Synthesis of HrpN-encapsulated chitosan nanoparticles for uptake in Carica papaya L. leaves

Carica papaya L. is a favourite tropical fruit worldwide and considered as an economically important crop grown in Malaysia. Unfortunately, outbreak of papaya dieback disease caused by a phytopathogenic bacteria, Erwinia mallotivora, had resulted a significant drop in market revenue. Discovery of effector proteins secreted by E. mallotivora, have brought a new hope to disease management strategy as they promptly induce defence mechanism like systemic acquired resistance (SAR) against pathogens attack. However, the protein formulations prone to instability which lead to loss of efficiency and efficacy. The application of nanobiotechnology through encapsulation in chitosan biopolymer may be a desirable way to deliver biological compounds into the crops. Chitosan was chosen for its ideal character like biocompatibility, biodegradability, antimicrobial properties, as well as its ability to increase plant immune responses on its own. Thus, this study aimed to synthesis, characterize, and deliver a delivery system of effector protein using chitosan nanoparticles (CNP) as a vector into papaya leaves. For these purposes, recombinant HrpN protein synthesis, HrpN encapsulation in CNP, physicochemical characterization, in vitro HrpN release profiling, fluorescence- based plant uptake assessment, and toxicity evaluation in zebrafish embryos were performed. A selected E. mallotivora Type III effector, termed HrpN, was produced as recombinant protein and subjected to encapsulation within the core of chitosan nanoparticles via ionic gelation method. The optimum HrpN- encapsulated chitosan nanoparticles (CNP-HrpN) formulation synthesized spherical CNP-HrpN which had high encapsulation efficiency (>80%) and colloidal stability, influenced by size (particle size distribution = 106.34 ± 2.053 nm) and high homogeneity (polydispersity index = 0.188 ± 0.011). Infrared spectroscopy portrayed the spectra of synthesized nanoparticles which involve bioactive compounds to their vacancies, successfully. In vitro HrpN release analysis demonstrated sustained release for over 72 hours. On another note, fluorescence imaging revealed the accumulation of CNP-HrpN in papaya leaves at 24 hour post-delivery. Additionally, CNP-HrpN was found non-toxic towards zebrafish embryos through toxicology evaluation. This study successfully synthesized HrpN-encapsulated chitosan nanoparticles using ionic gelation, characterized their physicochemical properties, and showed sustained HrpN release with successful uptake in papaya leaves, describing its potential as a plant defense delivery system. Further in vivo testing in greenhouse condition is recommended to reaffirm this possibility. Hopefully, this study will lay a foundation for developing conceivable biocompatible plant defence tool for better control of plant diseases in future.

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