www.jki.bund.de

1. Upper band A satellite virus associated withBrome mosaic virus isolated from winter wheat and triticale in the Russian Central Cernozem region 28 nm 18 nm 28 nm Frank Rabenstein1, Angelika Ziegler1, Annegret Wolf2,Hans-Peter Mock2, Bernd Schlieter3, and Nikolay Danilkin4 Lower band Density gradient Background Cereal plants displaying yellow dwarf symptoms were observed in several experimental plots of a German-affiliated breeding company located in the Central-Cernozem region of Russia (Fig. 1). In particular, barley, wheat and triticale were infested (Fig. 2 and 3) and either Barley yellow dwarf luteoviruses (BYDV-PAV, BYDV-MAV, CYDV-RPV) or Wheat dwarf geminiviruses (Wheat dwarf virus, Barley dwarf virus) were detected by ELISA-variants using polyclonal antisera or monoclonal antibodies. • Figure 1: Location of the German Seed Alliance (GSA) breeding station in the Lipezk oblast in the Central-Cernozem region of Russia However, samples collected from wheat and triticale plants contained high concentrations of Brome mosaic virus (BMV) as detected by DAS-ELISA and immuno-electron microscopy decoration tests. The occurrence of BMV in Russia has been known for a long time. The distribution and severity zones of this virus in Russia are shown in Fig. 4. Figure 3: Virus infected wheat plants displaying dwarfing symptoms in the field trial(Photo Nikolay Danilkin, Russia) Virus isolation and purification Two BMV isolates were obtained from winter wheat and triticale by mechanical inoculation on test plants (Fig. 5). In addition to BMV particles of 28-30 nm in diameter both isolates contained smaller spherical particles of ca.18 nm in diameter (Fig. 6). The wheat isolate (BMV-R-W) was selected for further investigation and propagated in winter barley cv. ‘Erfa’. Figure 4: Distribution and severity zones of BMV in Russia (A. E. Tsyplenkov & M. I. Saulich http://www.agroatlas.ru/en/content/diseases/Gramineae/Gramineae_Brome_mosaic_virus/map/) • Upper band • Lower band Figure 2: Diseased barley plants observed in fieldtrials located in the Central-Cernozem (Photo Nikolay Danilkin, Russia) • Figure 6: Typical 28-30 nm BMVparticles and smaller particles of approx. 17 to 18 nm in diameter after negative staining of BMV-R--W • Figure 7: Purification and separation of smaller particles of approx. 17 to 18 nm in diameter by density gradient centrifugation. The lower band contained normal BMV particles, 28-30 nm in size. BMV-R-W preparations after negative staining with uranyl acetate Virus particles were purified by density gradient centrifugation. A broad, more diffuse lower band was observed after ultracentrifugation. It contained typical BMV particles of approx. 30 nm and a minor upper band of smaller particles of ca 18 nm in size (Fig. 7). Figure 5: Symptoms of the wheat isolate (BMV-R-W) on wheat (left) and barley (right) about two weeks after mechanical inoculation RNA extraction and sequencing • RNA extracted from purified BMV-R-W consisted of four RNAs characteristic for normal BMV isolates,whereas the particles from the upper band contained RNA of approximately 1200 nucleotides. Sequencing revealed an open reading frame coding for a protein of 155 amino acids. Multiple alignments showed about 50 % identity to the P20 protein encoded by Bamboo mosaic virus satellite RNA and to the capsid protein of Panicum mosaic satellite virus. There are several identical amino acid motifs including an arginine-rich N-terminal domain (Fig. 8). MALDI-TOF analysis indicates that the BMV-sat 155 amino acid protein is indeed the satellite virus capsid protein (Fig. 9). MAGRRNRNKRSRGSSVPIGRNSDVMYGTLTLGSTTSWQRKSFPVLAGMGDRPFQITAVRYDVVSQGPMMFQVRLYNP DGNDVAASSGVQLAGTTPRSRWLPRVRGQNVWFNGNSPQTTVLVAVDGLAAVKGMKTPDNIVTLEVQYRLAPNELQSA BMV-sat -MAG—RRNRNKRSRGSSVPIGRNSDVMYGTLTLGSTTSWQRKSFPVLAGMGDRPFQITAVRYDVVSQGPMMFQVRLYNPD 78 SPMV MAPkRSRRSNRRAGSRAAATSLVYDTCYVTLTERATTSFQRQSFPTLKGMGDRAFQVVAFTIQGVSAAPLMYNARLYNPG 80 P20 ----MVGRRNRRQRSCV---SQMTDIMYGSLTLGSTTTWTRKNFPGLANMGDRPFQVISAKIVVSSASPMLYQARLYSPH 73 BMV-sat GNDVAASSGVQLAGTTPRSRWLPRVRGQNVWFNGNSPQTTVLVAVDGLAAVKGMKTPDNIVTLEVQYRLAPNELQSA- 155 SPMV DTDSVHATGVQLMGTVPRTVRLTPRVGQNNWFFGNTEEAETILAIDGLVSTKGANAPSNTVIVTGCFRLAPSELQSS- 157 P20 DDDNVGSTGLQMSGTTPRTHRMRALPGQNTWFSGNTSSTQVIVAIDGLKTKTSDVTPQNAVAVQISYRVAPSELQSAt 151 • Figure 9: Protein extracted from purified 18nm particles was treated with trypsin and the fragments were analyzed by MALDI-TOF. In red: fragments found to be identical to the amino acid sequence deduced from the nucleotide sequence of BMV-sat. This indicates that BMV-sat is encoding its own capsid protein. • Figure 8: Multiple alignment results of 3 protein sequences (155 letters) Bromoviridae Conclusion Taking together, our results show that a hitherto unknown satellite virus of BMV was identified. Further studies are necessary to support the conclusion that among RNA viruses with jelly roll capsid proteins such a helper virus - satellite virus interaction exists not only for members of Tombusviridae (STNV, MWLMV, and PMV) and Virgaviridae (STMV) but also for BMV, the type member of the genus Bromovirus within the family Bromoviridae. • Figure 10: Classification of satellite viruses (Krupovic& Cvirkaite-Krupovic (2012): Towards a more comprehensive classification of satellite viruses. Nature Reviews Microbiology 10, 234| doi:10.1038/nrmicro2676-c4) 1Julius Kuehn Institute, Federal Research Centre for Cultivated Plants (JKI), Institute of Epidemiology and Pathogen Diagnostics, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany 2Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), OT Gatersleben, Corrensstrasse 3, 6466 StadtSeeland, Germany 3Deutsche Saatveredelung AG, Saatzuchtstation Leutewitz, OT Leutewitz Nr. 26, 01665 Käbschütztal, Germany 4GSA Agro LLC, 399921, Township Roshinskiy, Tchapliginsky district, Lipezk region, Russia Contact: frank.rabenstein@jki.bund.de www.jki.bund.de