptome of 240,000 contigs. An abundant transcriptome assembled separately from 102,000 highly expressed contigs was annotated and analyzed for gene ontology and metabolic pathways. Organ-specific analysis showed significant variation of gene expression between plant organs, with the highest number of specific reads in inflorescences and flowers. Analysis of the enriched biological processes and molecular functions revealed characteristic patterns for stress response, flower development and photosynthetic activity. Orthologues of key flowering genes were differentially expressed, not only in reproductive tissues, but also in leaves and bulbs, suggesting their role in flower-signal transduction and the bulbing process. More than 100 variants and isoforms of enzymes involved in organosulfur metabolism were differentially expressed and had organ-specific patterns. In addition to plant genes, viral RNA of at least four garlic viruses was detected, mostly in the roots and cloves, whereas only 1-4% of the reads were found in the foliage leaves. CONCLUSIONS: The de novo transcriptome of fertile garlic represents a new resource for research and breeding of this important crop, as well as for the development of effective molecular markers for useful traits, including fertility and seed production, resistance to pests and neutraceutical characteristics.

Distribution of potato spindle tuber viroid in reproductive organs of petunia during its developmental stages.

Embryo infection is important for efficient seed transmission of viroids. To identify the major pattern of seed transmission of viroids, we used in situ hybridization to histochemically analyze the distribution of Potato spindle tuber viroid (PSTVd) in each developmental stage of petunia (flowering to mature seed stages). In floral organs, PSTVd was present in the reproductive tissues of infected female × infected male and infected female × healthy male but not of healthy female × infected male before embryogenesis. After pollination, PSTVd was detected in the developed embryo and endosperm in all three crosses. These findings indicate that PSTVd is indirectly delivered to the embryo through ovule or pollen during the development of reproductive tissues before embryogenesis but not directly through maternal tissues as cell-to-cell movement during embryogenesis.

Pollen transmission of asparagus virus 2 (AV-2) may facilitate mixed infection by two AV-2 isolates in asparagus plants.

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.

Pelargonium zonate spot virus is transmitted vertically via seed and pollen in tomato.

In autumn 2007, a new disease with unknown etiology was observed in open-field tomato (Solanum lycopersicum) in the Lachish region of Israel. The symptoms included mild mosaic, leaf malformation, and severe stunting of the plants. The causal agent was readily transmitted mechanically from the sap of infected plants to indicator plants. Viral particles were purified from infected plants and cDNA was synthesized from RNA isolated from the particles. Cloning and sequencing of the cDNA showed 95% identity to RNA 3 of Pelargonium zonate spot virus (PZSV). Using reverse-transcription polymerase chain reaction, PZSV was detected in both seed and pollen grains of infected tomato plants. Attempts to disinfect seed by using hydrochloric acid and trisodium phosphate failed to eliminate this PZSV detection. Seed from infected tomato plants gave rise to infected seedlings with a seed-transmission rate of PZSV of 11 to 29%. Pollen grains collected from flowers of infected plants were used to hand pollinate healthy mother tomato plants. Although none of the pollinated mother plants became infected with PZSV, 29% of the seedlings produced from seed harvested from these plants were found to be infected. This is the first demonstration that PZSV is transmitted vertically via both pollen and seed in tomato plants.

Genomic variability in potato virus M and the development of RT-PCR and RFLP procedures for the detection of this virus in seed potatoes.

Potato virus M (PVM, Carlavirus) is considered to be one of the most common potato viruses distributed worldwide. Sequences of the coat protein (CP) gene of several Canadian PVM isolates were determined. Phylogenetic analysis indicated that all known PVM isolates fell into two distinct groups and the isolates from Canada and the US clustered in the same group. The Canadian PVM isolates could be further divided into two sub-groups. Two molecular procedures, reverse transcription - polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) were developed in this study for the detection and identification of PVM in potato tubers. RT-PCR was highly specific and only amplified PVM RNA from potato samples. PVM RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 dormant tubers. Restriction analysis of PCR amplicons with MscI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by RFLP analysis may be a useful approach for screening potato samples on a large scale for the presence of PVM.

Analysis of cell-to-cell and long-distance movement of apple latent spherical virus in infected plants using green, cyan, and yellow fluorescent proteins.

Apple latent spherical virus (ALSV) expressing green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP) was constructed and used to analyze the local and systemic movement of the virus in infected plants. In Chenopodium quinoa plants inoculated with GFP-ALSV, the infection foci first appeared as small fluorescent spots 2-3 days post inoculation (dpi). The GFP spots expanded as rings from 5 dpi, then fused to each other, and most fluorescence faded out at 10-12 dpi. In upper uninoculated leaves, GFP fluorescence was first observed 6-7 dpi on the basal area of mature leaves and on the entire area of young developing leaves. The appearance of fluorescent flecks on young leaves was first found on and near the class III and IV veins. ALSV labeled with two different fluorescent proteins (CFP-ALSV and YFP-ALSV) were used to investigate the distribution of identical, but differently labeled viruses in mixed infection. Fluorescence from CFP and YFP was in each case observed in separate areas in both inoculated and upper uninoculated leaves, indicating that populations of identical, but differently labeled viruses were replicated and distributed in discrete areas of infected leaves.

The role of variability in evaluating ultra high dilution effects: considerations based on plant model experiments.

A series of experiments, performed on plant models with ultra high dilutions (UHD) of arsenic trioxide at 45th decimal potency has been reviewed with a particular focus on variability. The working variables considered are: the number of germinated seeds out of a fixed set of 33, the stem length of wheat seedlings and the number of necrotic lesions in tobacco leaf disks inoculated with tobacco mosaic virus (TMV). A thorough comparison between treatment and control group has been proposed, considering the two main sources of variability in each series of experiments: variability within and between experiments. In treated groups, a systematic decrease in variability between-experiments, as well as a general decrease, with very few exceptions, in variability within experiments has been observed with respect to control. Variability is traditionally considered as control parameter of model systems. Our hypothesis, based on experimental evidences, proposes a new role of variability as a target of UHD action. This hypothesis may help interpret unanswered questions that keep rising in basic and clinical research in homeopathy.

Decision making in controlling virus yellows of sugar beet in the UK.

Virus yellows is an important disease affecting yield in sugar beet in the UK. Myzus persicae (Sulzer) is the most effective and efficient aphid vector of the three viruses causing the disease: beet yellows virus, beet mild yellowing virus and beet chlorosis virus. Control of virus yellows disease is thus focused on the study and control of this aphid species. UK national surveys of virus yellows began in 1946 and these data helped to formulate disease forecasting schemes to optimise control. Over the years, in addition to improvements in farm hygiene, periodic changes and developments in control of the disease have occurred. To accommodate these important developments, virus yellows forecasting schemes have evolved accordingly. The most recent version has been adapted to take account of the current widespread use of imidacloprid seed treatment. Its application offers potential to optimise the rational use of aphicides such as imidacloprid so as to benefit beet growers and the environment by reducing prophylactic use of seed treatment.

Delivery of subunit vaccines in maize seed.

The use of recombinant gene technologies by the vaccine industry has revolutionized the way antigens are generated, and has provided safer, more effective means of protecting animals and humans against bacterial and viral pathogens. Viral and bacterial antigens for recombinant subunit vaccines have been produced in a variety of organisms. Transgenic plants are now recognized as legitimate sources for these proteins, especially in the developing area of oral vaccines, because antigens have been shown to be correctly processed in plants into forms that elicit immune responses when fed to animals or humans. Antigens expressed in maize (Zea mays) are particularly attractive since they can be deposited in the natural storage vessel, the corn seed, and can be conveniently delivered to any organism that consumes grain. We have previously demonstrated high level expression of the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis in corn, and have demonstrated that these antigens delivered in the seed elicit protective immune responses. Here we provide additional data to support the potency, efficacy, and stability of recombinant subunit vaccines delivered in maize seed.