<i>Phytophthora infestans</i>, the oomycete pathogen responsible for late blight of potato and tomato, is regarded as the biggest threat to global potato production and is thought to cost the industry around £6 billion annually. Traditionally, fungicides have been used to control the disease, but this is both economically and environmentally costly, as multiple chemical applications may be required during a single growing season. <i>P. infestans</i> has rapidly overcome genetic resistances introduced into cultivated potato from wild species. This provides the rationale for developing artificial resistance genes to create durable resistance to late blight disease.<i>Phytophthora</i> species secrete essential effectors into plant cells that target critical host cellular mechanisms to promote disease. One such <i>P. infestans</i> effector is AVR3a<sup>KI</sup> which is recognised by the potato R3a protein, a member of the CC-NB-LRR type resistance gene family. However, the closely related virulent form, AVR3a<sup>EM</sup>, which is homozygous in more than 70% of wild <i>P. infestans</i> isolates, evades this recognition. Domain swapping experiments have revealed that the LRR domain of R3a is involved in recognition of AVR3a<sup>KI</sup>, as the CC-NB domain of an R3a-paralog which does not mediate recognition of AVR3a<sup>KI</sup>, is able to induce a HR when combined with the LRR of wild-type R3a. However, a chimeric protein consisting of the CC-NB domain of a more distantly-related homolog of R3a and the LRR of domain of R3a, is unable to recognise AVR3a<sup>KI</sup>, suggesting that function is achieved only when the different domains of an R protein are attuned to recognition and signalling. Gain-of-function variants of <i>R3a</i> (<i>R3a*</i>), engineered by an iterative process of error-prone PCR, DNA fragmentation, re-assembly of the leucine rich repeat (LRR)-encoding region of <i>R3a</i>, are able to recognise both forms of AVR3a. This gain-of-recognition is accompanied by a gain-of-mechanism, as shown by a cellular re-localisation from the cytoplasm to prevacuolar compartments upon perception of recognised effector forms. However, R3a* variants do not confer resistance to AVR3a<sup>EM</sup>-carrying isolates of <i>P. infestans</i>.Future efforts will target the NB-ARC domain of R3a, in a bid to fine-tune the intra-cellular signalling of gain-of-recognition R3a* variants. It is hoped that a shuffled <i>R3a*</i> gene, capable of conferring resistance to <i>P. infestans</i> isolates harbouring AVR3a<sup>EM</sup>, will provide durable late blight resistance when deployed in the field in combination with other mechanistically different R proteins.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:716203 |
| Date | January 2016 |
| Creators | Stevens, Laura J. |
| Contributors | Birch, Paul ; Chapman, Sean ; Hein, Ingo |
| Publisher | University of Dundee |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://discovery.dundee.ac.uk/en/studentTheses/27fe2bc9-ac18-4000-a3cf-9bb895cabe3a |
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