Subterranean Clover Stunt Virus Revisited: Detection of Two Missing Genome Components

Subterranean clover stunt virus (SCSV) is a type species of the genus Nanovirus in the family Nanoviridae. It was the first single-stranded DNA plant virus with a multipartite genome, of which genomic DNA sequences had been determined. All nanoviruses have eight genome components except SCSV, for which homologs of two genome components present in all other nanovirus genomes, DNA-U2 and DNA-U4, were lacking. We analysed archived and more recent samples from SCSV-infected legume plants to verify its genome composition and found the missing genome components. These results indicated that SCSV also has eight genome components and is a typical member of the genus Nanovirus.

Genetic improvement of subterranean clover (Trifolium subterraneum L.). 2. Breeding for disease and pest resistance

Subterranean clover (Trifolium subterraneum L.) is the most widely sown pasture legume in southern Australia and resistance to important diseases and pests has been a major plant-breeding objective. Kabatiella caulivora, the cause of clover scorch, is the most important foliar fungal pathogen, and several cultivars have been developed with resistance to both known races. Screening of advanced breeding lines has been conducted to prevent release of cultivars with high susceptibility to other important fungal foliar disease pathogens, including rust (Uromyces trifolii-repentis), powdery mildew (Oidium sp.), cercospora (Cercospora zebrina) and common leaf spot (Pseudopeziza trifolii). Several oomycete and fungal species cause root rots of subterranean clover, including Phytophthora clandestina, Pythium irregulare, Aphanomyces trifolii, Fusarium avenaceum and Rhizoctonia solani. Most breeding efforts have been devoted to resistance to P. clandestina, but the existence of different races has confounded selection. The most economically important virus diseases in subterranean clover pastures are Subterranean clover mottle virus and Bean yellow mosaic virus, while Subterranean clover stunt virus, Subterranean clover red leaf virus (local synonym for Soybean dwarf virus), Cucumber mosaic virus, Alfalfa mosaic virus, Clover yellow vein virus, Beet western yellows virus and Bean leaf roll virus also cause losses. Genotypic differences for resistance have been found to several of these fungal, oomycete and viral pathogens, highlighting the potential to develop cultivars with improved resistance. The most important pests of subterranean clover are redlegged earth mite (RLEM) (Halotydeus destructor), blue oat mite (Penthaleus major), blue-green aphid (Acyrthosiphon kondoi) and lucerne flea (Sminthurus viridis). New cultivars have been bred with increased RLEM cotyledon resistance, but limited selection has been conducted for resistance to other pests. Screening for disease and pest resistance has largely ceased, but recent molecular biology advances in subterranean clover provide a new platform for development of future cultivars with multiple resistances to important diseases and pests. However, this can only be realised if skills in pasture plant pathology, entomology, pre-breeding and plant breeding are maintained and adequately resourced. In particular, supporting phenotypic disease and pest resistance studies and understanding their significance is critical to enable molecular technology investments achieve practical outcomes and deliver subterranean clover cultivars with sufficient pathogen and pest resistance to ensure productive pastures across southern Australia.

First Report of a Nanovirus Disease of Pea in Germany

During the growing season of 2009, a disease consisting of leaf rolling, top yellows, and plant stunting affected pea (Pisum sativum) in fields near Aschersleben, Saxony-Anhalt, Germany. Samples from symptomatic plants collected in July 2009 were analyzed at the JKI in Braunschweig for infections by various legume viruses by ELISA, immunoelectron microscopy, and transmission assays by sap and aphids. Of 23 samples, 9 were shown to contain Pea enation mosaic virus and three samples each contained Bean leafroll virus and Soybean dwarf virus. From two further samples that had tested negative for the aforementioned viruses, we succeeded in transferring a disease agent to faba bean (Vicia faba) seedlings by giving 50 to 100 individuals of the pea aphid (Acyrthosiphon pisum) acquisition and inoculation access feedings each of ~48 h. Following vector transmission, the agent caused severe yellowing and stunting in pea and faba bean, sometimes followed by necrosis. Attempts at mechanical transmission of the agent failed, and isolation of double-stranded RNA from infected tissue was not successful. Therefore, we considered the possible presence of a nanovirus (4). When using polyclonal antibodies (PAbs) against Faba bean necrotic yellows virus (FBNYV) for double-antibody sandwich (DAS)-ELISA analysis of the two isolates of the disease agent we observed weak but clearly positive reactions. To confirm these weak DAS-ELISA reactions, we used all available monoclonal antibodies (MAbs) raised against FBNYV (1) and faba bean necrotic stunt virus (FBNSV) (3) individually in triple-antibody sandwich (TAS)-ELISA in combination with the FBNYV PAbs for plate coating. Six of 26 MAbs reacted from weak to strong with the two pea isolates, with MAbs FBNYV-3-1F7 and FBNSV-5-1G8 giving the strongest reactions and none of the MAbs giving a differential reaction with the two pea isolates. Employing rolling circle amplification of total DNA extracted from symptomatic leaves of one of the pea isolates yielded a substantial amount of high molecular weight DNA, whereas little or no amplification occurred when using DNA from noninoculated pea leaves. Restriction of the amplified DNA in a nanovirus iteron-specific manner by AatII endonuclease yielded a predominant and abundant product of ~1 kb (3). Sequence comparisons of eight cloned DNAs of 1,002 nucleotides long unequivocally identified them as complete DNA-R component of a new member of the genus Nanovirus (2,4). Its DNA-R sequence (GenBank No. GU553134) is nearly equidistant from the DNA-R sequences of FBNYV (Y11405), FBNSV (GQ150778), Milk vetch dwarf virus (MDV) (AB027511) and Subterranean clover stunt virus (SCSV) (AJ290434), sharing with them respective sequence identities of 79, 78, 79, and 73%. Moreover, it is more distinct from the DNA-R sequences of FBNYV, FBNSV, and MDV than the three latter are from each other (86 to 91%). This together with the serological data relating to the capsid protein properties of this virus strongly suggest that it is distinct from the hitherto described nanoviruses FBNYV, MDV, FBNSV, and SCSV. Therefore, we propose the name pea necrotic yellow dwarf virus (PNYDV) for this new nanovirus naturally infecting pea in Germany. References: (1) A. Franz et al. Ann. Appl. Biol. 128:255, 1996. (2) I. Grigoras et al. J. Gen. Virol. 89:583, 2008. (3) I. Grigoras et al. J. Virol. 83:10778, 2009. (4) H. J. Vetten et al. Page 343 in: Virus Taxonomy. Elsevier/Academic Press, London, 2005.

First Report of Faba bean necrotic yellows virus Affecting Legume Crops in Azerbaijan

A total of 482 chickpea (Cicer arietinum L.), 182 lentil (Lens culinaris Medik.), 12 vetch (Vicia sativa L.), 5 field pea (Pisum sativum L.), and 3 faba bean (Vicia faba L.) samples were collected from plants with symptoms suggestive of a viral infection (leaf rolling, yellowing, and stunting) from the major legume-production areas of Azerbaijan in the 2007 and 2008 growing seasons. All samples were tested by the tissue-blot immunoassay (3) at the Virology Laboratory of ICARDA, Syria using 11 specific legume virus antisera including a monoclonal antibody (2-5H9) (1) for Faba bean necrotic yellows virus (FBNYV). Laboratory tests showed that FBNYV was detected in 73, 61, 11, 3, and 2 samples of chickpea, lentil, vetch, field pea, and faba bean, respectively. Total DNA was extracted from six FBNYV-positive samples (two chickpea, two lentil, and two vetch) and tested by PCR with the following four primer sets (FBNYV, Milk vetch dwarf virus [MDV], Subterranean clover stunt virus [SCSV], and nanovirus DNA-R primers [F103 and R101]) (2). All six Azeri samples as well as the reference nanovirus isolates (SCSV-Australia, MDV-Japan, and FBNYV-Syria) generated amplicons of the expected size (~770 bp) using the nanovirus DNA-R primers (F103 & R101). In addition, Azeri samples and FBNYV-Syria yielded a PCR amplicon of the expected size (666 bp) with the FBNYV primer pair. The MDV- and SCSV-specific primers did not generate amplicons with these six samples. Sequence analysis of the FBNYV amplicons from two isolates (AzL 282-07 from lentil [GenBank Accession No. GQ351600] and AzV 277-07 from vetch [GenBank Accession No. GQ371215]) showed that they were 99% identical with each other. Comparing the sequence of AzL 282-07 with that of other nanoviruses revealed identities of 97% (FBNYV-Spain; DQ830990), 96% (FBNYV-Iran; AM493900), 92% (FBNYV-Syria; Y11408), 92% (FBNYV-Egypt; AJ132183), 78% (MDV; AB044387) and 69% (SCSV-Australia; U16734). FBNYV has been reported to infect food legumes in many countries in West Asia and North Africa and cause economic losses on faba bean in Egypt, Jordan, and Syria. To our knowledge, this is the first record of FBNYV infecting legume crops in Azerbaijan. References: (1) A. Franz et al. Ann. Appl. Biol. 128:255, 1996. (2) S. G. Kumari et al. Phytopathol. Mediterr. 47:42, 2008. (3) K. M. Makkouk and A. Comeau. Eur. J. Plant Pathol. 100:71, 1994.

Transcripts encoding the nanovirus master replication initiator proteins are terminally redundant

The multicomponent single-stranded DNA plant nanoviruses encode unique master replication initiator (Rep) proteins. We have mapped the 5′ and 3′ termini of the corresponding polyadenylated mRNAs from faba bean necrotic yellows virus (FBNYV) and subterranean clover stunt virus and found that these are terminally redundant by up to about 160 nt. Moreover, the origin of viral DNA replication is transcribed into RNA that is capable of folding into extended secondary structures. Other nanovirus genome components, such as the FBNYV DNA encoding the protein Clink or an FBNYV DNA encoding a non-essential para-Rep protein, are not transcribed in such a unique fashion. Thus, terminally redundant mRNAs and the resulting transcription of the replication origin appear to be restricted to nanovirus master Rep DNAs. We speculate that this may be a way to regulate the expression of the essential master Rep protein.

A suite of novel promoters and terminators for plant biotechnology

The gene regulation signals from subterranean clover stunt virus (SCSV) were investigated for their expression in dicot plants. The SCSV genome has at least eight circular DNA molecules. Each circular DNA component contains a promoter element, a single open reading frame and a terminator. The promoters from seven of the segments were examined for their strength and tissue specificity in transgenic tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and cotton (Gossypium hirsutum L.) using a GUS reporter gene assay system. While the promoters of many of the segments were poorly expressed, promoters derived from segments 4 and 7 were shown to direct high levels of expression in various plant tissues and organs. The segment 1 promoter directs predominantly callus-specific expression and, when used to control a selectable marker gene, facilitated the transformation of all three species (tobacco, potato and cotton). From the results, a suite of plant expression vectors (pPLEX) derived from the SCSV genome were constructed and used here to produce herbicide- and insect-resistant cotton, demonstrating their utility in the expression of foreign genes in dicot crop species and their potential for use in agricultural biotechnology.

A suite of novel promoters and terminators for plant biotechnology. II. The pPLEX series for use in monocots

A suite of plant expression vectors (pPLEX), constructed from the gene regulation signals from subterranean clover stunt virus (SCSV) genome, has previously been used in dicot transformation for a variety of applications in plant biotechnology. To assess their use for the transformation of monocots, a number of modifications were made to the basic vector series and assessed in rice. In their unmodified forms, the SCSV promoters directed low levels of gene expression, however, insertion of an intron between the promoter and the transgene open reading frame (analogous to the rice actin and maize ubiquitin promoter systems) increased transgene expression 50-fold. The expression patterns from the intron-modified SCSV (segments 4 and 7) promoters were very similar to those directed by the actin or ubiquitin promoters. All promoter systems investigated directed expression that appeared to be constitutive within leaf tissue, and localised to the epidermal and vascular tissues of the root. The pPLEX vectors described here are an important counterpart to the dicot pPLEX series and have the potential to be useful in monocot research and biotechnology.