Natural and engineered resistance to wheat streak mosaic virus (Tritimovirus: Potyviridae)
Creators: name: Fahim, Muhammad
Publisher: name: The Australian National University
Year: 2011
Subjects: subject: QR411.F34 2011
subject: Wheat streak mosaic virus Australia
subject: Wheat Disease and pest resistance Australia
subject: Wheat Genetics

description: Wheat streak mosaic virus (WSMV) is a new virus of wheat crop in Australia. Discovered in the Australian Capital Territory (ACT) in 2003, the virus has put Australian commercial bread wheat at a risk of major losses. Although, the virus is naturally transmitted by Wheat curl mites (WCM), some of the Australian farming community expressed concerns that grazing of early sown, dual-purpose wheat for winter forage may have a role in the spread of WSMV. We probed this issue in a series of experiments with housed sheep grazing on WSMV infected wheat plants. However, we find no evidence for the suggestion that grazing sheep spread the WSMV between plants in a grazed wheat crop as a consequence of the grazing process itself. We tested for natural resistance against WSMV in diverse germplasm including three different known resistance sources in cultivated wheat. Previously reported resistances were effective against the Australian isolate of WSMV. Some accessions of these resistances were ineffective at higher temperatures (all Wsm1 and most Wsm2 accessions); some were reported to have linked negative agronomic traits (most accessions of Wsm1). Two exceptions were c2652 and Wsm2 accession CA745 which were very effective at controlled higher temperatures (28{u00B0}C), in the glasshouse, and also protected plants from symptoms and yield loss following WSMV mechanical inoculation in the field, making these two sources particularly useful in the relatively warm Australian agro-climate. New molecular markers were developed for the various derivatives of Wsm1 resistance that should help speed up the breeding of resistance into wheat cultivars. These Wsm1 markers are now being used by CSIRO for breeding Wsm1-resistance into elite wheat cultivars. Furthermore, we developed and tested two independent transgenic strategies based on intron-hairpin RNA (ihpRNAi) and artificial microRNAs (amiRNA). Both strategies were effective in conferring immunity in transgenic wheat to mechanically inoculated WSMV. We classified this resistance as immunity by four criteria: no disease symptoms were produced; Enzyme linked immunosorbent assay (ELISA) readings were as in un-inoculated plants; viral sequences could not be detected by RT-PCR from leaf extracts; and leaf extracts failed to give infections in susceptible plants when used in test-inoculation experiments. We developed ihpRNA or RNAi based immune transgenic wheat by designing an RNAi construct to target the Nuclear inclusion protein 'a' (NIa) gene of WSMV. The Northern and Southern blot hybridization analysis indicated the ihpRNA transgene integrated into the wheat genome and was processed into typical 21-24 nucleotide long siRNAs and correlated with immunity in transgenic plants. In order to achieve amiRNA immunity, we designed five artificial microRNAs (amiRNA) against different portions of the WSMV genome, utilising published miRNA sequence and folding rules; these amiRNAs were incorporated into five duplex arms of the polycistronic rice primary microRNA (pri-miR395) and transformed into wheat. Southern blot hybridisation showed that the transgene was stably integrated into the wheat genome and processed into small RNAs, both correlating with transgenic resistance against WSMV. As a consequence of the work described in this thesis, the wheat industry in Australia and abroad has both conventional and transgenic options for the control of this serious viral pathogen.