Plantain Virus X: a New Potexvirus from Plantago Lanceolata SUMMARY
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J. gen. ViroL (1981), 54, 75-90 75 Printed in Great Britain Plantain Virus X: a New Potexvirus from Plantago lanceolata By J. HAMMONDt AND R. HULL* John Innes Institute, Colney Lane, Norwich, U.K. (Accepted 22 December 1980) SUMMARY Plantain virus X (P1VX), a previously undescribed potexvirus, is common in Plantago lanceolata in Great Britain. P1VX infected 22 species from six families, out of 48 species from 10 families tested. Nicotiana × edwardsonii was a useful diagnostic host, although no suitable host is known for lesion assays. Sap from infected N. clevelandii was infective after 10 min at 60 °C, but not 70 °C. Infectivity in dried leaf material of N. x edwardsonii was retained for at least 26 months, and a purified preparation was infective after freezing for at least 3 months. In negatively stained preparations P1VX has flexuous particles of 570 to 580 by about 12 nm, with clearly visible cross-banding. The pitch of the particles was 3.4 nm, with a true repeat of five turns and probably 8~ subunits per turn. Purification of P1VX from N. clevelandii leaves yielded up to about 30 mg pure unaggregated virus/kg of leaf. Purified virus sedimented as a single component with a sedimentation coefficient (s°0.w) of 119S. The buoyant density in CsC1 was 1.31 g/ml, and in Cs2SO 4 was 1.259 g/ml. Purified virus contained a single coat protein species which did not migrate anomalously in SDS-polyacrylamide gel electro- phoresis, and had an apparent mol. wt. of 28 858 to 26 000 depending on the method of analysis. PIVX coat protein migrated more slowly in slab-gel electrophoresis than did the coat proteins of six other potexviruses. A single nucleic acid species of about 2.1 x 106 mol. wt. was detected by polyacrylamide gel electrophoresis. No serological relationship to P1VX was detected in tests with the coat proteins of six, and the antisera to seven, other potexviruses. All of the properties described are consistent with P1VX being a distinct member of the potexvirus group. INTRODUCTION In a survey of the viruses endemic in Plantago species in Great Britain, 51 out of 130 plants ofPlantago lanceolata were found to be infected with a virus with flexuous rod-shaped particles. The properties of the virus suggest that it is a previously unrecorded virus and that it is similar to the potexviruses. It is provisionally named plantain virus X (P1VX). This paper describes its host range, some of its properties and its relationship to other members of the potexvirus group. METHODS Virus isolates. The isolate of P1VX, which was free from other viruses, was found near Cambridge and was maintained by subculture in Nicotiana clevelandii, with occasional passage through P. lanceolata. Potato virus X (PVX) strain X n ringspot (Matthews, 1949; from the John Innes Institute plant virus collection) was propagated in Nicotiana tabacum cv. White Burley. Purified papaya mosaic virus (PaMV) and purified coat proteins of narcissus 4" Present address: Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907, U.S.A. 0022-1317/81/0000-4424 $02.00 © 1981 SGM 76 J. HAMMOND AND R. HULL mosaic virus (NMV) and clover yellow mosaic virus (CYMV) were gifts of Professor J. B. Bancroft. Purified proteins of white clover mosaic virus (WCMV) and nerine virus X (NeVX) were gifts of Miss M. N. Short. Host range studies. Test plants were grown at about 18 °C, and were placed in the dark for 24 h before inoculation. They were mechanically inoculated with leaf tissue ofN. elevelandii or P. lanceolata ground in 0.1 M-acetate pH 6 (1:1, w/v). The infectivity of the inoculum was tested by the inclusion of Nicotiana x edwardsonii [formerly N. clevelandil x N. glutinosa (Christie & Hall, 1979)] and/or P. lanceolata among the plants inoculated. Detection and identification of infection was confirmed by back inoculation (from both inoculated and non-inoculated leaves, 10 to 15 days after inoculation) to N. x edwardsonii. In vitro properties. The thermal inactivation point was determined by grinding infected N. clevelandii leaves in an equal weight of 0.1 M-acetate pH 6, and heating aliquots to 50, 60, 70 or 80 °C for 10 min in a water bath before inoculating the sap into plants of N. x edwardsonii. To demonstrate the retention of infectivity in dried material, infected leaf tissue of N. clevelandii and N. x edwardsonii was shredded and dried in sealed containers over anhydrous CaC12. Inoculations were made using the dried material, ground in 0.1 M-acetate pH 6. Virus purification. The method used was adapted from those of Goodman (1975) for PVX, and of van Oosten (1972) for plum pox virus. Leaves of N. clevelandii were harvested 12 to 15 days after inoculation, and stored frozen. The tissue was then thawed at 4 °C, and all subsequent steps carried out at 4 °C. The tissue was homogenized in 0.5 M-(K2H/NaH2)PO 4 buffer pH 7.6 containing 10 mM-EDTA at a rate of 2 ml buffer/g of leaf. Triton X-100 was added to give 2 to 2.5 % (v/v), the mixture gently stirred for 1 h and then centrifuged at 7000 rev/min for 10 rain (Sorval GSA rotor). The supernatant was centrifuged for 3 h at 27 000 rev/min in a Beckman type 30 rotor, after which the tubes were drained, gently rinsed in distilled water and the pellets resuspended overnight in 2 ml 5 mM-EDTA pH 7.6 per tube. The resuspended pellets were clarified by low-speed centrifugation (10000 rev/min, 10 min; Sorval SS-34 rotor) and the virus in the supernatant was centrifuged through a 7 ml cushion of 30% (w/v) sucrose in 0.1 M-phosphate pH 7.6, 2 mM-EDTA in the type 30 rotor. The tubes were drained, rinsed in distilled water and the pellets resuspended overnight in 1 ml 5 mM-EDTA per tube. After low-speed centrifugation of the resuspended pellets, 1 to 2 ml of the supernatant was layered on to each gradient of 10 to 40% (w/v) sucrose in 0.1 M-phosphate, 2 mM-EDTA, which was then centrifuged for 2.25 h at 24 000 rev/min in a Beckman SW27 rotor. The gradients were fractionated by upward displacement through an ISCO UA2 u.v. analyser recording at 254 nm. The virus fractions were collected manually, diluted with an equal volume of distilled water, and concentrated by high-speed centrifugation. The purified virus was resuspended in 5 mM-EDTA pH 7.6, and was used immediately or stored frozen. Electron microscopy. Epidermal dip preparations were made by the method of Hitchborn & Hills (1965), using 3% sodium tungstate, adjusted to pH values between 5.5 and 7 with formic acid, as the negative stain. Purified preparations of P1VX were stained with sodium tungstate, or 2% aqueous uranyl acetate, or 2% aqueous uranyl formate. For ultrathin sections of Nicotiana benthamiana and N. × edwardsonii, small pieces of leaf tissue were immersed in 2 % glutaraldehyde in 0.05 M-cacodylate buffer pH 7 for at least 3 h at room temperature. The pieces were then washed three times in cacodylate buffer, post-fixed for 1 to 3 h in 1% osmium tetroxide in cacodylate buffer at room temperature, washed in distilled water and dehydrated through an alcohol series saturated with uranyl acetate, before leaving overnight in uranyl acetate-saturated acetone. After two washes in propylene oxide the pieces were embedded in Epon 812. Sections about 70 nm thick were cut using an LKB Ultratome 1, post-stained with lead acetate and examined in a Siemens 1A electron microscope. Plantain virus X 77 Analytical ultracentrifugation. Virus preparations at 0.2 to 6.5 mg/ml were examined by moving boundary sedimentation using the An-D rotor of a Beckman model E analytical ultracentrifuge equipped with Schlieren optics. Sedimentation coefficients were estimated by the graphical method of Markham (1960) at several virus concentrations and extrapolated to zero concentration. To measure the buoyant density, approx. 100 pg P1VX was centrifuged in the An-D rotor for at least 16 h at 30000 rev/min and 20 °C in gradients of CsC1 or Cs2SO4 buffered in 0-1 M-phosphate buffer pH 7. The position of the virus band was recorded using Schlieren optics, and the buoyant densities calculated by the method of lift et al. (1961) for C sC1, and that of Ludlum & Warner (1965) for Cs2SO4. Diffraction pattern. Electron micrographs of a mixture of purified PIVX and catalase crystals stained in uranyl formate were used to produce diffraction patterns using the method described by Horne& Markham (1972). The diffraqtion pattern of catalase (Luftig, 1967) was used to calibrate the measurements from the P1VX diffraction pattern. Mol. wt. estimation of coat protein subunits. Protein samples were prepared from virus preparations at approx. 1 mg/ml by the addition of an equal volume of the dissociation buffer of Hull & Lane (1973) and heating to 60 °C for i0 min. The following marker proteins were used, and similarly treated: lysozyme (mol. wt. 14300), sperm whale myoglobin (17200), chymotrypsinogen A (25 700), ~-globulins (23 500 and 50000), carbonic anhydrase (29 000), aldolase (40000) ovalbumin (43000) and bovine serum albumin (67000). Electrophoresis was in 8 x 0-5 cm cylindrical gels of 3, 5, 6, 7.5 or 10% total acrylamide, or in slab gels 12 x 17 x 0.15 cm of 5, 7-5, 10 or 12.5% total acrylamide. In each case gels were 2% cross-linked with methylene bisacrylamide, and contained 0.1% (w/v) sodium dodecyl sulphate (SDS).