Abstract
The Russian wheat aphid (Diuraphis noxia, Kurdj.) is an economically important pest of wheat and barley that has spread to all major wheat producing countries on the planet. This phloem-feeding insect causes large-scale plant damage by inducing a breakdown of chloroplast and cellular membranes, thereby reducing yield. On susceptible plant cultivars, infestation eventually leads to the death of the plant. Resistant wheat genotypes currently represent the most effective long term solution to control D. noxia infestations; however the genetically homogenous nature of modern monoculture has led to the development of resistance breaking D. noxia biotypes. These new biotypes are systematically and rapidly overcoming the incorporated resistance genes under field conditions. While numerous studies have been conducted on the wheat – D. noxia interaction, few of the genes implicated in resistance have been functionally validated. In this study, two miRNAs involved in the wheat – D. noxia interaction were studied and their targets were investigated. Both TaDn-miR78 and tae-miR7757 were found to target a number of nucleotide-binding leucine-rich repeat genes (NLRs) and an analysis of 24 904 NB-ARC domain containing sequences indicated that the tae-miR7757 binding site translates to an overlooked, conserved motif in plant resistance proteins.
Plants respond in a similar manner to aphid feeding as to pathogen attack. This led to the discovery of two targets that exhibited similarity to NLR genes with integrated domains (NLR-IDs). NLR-IDs in Arabidopsis have been shown to detect the effectors of attacking pathogens that would otherwise subvert the plant immune system. The full-length sequence of the first target, 5AL-B4, was amplified in order to confirm the predicted domains. The confirmed domains included an N-terminal Ankyrin integrated domain, followed by a traditional coiled-coil NLR structure with a C-terminal WRKY integrated domain. Following this, virus-induced gene silencing (VIGS) using a Barley stripe mosaic virus vector was used to target and silence 5AL-B4 in a wheat cultivar carrying the Dn1 resistance gene (Tugela DN). Knockdown of the NLR-ID using VIGS resulted in the resistant cultivar developing a susceptible phenotype, displaying longitudinal chlorotic streaking similar to the susceptible control. There was an increase in aphid fitness on silenced plants, with the number of aphids produced comparable to susceptible controls and higher than in resistant controls...
Ph.D.