Characterization of the non-structural protein p4 of the recently detected new virus European mountain ash ringspot-associated virus in European mountain ash (Sorbus aucuparia L.)

• Applicants: Professorin Dr. Carmen Büttner; Professor Dr. Hans-Peter Mühlbach
• Subject Area: Plant Breeding and Plant Pathology
• Term: from 2013 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 238781695

The European mountain ash ringspot-associated virus (EMARaV) infects the European mountain ash (Sorbus aucuparia L.) and is widespread throughout Northern- and Central Europe. It leads from degeneration up to die back of the plant. EMARaV has been associated with the ringspot disease of Sorbus aucuparia L. So far, modes of virus transmission, possible host range and economic importance of EMARaV are unknown. We characterized EMARaV as a novel negative orientated single strand RNA virus that can be transmitted by grafting (Führling and Büttner, 1995). Each of the four identified viral RNAs contains one open reading frame. Sequence comparisons revealed a possible function of the RNA1, RNA2 and RNA3 encoded proteins as a RNA dependent RNA polymerase (p1), a glycoprotein precursor (p2) and a nucleocapsid protein (p3) respectively. So far, the function of the RNA4 encoded p4 protein is unknown. The open reading frame of this RNA shows no sequence homologies to known proteins. We hypothesize that this protein might act as a gene silencing suppressor and/or a movement protein. In plants, cell-to-cell spread as well as systemic distribution of EMARaV requires a viral movement protein. In this proposal we aim to investigate the possible movement function of the p4 protein.The localization of the p4 protein will be identified in p4-transfected protoplasts and in cross sections of EMARaV-infected European mountain ash leaves using a p4 specific antibody. We will investigate whether p4 colocalizes with cell walls taking advantage of specific plasmodesmal markers. EMARaV is presumably moving as nucleocapsid coated genomic RNA (ribonucleoprotein complex) along p4 tubular structures formed as a result of the aggregation of viral movement proteins. We will examine whether tubule formation takes place in p4-transfected protoplasts. The formation of tubuli and the transport of ribonucleoprotein complexes along those structures will be verified in protein-protein interaction experiments. We will analyze whether the p4 protein interacts with itself and nucleocapsid proteins using bimolecular fluorescence complementation (BIFC).The gall mite Phytoptus pyri has been identified as a potential vector of this virus and replication of EMARaV in this vector is likely. The detection of the non-structural protein p4 in the gall mite would further confirm virus transmission in a circulative propagative manner. The main objective of this study is to investigate and understand the function of the p4 protein of EMARaV and will thus deepen our much needed knowledge about spread and epidemiology of EMARaV.

• DFG Programme: Research Grants