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Title:   Author  item
                                            Lim, Hyoun-Subitem
                                            VAIRA, ANNA MARIA - CNR, IVV, ITALYitem
                                            Reinsel, Michaelitem
                                            Bae, Hanhongitem
                                            Bailey, Bryanitem
                                            Domier, Leslieitem
                                            Hammond, John
  Submitted to: Journal of General Virology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 9/24/2009
                                    Publication  Date: 12/15/2009
                                    Citation: Lim, H.S., Vaira, A.M., Reinsel, M.D., Bae, H., Bailey, B.A., Domier, L.L., Hammond, J. 2010. Localization of Althernanthera mosaic virus patholgenicity determinants to RdRp and TGB1, and separation of TGB1 silencing suppression from movement functions. Journal of General Virology. 91:277-287.Interpretive Summary: Different isolates of a virus often induce distinct symptoms, or differ in host range, as a consequence of variations between viral genes affecting these biological characteristics. We have previously examined isolates of Alternanthera mosaic virus (AltMV) from creeping phlox and portulaca that differed in symptom expression, including induction of necrosis, in the experimental host Nicotiana benthamiana. Here we have made a detailed examination of infectious clones derived from a phlox isolate of AltMV, and identified two biologically distinct sub-isolates present in the original viral population. Chimeric viruses created by gene exchange between the infectious clones identified three viral genes that contribute towards necrotic symptoms that are induced by some sequence variants, especially at low temperature. The mixture of sub-isolates present in the original viral population appears to ameliorate symptoms, and may benefit survival of the more severe sequence type as well as of the host plant. The viral coat protein gene was identified as the most important contributor to necrosis, as substitution of the coat protein gene of the portulaca isolate resulted in loss of the ability to induce necrosis in N. benthamiana. These results help to explain the different responses of ornamental hosts to various isolates of AltMV, and may lead to better protection of ornamental crops from AltMV infection.
                                Technical Abstract: The potexvirus Alternanthera mosaic virus (AltMV) naturally infects several ornamental plants; AltMV-SP was isolated from Phlox stolonifera and maintained in Nicotiana benthamiana. The 5’ and 3’ regions of the genome were separately amplified by PCR and cloned; four full-length infectious clones were created from AltMV-SP by ligation of two independent clones of each of the 5’ half [containing most of the RNA-dependent RNA polymerase (RdRp) (5’ clones 3 or 4)] and the 3’ half [RdRp polymerase domain (Pol) + triple gene block (TGB) + coat protein (CP) (3’ clones 1 or 7)] of the genome, and designated 3-1, 3-7, 4-1, and 4-7. Each clone produced distinct symptoms in N. benthamiana and all clones induced more severe symptom at 15 oC than at 25 oC. AltMV3-7 and AltMV 4-7 induced severe symptoms, and induced necrosis at 15 oC. However, a mixed infection of all four clones was maintained at 15 oC without eliciting necrosis. In plants infected with the original AltMV-SP, sequences represented by severe clone 4-7 and mild clone 3-1 co-existed in a ratio of about 10:1 at 25 oC, and about 6:4 at 15 oC. Sequence analysis revealed few amino acid differences between the mild clone (3-1) and severe clone (4-7), with four changes in the RdRp Pol domain [R(1110)P, K(1121)R, R(1255)K, and S(1535)P], and one in TGB1 [P(88)L]; no amino acid differences were detected in TGB2, TGB3,  or CP. Gene exchanges identified effects of both the RdRp Pol domain and TGB1 on symptom expression and virus accumulation. Real-time quantitative polymerase chain reaction (QPCR) using CP- or RdRP-specific primers showed that AltMV(4-7) was replicated to levels of >100 x that of AltMV(3-1). When these two clones co-infected N. benthamiana at 25 oC, the CP RNA copy number of AltMV(3-1) was always below that of AltMV(4-7) alone. Substitution of coat protein from AltMV-Po strain to severe AltMV clone 3-7 resulted in milder symptoms including absence of necrosis at 15 oC, at a similar level of replication. Agroinfiltration, electron microscopy, and yeast two hybrid assays all indicated strong interactions between AltMV-SP, but not AltMV-Po, CP subunits, suggesting that CP interactions have a major influence on induction of necrotic symptoms.


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