Downloaded from www.microbiologyresearch.org by IP: 54.162.133.179 On: Mon, 01 Feb 2016 07:19:05 Journal of General Virology (1994), 75, 2559-2565. Printed in Great Britain 2559 Endosymbiotic bacteria associated with circulative transmission of potato leafroll virus by Myzus persicae Johannes F. J. M. van den Heuvel,* Martin Verbeek and Frank van der Wilk DLO Research Institute for Plant Protection (IPO-DLO), P.O. Box 9060, 6700 GW Wageningen, The Netherlands In order to understand the molecular mechanisms underlying circulative transmission of potato leafroll virus (PLRV) by aphids, we screened Myzus persicae proteins as putative PLRV binding molecules using a virus overlay assay of protein blots. In this way, we found that purified PLRV particles exhibited affinity for five aphid proteins. The one most readily detected has an M r of 63K, and was identified as symbionin. This is the predominant protein synthesized by the bacterial endo- symbiont of the aphid and is released into the hae- molymph. Since further studies clearly showed that PLRV particles also bind to native symbionin, it was envisaged that virus particles when acquired into the haemocoel of an aphid interact with symbionin. In- hibition of prokaryotic protein synthesis by feeding M. persicae nymphs on an antibiotic-containing artificial diet prior to PLRV acquisition reduced virus trans- mission by more than 70 %. The major coat protein of the virus was found to be degraded in the antibiotic- treated aphids; this would obviously have resulted in an increased exposure of viral RNA to enzymic breakdown and concomitant loss of infectivity. For these reasons we conclude that endosymbiotic bacteria play a crucial role in determining the persistent nature of PLRV in the aphid haemolymph and that symbionin is probably the key protein in this interaction. Introduction Potato leafroll virus (PLRV) is a single-stranded RNA virus that belongs to the luteovirus group (Waterhouse et al., 1988). Members of this group infect a wide range of mono- and dicotyledonous plants in which they replicate almost exclusively in the phloem tissue. Luteoviruses are transmitted by aphids in a circulative manner (Harrison, 1958; Sylvester, 1980). Briefly, this means that virus particles are ingested along with phloem sap from infected host plants and transcellularly transported through the hindgut into the haemocoel. The acquired virus particles are retained in an infective form in the haemolymph for the lifespan of the aphid, apparently without replication (Eskandari et al., 1979). Upon contacting the accessory salivery glands, they may be transported through this gland, eventually arriving in the salivary duct from which they are excreted with the saliva when the aphid feeds (Gildow & Gray, 1993). Luteo- viruses display a high degree of vector specificity among aphid species. These well-developed specificities suggest an intimate association between a luteovirus and its vectors in which both surface domains of the viral capsid and sites or substances in the aphid are involved (Gildow, 1987). The role of the viral capsid in conferring aphid transmissibility to a luteovirus has been convincingly demonstrated (Rochow, 1970, 1982). However, aphid- derived components suggested to be involved in virus transmission have not yet been identified. Therefore, the main objective of our work was to detect these components and to determine their role in virus transmission. To this end we developed an immunoblot- based virus overlay assay to ascertain whether particles of PLRV bind to proteinaceous components from PLRV's major aphid vector, Myzus persicae. Methods Viruses and antibodies. PLRV-Wageningen (Van der Wilk et al., 1989) was maintained on Physalis floridana as previously described (Van den Heuvel & Peters, 1990). Beet western yellows virus (BWYV) was provided by Dr V. Brault (Institut de Biologic Mol6culaire des Plantes du CNRS, Strasbourg, France) and transferred to P.floridana by M. persieae. Both viruses were purified from leaf material by a modified enzyme-assisted procedure (Van den Heuvel et al., 1990). Purified virus suspensions of blackeye cowpea mosaic virus (BICMV), tomato spotted wilt virus (TSWV), cowpea mosaic virus (CPMV) and beet necrotic yellowvein virus (BNYVV), together with their respective antibodies, were generouslyprovided by colleagues at the Department of Virology, Wageningen Agricultural University (WAU) and IPO- DLO. Anti-idiotypic antibodies (AiAbs) were raised to PLRV-specific MAbs (monoclonal antibodies) (Van den Heuvel et aL, 1990) by injecting rabbits three times at biweeklyintervals with 1 mg of partially purified MAbs derived from mouse ascitic fluids. Antibodies against non-idiotypic sites of the MAbs were removed from the rabbit sera by incubating with mouse anti-thyroglobulin antibodies and subsequent centrifugation essentially following Hu & Rochow (1988). Aphids and other vectors of plant viruses. M. persicae biotype WMp2 was reared on Brassica napus L. subsp, oleifera (oilseed rape) in a 0001 2519 © 1994 SGM