Molecular Characterization of Beet Necrotic Yellow Vein Virus in Greece and Transgenic Approaches towards Enhancing Rhizomania Disease Resistance Ourania I. Pavli Thesis committee Thesis supervisor Prof.dr. J.M. Vlak Personal Chair at the Laboratory of Virology Wageningen University Prof.dr. G.N. Skaracis Head of Plant Breeding and Biometry Department of Crop Science Agricultural University of Athens, Greece Thesis co-supervisors Dr.ir. M. Prins Program Scientist KeyGene, Wageningen Prof.dr. N.J. Panopoulos Professor of Biotechnology and Applied Biology Department of Biology University of Crete, Greece Other members Prof.dr. R.G.F. Visser, Wageningen University Prof.dr.ir. L.C. van Loon, Utrecht University Dr.ir. R.A.A. van der Vlugt, Plant Research International, Wageningen Prof.dr. M. Varrelmann, Göttingen University, Germany This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences. 2 Molecular Characterization of Beet Necrotic Yellow Vein Virus in Greece and Transgenic Approaches towards Enhancing Rhizomania Disease Resistance Ourania I. Pavli Thesis submitted in partial fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof.dr. M.J. Kropff in the presence of the Thesis Committee appointed by the Doctorate Board to be defended in public on Monday 11 January 2010 at 1.30 PM in the Aula 3 Pavli, O.I. Molecular characterization of beet necrotic yellow vein virus in Greece and transgenic approaches towards enhancing rhizomania disease resistance 166 pages Thesis, Wageningen University, Wageningen, NL (2010) With references, with summaries in Dutch, English and Greek ISBN 978-90-8585-547-7 4 Contents Abstract 7 Chapter 1 General Introduction 9 Chapter 2 Performance of Rz1-based Resistance against 39 Sugar Beet Rhizomania Disease in Greece Chapter 3 Molecular Characterization of Beet Necrotic 49 Yellow Vein Virus in Rhizomania Diseased Sugar Beet throughout Greece Chapter 4 Survey for the Detection of Rhizomania 63 Related Soil-borne Viruses in Sugar Beet Cultivation Zones in Greece Chapter 5 A High Frequency Agrobacterium rhizogenes - 73 Mediated Sugar Beet Transformation Protocol Chapter 6 BNYVV Replicase-Derived dsRNA Confers 83 Resistance to Rhizomania Disease of Sugar Beet as Evidenced by a Novel Transgenic Hairy Root Approach Chapter 7 Expression of HrpZPsph in Transgenic Nicotiana 97 benthamiana Results in BNYVV - Induced Tissue Necrosis and Enhanced Rhizomania Resistance Chapter 8 General Discussion 115 Summary 131 Samenvatting 135 Περίληψη 141 Appendix 147 Acknowledgements 161 5 ABBREVIATIONS aa amino acid BNYVV Beet necrotic yellow vein virus bp Base pair BSBMV Beet soilborne mosaic virus BSBV Beet soilborne virus BVQ Beet virus Q CP coat protein dpi days post inoculation dpt days post transformation ds double stranded ELISA sandwich enzyme-linked immunosorbent assay GM genetically modified/genetic modification HCD hypersensitive cell death HR hypersensitive response Hrp harpin Hyb hybrid IR inverted repeat kb kilo base kDa kilo dalton MP movement protein mRCP multiplex PCR nptII neomycin phosphotransferase II nt nucleotide OD optical density ORF open reading frame p protein PCR polymerase chain reaction PDR pathogen-derived resistance PTGS post-transcriptional gene silencing RB resistance breaking RC resistant commercial Ri root-inducing RdRp RNA-dependent RNA polymerase rep replicase RFLP restriction fragment length polymorphism RT-PCR reverse-transcription PCR SAR systemic acquired resistance SC susceptible commercial si small interfering SP signal peptide ss single stranded TGB triple gene block TILLING targeted induced local lesions in genomes TTSS type III Secretion System V valine vir virulence 6 ABSTRACT Rhizomania disease of sugar beet, caused by Beet necrotic yellow vein virus (BNYVV), is responsible for severe economic losses. Due to the widespread occurrence of BNYVV and the absence of other practical and efficient control measures, economic viability of the crop is to the largest extent dependent on the use of varieties genetically resistant to the disease. Recent reports on the emergence of virus strains capable of compromising the Rz1-based resistance as well as on the spread of highly pathogenic RNA 5-containing BNYVV isolates have necessitated a detailed investigation of the situation as it evolves in Greece. The study revealed the widespread occurrence of BNYVV throughout the country as well as the prevalence of pathotype A isolates in all sugar beet growing regions. Sequence determination of the p25 protein, responsible for symptom development, pointed to the amino acid motifs ACHG/VCHG in the hypervariable amino acid region 67-70. However, the presence of valine (V) in position 67 was not associated with increased pathogenicity and resistance breaking properties. Disease severity appeared mostly dependent on agroclimatic conditions influencing the progress of the disease. A survey for a possible occurrence of Beet Soilborne Virus (BSBV) and Beet Virus Q (BVQ) in rhizomania infested fields revealed the co-existence of both viruses, with BVQ being systematically found in co-infections with BNYVV, while BSBV was in all cases only found in triple infections. Towards the exploitation of the antiviral properties of RNA silencing, three intron hairpin constructs carrying parts of the BNYVV replicase gene, were evaluated for their potential to confer rhizomania resistance in Ri T-DNA-transformed sugar beet roots. The results show that transgenic hairy roots were effectively protected against the virus disease and further indicate that the developed methodology for Agrobacterium rhizogenes-mediated transformation can be employed as a suitable platform to study transgene expression in sugar beet and other transformation recalcitrant crop species. In parallel, the potential to exploit the HrpZPsph protein from Pseudomonas syringae pv. phaseolicola for engineering rhizomania resistance in sugar beet against BNYVV was demonstrated by the successful engineering and protection against BNYVV in the model plant Nicotiana benthamiana. 7 CHAPTER 1 8 GENERAL INTRODUCTION Sugar beet (Beta vulgaris L. ssp. vulgaris) is one of the most important industrial crop species, occupying globally a cultivated area of approximately 8.1 million hectares spread over 41 countries (Rush et al., 2006). From a historical view point, it represents a European bred crop with a short but outstanding evolution characterized by pronounced improvements in breeding and cultivation which along with advanced processing technologies, have placed sugar beet as world’s second to sugar cane in raw sugar production worldwide. Sugar beet, whose root sugar content potential has throughout the years reached 15-20%, today provides approximately 25% of world sugar consumption. In addition, considerable quantities of sugar beet-based ethanol are also being produced for years, mostly in France. The recent interest for bioethanol production, as a replacement of fossil fuels in the transportation sector, has triggered significant research efforts in exploiting the crop’s potential towards this biofuel and for the production of biogas as well. Relevant business endeavours are being seriously pursued in several European countries, including Greece. Worldwide, economic viability of sugar beet growing is to a large extent depended on the successful protection against pathogens, of which Rhizomania, a viral root disease caused by Beet necrotic yellow vein virus (BNYVV), exerts a very high impact. Although several agronomic practices may help to a small degree, the only practical means of confronting this most devastating disease is by employing genetically resistant varieties. Due to the limited availability of useful natural genetic sources of resistance against the prevailing virus strains (Grimmer et al., 2007) as well as the recent emergence of novel resistance breaking strains, all relevant breeding activities become of paramount importance. HISTORY OF THE SUGAR BEET CROP Sugar beet belongs to the genus Beta of the Chenopodiaceae family, a genus also including all wild beet relatives. Mainly based on the presence of the sea beet (Beta vulgaris L. ssp. maritima), considered as sugar beet’s most probable wild progenitor, the genus Beta has originated in a widespread area ranging from the British Isles and the North Atlantic coast across Europe and the Mediterranean and the Black Sea to the Persian Gulf and the mouth of the Indus River (Ulbrich, 1934; Biancardi, 2005). In prehistoric times, sea beet is believed to have been domesticated around the Persian Gulf as early as wheat and barley, about 12,000 years ago (Simmonds, 1976). Sugar beet cultivation is far more recent however, dating back in ancient times when the first cultivars resembling spinach beet and Swiss chard that were grown as garden vegetables during the Greek and the Roman era. The earliest references to the beet plant 9 were provided by Aristophanes (445-385 BC) and Euripides (480-406 BC) and throughout the years several names, all of Greek origin, have been used. Its first name was teutlon, a name still being used in modern Greek language. Theophrastos (372-287 BC) used the term sicula to describe beet from the old Greek colony of Sicily. Upon the end of the second century BC, it was mentioned as Beta by Roman writers as well as by the Roman military physician Dioscuridis, whose interest was in the medical value of the plant (Lippmann, 1929; Ford-Lloyd & Williams, 1975; Winner,