Molecular characterization of beet necrotic yellow vein virus in Greece and transgenic approaches towards enhancing rhizomania disease resistance

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 benthamian