Heiko Ziebell

The role of the Cucumber mosaic virus 2b protein in viral movement and symptom induction.

The Cucumber mosaic virus (CMV) 2b protein is a counter-defense factor and symptom determinant. Conserved domains in the 2b protein sequence were mutated in the 2b gene of strain Fny-CMV. The effects of these mutations were assessed by infection of Nicotiana tabacum, N. benthamiana, and Arabidopsis thaliana (ecotype Col-0) with mutant viruses and by expression of mutant 2b transgenes in A. thaliana. We confirmed that two nuclear localization signals were required for symptom induction and found that the N-terminal domain was essential for symptom induction. The C-terminal domain and two serine residues within a putative phosphorylation domain modulated symptom severity. Further infection studies were conducted using Fny-CMVdelta2b, a mutant that cannot express the 2b protein and that induces no symptoms in N. tabacum, N. benthamiana, or A. thaliana ecotype Col-0. Surprisingly, in plants of A. thaliana ecotype C24, Fny-CMVdelta2b induced severe symptoms similar to those induced by the wild-type virus. However, C24 plants infected with the mutant virus recovered from disease while those infected with the wild-type virus did not. Expression of 2b transgenes from either Fny-CMV or from LS-CMV (a mild strain) in Col-0 plants enhanced systemic movement of Fny-CMVdelta2b and permitted symptom induction by Fny-CMVdelta2b. Taken together, the results indicate that the 2b protein itself is an important symptom determinant in certain hosts. However, they also suggest that the protein may somehow synergize symptom induction by other CMV-encoded factors.

Cucumber mosaic virus and its 2b RNA silencing suppressor modify plant-aphid interactions in tobacco.

The cucumber mosaic virus (CMV) 2b protein not only inhibits anti-viral RNA silencing but also quenches transcriptional responses of plant genes to jasmonic acid, a key signalling molecule in defence against insects. This suggested that it might affect interactions between infected plants and aphids, insects that transmit CMV. We found that infection of tobacco with a 2b gene deletion mutant (CMVΔ2b) induced strong resistance to aphids (Myzus persicae) while CMV infection fostered aphid survival. Using electrical penetration graph methodology we found that higher proportions of aphids showed sustained phloem ingestion on CMV-infected plants than on CMVΔ2b-infected or mock-inoculated plants although this did not increase the rate of growth of individual aphids. This indicates that while CMV infection or certain viral gene products might elicit aphid resistance, the 2b protein normally counteracts this during a wild-type CMV infection. Our findings suggest that the 2b protein could indirectly affect aphid-mediated virus transmission.

Cross-Protection: A Century of Mystery

Cross-protection is a phenomenon in which infection of a plant with a mild virus or viroid strain protects it from disease resulting from a subsequent encounter with a severe strain of the same virus or viroid. In this chapter, we review the history of cross-protection with regard to the development of ideas concerning its likely mechanisms, including RNA silencing and exclusion, and its influence on the early development of genetically engineered virus resistance. We also examine examples of the practical use of cross-protection in averting crop losses due to viruses, as well as the use of satellite RNAs to ameliorate the impact of virus-induced diseases. We also discuss the potential of cross-protection to contribute in future to the maintenance of crop health in the face of emerging virus diseases and related threats to agricultural production.

Effects of dicer-like endoribonucleases 2 and 4 on infection of Arabidopsis thaliana by cucumber mosaic virus and a mutant virus lacking the 2b counter-defence protein gene

In tobacco and Nicotiana benthamiana, limited cross-protection against cucumber mosaic virus strain Fny (Fny-CMV) was provided by prior inoculation with a deletion mutant lacking the 2b silencing-suppressor protein gene (Fny-CMVDelta2b). Cross-protection by Fny-CMVDelta2b did not result from induction of systemic RNA silencing. We investigated whether protection occurs through induction of localized RNA silencing by using Arabidopsis thaliana plants harbouring mutations in genes encoding the dicer-like (DCL) endoribonucleases 2, 3 and 4 involved in antiviral silencing. In wild-type A. thaliana (Col-0) plants, Fny-CMVDelta2b was symptomless and cross-protected against Fny-CMV infection. Cross-protection by Fny-CMVDelta2b against Fny-CMV infection was not abolished in dcl2, dcl3 or dcl4 mutant plants and was strongest in dcl2/4 double mutants, although in these plants and in dcl4 mutants, Fny-CMVDelta2b replicated to high levels and induced strong symptoms. The results suggest that Fny-CMVDelta2b/Fny-CMV cross-protection is not completely dependent on RNA silencing and also involves competition between these viruses.

Genome diversity and evidence of recombination and reassortment in nanoviruses from Europe

The recent identification of a new nanovirus, pea necrotic yellow dwarf virus, from pea in Germany prompted us to survey wild and cultivated legumes for nanovirus infections in several European countries. This led to the identification of two new nanoviruses: black medic leaf roll virus (BMLRV) and pea yellow stunt virus (PYSV), each considered a putative new species. The complete genomes of a PYSV isolate from Austria and three BMLRV isolates from Austria, Azerbaijan and Sweden were sequenced. In addition, the genomes of five isolates of faba bean necrotic yellows virus (FBNYV) from Azerbaijan and Spain and those of four faba bean necrotic stunt virus (FBNSV) isolates from Azerbaijan were completely sequenced, leading to the first identification of FBNSV occurring in Europe. Sequence analyses uncovered evolutionary relationships, extensive reassortment and potential remnants of mixed nanovirus infections, as well as intra- and intercomponent recombination events within the nanovirus genomes. In some virus isolates, diverse types of the same genome component (paralogues) were observed, a type of genome complexity not described previously for any member of the family Nanoviridae. Moreover, infectious and aphid-transmissible nanoviruses from cloned genomic DNAs of FBNYV and BMLRV were reconstituted that, for the first time, allow experimental reassortments for studying the genome functions and evolution of these nanoviruses.

The complete genome sequences of a Peruvian and a Colombian isolate of Andean potato latent virus and partial sequences of further isolates suggest the existence of two distinct potato-infecting tymovirus species

The complete genomic RNA sequences of the tymovirus isolates Hu and Col from potato which originally had been considered to be strains of the same virus species, i.e. Andean potato latent virus (APLV), were determined by siRNA sequencing and assembly, and found to share only c. 65% nt sequence identity. This result together with those of serological tests and comparisons of the coat protein gene sequences of additional tymovirus isolates from potato suggest that the species Andean potato latent virus should be subdivided into two species, i.e. APLV and Andean potato mild mosaic virus (APMMV). Primers were designed for the broad specificity detection of both viruses.

Plant Defence and Viral Interference

Plants face attack by a number of pathogens, pests and abiotic stresses. Due to their sessile nature, they cannot flee but have to fight their attackers. They have developed numerous mechanisms to fight off pathogens such as viruses. Some of these are genetically determined by resistance genes, coding for factors that might be required for the replication of a virus (recessive resistance genes) or that trigger an active defence via the hypersensitive response to restrict the invading attacker (such as dominant resistance genes). Additionally, adaptive responses such as induced resistance or RNA silencing are further obstacles that might prevent successful infection of a host. In return, viruses have developed several countermeasure strategies in order to infect plants successfully. Some of these strategies are presented in this review.

Prospects for engineering and improvement of cross-protective virus strains

Mild strain cross-protection is currently an important method for the production of high quality plant products; despite challenge from severe virus isolates the initial protecting strain precludes symptom development. The mechanism of cross-protection is not yet resolved as RNA silencing does not sufficiently explain the phenomenon. Six requirements have been put forward to ensure long-lasting protection. We propose two additional requirements for effective and durable mild strain cross-protection; mild strains based on knowledge of the mechanism and consideration of impacts to consumers. Future research on predicting phenotype from genotype and understanding virus-plant and virus-vector interactions will enable improvement of cross-protective strains. Shared international databases of whole ecosystem interactions across a wide range of virus patho- and symbiotic-systems will form the basis for making step-change advances towards our collective ability to engineer and improve mild strain cross-protection.

Red clover vein mosaic virus—A Novel Virus to New Zealand that is Widespread in Legumes

Red clover vein mosaic virus (RCVMV) is an important virus of leguminous crops that can cause devastating losses. During a routine survey of legumes conducted on the South Island of New Zealand, RCVMV was found in mixed infections in clover plants with Alfalfa mosaic virus and White clover mosaic virus. The full-length sequence of the New Zealand isolate RCVMV-NZ from clover shared 96% nucleotide sequence identity with a chickpea isolate previously described from Washington (United States). Targeted surveys of pea, faba bean, and pasture crops showed that RCVMV-NZ is widespread on the South Island in New Zealand. This isolate is causing mild if any symptoms on experimental hosts and naturally infected plants.

Nanovirus DNA-N encodes a protein mandatory for aphid transmission

Nanoviruses possess a multipartite single-stranded DNA genome and are naturally transmitted to plants by various aphid species in a circulative non-propagative manner. Using the cloned genomic DNAs of faba bean necrotic stunt virus (FBNSV) for reconstituting nanovirus infections we analyzed the necessity of different virus components for infection and transmission by aphids. We found that in the absence of DNA-U1 and DNA-U2 symptom severity decreased, and in the absence of DNA-U1 the transmission efficiency decreased. Most significantly, we demonstrated that the protein encoded by DNA-N (NSP) is mandatory for aphid transmission. Moreover, we showed that the NSP of FBNSV could substitute for that of a distantly related nanovirus, pea necrotic yellow dwarf virus. Altering the FBNSV NSP by adding 13 amino acids to its carboxy-terminus resulted in an infectious but non-transmissible virus. We demonstrate that the NSP acts as a nanovirus transmission factor, the existence of which had been hypothesized earlier.