1.12.5
TOWARDS TRANSGENIC FIJI DISEASE-RESISTANT SUGARCANE

JA HANDLEY1, P BURNS1, HM SOO1, MM MAUGERI1, GR SMITH2, JM DALE1 and RM HARDING1

1Queensland University of Technology, Brisbane, Queensland, Australia; 2Bureau of Sugar Experiment Stations, Indooroopilly, Queensland, Australia

Background and objectives
Sugarcane Fiji disease, caused by sugarcane Fiji disease reovirus (FDV), is one of the most important diseases affecting sugarcane in Queensland. FDV is the sole member of serogroup 1 within the genus Fijivirus of the family Reoviridae. Virions consist of a double icosahedral shell with outer spikes, and are transmitted by the planthopper Perkinsiella spp. The FDV genome consists of ten segments of dsRNA, approximately 30 kbp in total. FDV has been traditionally controlled through the use of conventionally bred cultivars; however, backcrossing for the introduction of resistance genes is extremely difficult due to the complex nature of the sugarcane genome. The overall aim of this study is to produce transgenic sugarcane resistant to FDV using pathogen-derived resistance strategies. Characterization of the FDV genome to identify potential transgenes is an essential step in this process.

Materials and methods
A random primed cDNA library was generated from purified viral dsRNA. Segment-specific clones were identified by Northern blot analysis and selected clones were sequenced. RT-PCR was used to amplify intervening regions between the sequence-specific clones, and anchor-ligated PCR was used to identify the terminal sequences. The pMAL bacterial expression system was used to express selected ORFs.

Results and conclusions
We have generated sequence data for approximately 80% of the FDV genome. Analyses of the FDV sequences have revealed that FDV has (i) a similar genome organisation to other Fijiviruses, and (ii) significant homology to maize rough dwarf (MRDV), rice black streak dwarf (RBSDV) and Nilaparvata lugens (NLRV) reoviruses. The 5' and 3' terminal sequences of FDV were identical to those reported for MRDV and RBSDV. Segments 7 and 9 are both bicistronic with two non-overlapping ORFs, while the other segments each encode a single ORF. Putative functions have been assigned to a number of segments based on homologies to other Fijiviruses: segment 1 encodes the viral RNA-dependent RNA polymerase, segment 3 encodes the major core capsid, segment 7 (ORF 2) encodes a GTP-binding protein, and segment 8 encodes an NTP-binding protein. The proteins encoded by ORF1 of segments 7 and 9 have been expressed in bacteria. Both proteins were recognized by antiserum produced against intact and subviral FDV, suggesting that these proteins are structural. We have cloned segment 9 into a plant expression vector and used this vector in a pilot study to develop transgenic sugarcane.