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Engineering resistance to maize lethal necrosis 10.17863/CAM.20731 Braidwood, Luke Anthony 0000000202333605 Baulcombe, David Charles 0000000307806878 Modern agriculture is dependent on both global supply chains and crop monocultures. These features aid the evolution and spread of novel plant pathogens. Limited genetic diversity in commercial crop lines can result in widespread susceptibility to emerging pathogens. Pathogen resistance may be developed through conventional breeding approaches, or a number of transgenic strategies. This thesis focuses on the characterisation of an emerging maize disease, Maize lethal necrosis (MLN), and engineering resistant maize lines using an artificial microRNA (amiRNA) approach. MLN is a synergistic viral disease caused by the interaction of Maize chlorotic mottle virus (MCMV) with any maize-infecting member of the potyviridae. I used next-generation RNA sequencing to characterise the MLN outbreak in East Africa, discovering that local and Chinese strains of the potyvirus Sugarcane mosaic virus (SCMV) typically coinfect with MCMV. A first global MCMV phylogeny was constructed using these samples combined with new Sanger sequencing of samples in Ecuador and Hawaii. The phylogeny supported previous hypotheses of a link between the Chinese and African outbreaks, and suggested a novel link between the Hawaiian and Ecuadorian outbreaks. The SCMV sequences generated demonstrated strong evidence of extensive recombination, in line with previous reports on SCMV and potyviruses. These data also produced first reports of a number of RNA viruses in East Africa, and five novel viral-like sequences, with their presence confirmed by RT-PCR. RNA silencing is an important component of the plant immune response to viral infection. amiRNAs can be used to generate specific and effective viral resistance through Watson- Crick base pairing between the amiRNA and the (RNA) viral genome. Previous amiRNA approaches have targeted invariable genomic regions using consensus sequences. However, the high mutation rate of RNA viruses means single cells contain a variety of mutant genomes, collectively called a quasispecies. To deter the evolution of resistance breaking I devised a novel strategy to include intra-sample variation from NGS data in amiRNA design, and constructs, each containing five of these amiRNAs, were transformed into tropical maize lines. RNA silencing may be hampered by the expression of viral suppressors of silencing (VSRs). Local VSR assays demonstrated that there are no local VSRs in the MCMV genome, while systemic VSR assays showed a possible systemic VSR role for the unique P32 protein, and an interesting link between photoperiod and systemic silencing more generally. University of Cambridge 2017-09-29 Thesis Doctoral Doctor of Philosophy (PhD) en BBSRC DTP https://www.repository.cam.ac.uk/handle/1810/273678 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/261b4b98-2ddd-4679-b073-a54188293aae/download 87eda9de84448d1f82354d60eee3eb5f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/6ca55a45-0d55-4d49-ba40-475ebffc764d/download 7c4b0f3507df615b3ab374dde03fdd22 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/8ef4dcb4-f21e-4a93-a592-3f33cf047208/download c139b882d289944b1199cf37aac090f5 https://creativecommons.org/licenses/by/4.0/ maize chlorotic mottle virus maize virology molecular biology NGS