Engineering durable late blight resistance to protect solanaceous plants
Phytophthora infestans, 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. P. infestans 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.Phytophthora species secrete essential effectors into plant cells that target critical host cellular mechanisms to promote disease. One such P. infestans effector is AVR3aKI 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, AVR3aEM, which is homozygous in more than 70% of wild P. infestans isolates, evades this recognition.Domain swapping experiments have revealed that the LRR domain of R3a is involved in recognition of AVR3aKI, as the CC-NB domain of an R3a-paralog which does not mediate recognition of AVR3aKI, 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 AVR3aKI, 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 R3a (R3a*), engineered by an iterative process of error-prone PCR, DNA fragmentation, re-assembly of the leucine rich repeat (LRR)-encoding region of R3a, 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 AVR3aEM-carrying isolates of P. infestans.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 R3a* gene, capable of conferring resistance to P. infestans isolates harbouring AVR3aEM, will provide durable late blight resistance when deployed in the field in combination with other mechanistically different R proteins.
Resistance gene, Artificial evolution, Effectors, Phytophthora infestans, Potato, Plant pathology, Oomycete, PCR shuffling
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