Identification of Phytophthora capsici nuclear effector targets to bolster host immune signalling and resistance pathways
Phytophthora capsici is a plant pathogenic oomycete that causes huge damage to agriculture worldwide. Its virulence is driven by effectors, which target various host cell components in order to suppress plant immune responses. To enhance plant immunity and to generate new resistances in crops, it is essential to develop new strategies. Synthetic biology approaches emerged as a tool to bolster plant immunity. There is a great deal of evidence that modification of plant proteins can affect plant-pathogen dialogue. For instance, modification of plant resistance proteins can lead to effector gain of recognition specificity whereas disruption of effector-target interaction via target modification leads to enhanced immunity. However, to achieve that, we need to better understand plant-pathogen interactions and identify effector virulence targets. In this work we Identified Importin α1 and α2 nuclear transporters as targets of P. capsici nuclear effector CRN1_719. In the effort to characterise these interactions we determined that Importin α1 and α2 may be implicated in nucleolar trafficking, have a role in plant immunity and are involved in PTI. We demonstrated that truncation of the IBB domain from Importin α1 results in higher binding affinity to CRN1_719, as well as its mislocalisation from the nucleolus. An affinity purification mass spectrometry approach aimed to better understand the role of Importin α1 and α2 upon infection showed that Importin α1 and α2 proteomes are drastically affected by P. capsici infection. In addition, we identified a set of Importin α1 and α2 putative interactors, and potentially the pathways that these nuclear transporters may be involved in. Our results demonstrate that nuclear and nucleolar trafficking have an important role in the context of plant immune signalling. Moreover, this data could potentially be used in the effort to modulate pathogen effector nuclear import. In addition, we explored R gene (LRR domain) mutagenesis in order to generate new recognition specificities of effectors. Although the size of the generated library did not allow us to achieve that, it is likely that by increasing library diversity, this approach could be applied to any pathosystem.
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