Degradation of Wheat Streak Mosaic Virus Capsid Protein During Leaf Senescence

Brakke, M. K., Skopp, R. N., and Lane, L. C. 1990. Degradation of wheat streak mosaic virus capsid protein during leaf senescence. Phytopathology 80:1401-1405. Wheat streak mosaicvirwscapsid protein degraded in vivo by proteolysis rnented slightly more slowly than those with 45 kDa protein. Antiserum as leaves senesced. The capsid protein of virus purified from young systemto intact virions, containing predominantly 45 kDa protein, reacted with ically infected leaves had an apparent size or 45 kDa in 12% sodium both 45 and 3 1 kDa proteins on Western blots. Thirteen isolates of WSMV dodecyl sulfate-polyacrylamide gels with minor amounts of 43, 42, 33, could be classified into two groups with major capsid proteins of 47 and 3E kDa proteins. The proportion of smaller proteins increased with and 45 kDa, respectively, as well as accompanying minor proteins. Most the age of the leaf. In some virus preparations only 3 1 kDa capsid protein natural isolates, including Sidney 81, contained 45 kDa protein, but the was detected. In vitro proteolysis of virions with 45 kDa protein produced type strain contained 47 kDa protein. virions with 31 kDa protein. Virions with 31 kDa capsid protein sediAdditionalkeywords: gel electrophoresis, potyviruses. Wheat streak mosaic virus (WSMV) causes a serious disease of winter wheat in North America, Jordan, and Europe (5). The virus resembles potyviruses in morphology and by inducing cylindrical or pinwheel-shaped cytoplasmic incIusions containing a 66 kDa protein, but differs from cIassica1 potyviruses in being transmitted by an eriophyid mite, Eriaphyes ~ulipae Keifer (4,7). Potyviruses usually have a capsid protein ranging in size from 28 to 40 kDa accompanied by varying amounts of smaller proteins (13,201. Detailed examination of the smaller proteins indicates they arise from degradation of the largest protein (12,20). Our preliminary estimates of capsid protein size by sodium dodecyl sulfate-polyacrylarnide gel electrophoresis varied. A 45 kDa protein predominated in most virus preparations, but a 3 Z kDa protein predominated in others. Most preparations had both 45 and 31 kDa proteins, as well as proteins of intermediate size. Proportions of the various proteins were erratic. To investigate these iaconsistencies we systematically studied the protein composition of several strains of WSMV and investigated the effect of proteases on the virion. MATERIALS AND METHODS Materials. Enzymes, enzyme inhibitors, enzyme-coupled antibody, and chemicals for developing Western blots were obtained from Sigma Chemical Co., St. Louis, MO. Virus maintenance and assay. Most experiments were done with the type strain of WSMV (PV57; ATCC, 1981) and an isolate collected from western Nebraska in 1981 (Sidney 81). The type isolate was collected in Kansas in 1932 and maintained by H. A. McKinney until 1955, when we obtained it from him. This isolate was transmitted by mites (E. tulipae) with the assistance of R. Staplesjust before it was contributed to the ATCCcollection. This article is In the public domain and not copyrlghtable. It may be freely reprinted with customary crediting of the source. The American Phytopalhologlcal Soc~efy, 1990. One experiment employed 1 1 additional isolates collected in Nebraska between 1956 and 1985. These isolates were kept in dried leaves at 2 C over CaCl, and rejuvenated by passage through plants every 4-5 yr. Virus was grown and purified from Michigan Amber wheat. It was generally inoculated at the two-leaf stage and plants were harvested when five or six leaves were fully expanded. Infectivity assays of WSMV and its RNA were based on systemic infection of wheat (Triricum aesrivum L. 'Michigan Amber') plants inoculated with a series of dilutions (2). The sires of virions and viral RNA were estimated by density gradient centrifugation (3,6,8). Leaf age. Leaves were harvested for purification from plants with rive to six fully expanded leaves. Where age of the infection was important, we used plants that were simultaneously inoculated and combined leaves of similar physiological ages from several plants. For this purpose the youngest fully expanded leaf is designated as the first leaf and older leaves are numbered downward in order of increasing age. Virion and RNA purification. Virions were purified by grinding infected wheat leaves in a blender with two volumes (ml/g) of 10 mM K2HP04, adjusting the pH to 6.1 with 1 M acetic acid, and centrifuging the extract at 10,000 rpm in a Sorvall SS34 rotor for I5 min. The supernatant was adjusted to pH 7.5-8.0 with NaOH and brought to 10 mM in trisodium citrate and 1% (vIY) in Triton X-100. The extract was centrifuged for 1.8 hr at 27,000 rpm at 5 C in a Beckman No. 30 rotor through a 4-ml pad of 20% (wlv) sucrose in 10 mM trisodium citrate, pH 8.0. Pellets were suspended in 10 mM trisodium citrate (pH 8) and clarified by centrifugation for 5 min at 5,000 rpm in a Sowall SS34 rotor. For density gradient centrifugation of virions, samples (24 ml) were floated on linear 1040% (wlv) sucrose gradients prepared in I0 m M trisodium citrate and centrifuged for 3 hr at 25,000 rpm at 5 C in a Beckman SW27 rotor. To extract R N A from the virus, portions of the preparation were diluted with equal volumes of 2X AAE buffer (IX AAE is 100 mM [NH4ltC0,, 300 mM NH,Cl, 2 m M ethylene diamine tetraacetic acid [EDTA], adjusted to pH 9.3 with NaOH) Vol. 80. No. 12, 199