Interactomics: Development of an efficient and improved Agrobacterium tumefaciens-mediated transformation method for transient expression of heterologous protein in recalcitrant plant tissues in planta

Abstract Background: Since more researches leaned towards a holistic approach of “omics” to understand PPI, interactomics studies pivoting on AP-MS approach are getting popular amongst plant scientists. However, to date, AP-MS approach has yet to be adopted to study plant-pathogen interaction even in model plants, hence, the needs to design and develop an optimised transformation procedure for successful isolation of pathogen-targeted plant proteins. Methods: Banana plants were grown in soil or hydroponic media for 2-3 months before subjected to transformation procedure. As a bait, m16D10 , a mature parasitism gene isolated from Meloidogyne incognita was transiently expressed in banana root tissues cv. Grand naine (ITC1256) to prey for interacting plant proteins. Subsequently, the plant-pathogen interacting protein complex was “pulled” at the purification step using histidine affinity column, electrophoresed in SDS-PAGE gel and subjected to western blot assay. Results: We first compared Agrobacterium tumefaciens -mediated transformation quality on root tissues grown in soil and hydroponic media and found that the latter yielded higher area of transformed tissues (99% - 100%). We found hydroponic-grown root tissues were amenable to syringe-based agroinfiltration method and required lesser acclimatisation period compared to the soil-grown root tissues. Our result showed that western blot minimum detection limit of heterologous protein amount in the purified root protein product was at 1:1 ratio, with minimum concentration of 9 mg/mL of purified protein. Using these parameters, western blot assay confirmed the expression of m16D10 gene in transformed plant root tissues and SDS-PAGE gel revealed that we had isolated putative plant proteins interacting with the bait protein. Conclusion: An improved, efficient, and cost-effective Agrobacterium tumefaciens -mediated transformation protocol was developed for transient expression of heterologous protein in recalcitrant plant tissues, suitable for AP-MS-based plant-pathogen interaction studies. Confirming our hypothesis, we demonstrated that the improved method had successfully “pulled-out” banana root proteins interacting with m16D10 protein using histidine affinity column.