“Deciphering the inter-kingdom RNA exchanges in the Alternaria-Tomato Pathosystem”

Norman, Christopher (2022) “Deciphering the inter-kingdom RNA exchanges in the Alternaria-Tomato Pathosystem”. PhD thesis, University of Worcester.

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Abstract

Fungal infections make up most plant crop losses in the agricultural industry. This contrasts with mammals, in which the major threat to our existence is viruses and bacteria with fungi making up only a small minority of the infections we suffer. Due to this there has been a large investment in the prevention of fungal infection, mainly using chemical fungicides. However, chemical fungicides have many disadvantages, most notably the runoff resulting in damage to wildlife and other forms of ecological impact, occurrence of resistant pathogen isolates and health risks for consumers and farmers using them. This has gradually led to public rejection of chemical fungicides as shown by the growth of the organic food industry. The growing technology of RNA silencing shows great potential in alleviating this problem. Host induced gene silencing (HIGS), naturally exists in plants as a means of protection from pathogens and has already become cited as a potential new resistance engineering method in crops. Either by spraying the plant directly with RNA molecules or inducing the RNA production in the plant using gene delivery vectors like Agrobacterium and viruses. The more we understand these interactions the more we can perfect this technology. Here the A. alternata-tomato pathosystem will be used to evaluate the role of sRNA in infection and defence of the plant and evaluate genes that may have value as a target for sRNA. In addition, this project will look at the potential of certain genes as targets for targeted RNA silencing to protect tomato from Alternaria alternata. Specifically, the Chitin synthase genes (CHS) and the effect silencing RNAs has on these genes has and whether they inhibit fungal infection. The methods used in this project focused on two areas, determining if CHS is a viable target for spray induced silencing through treating spores with small RNA solutions and determining any effect on germination rates. These experiments found limited effect on germination however did discover a significant effect on germination tube size and spore production, with tube size increasing significantly on treatment with sRNA and spore production occuring earlier and in larger quantities. sRNA produced by the plant during infection was also evaluated using sRNA sequencing, to identify novel miRNA. My conclusion for this project are that CHS is likely a poor candidate for sRNA silencing however with the number of miRNA identified to show upregulation during infection it can be concluded that natural miRNA play a role in infection and many of these miRNA could be the targets of future research to improve the defence response of the tomato plant or to develop new SIGS or HIGS systems.

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