Interpolyelectrolyte complexes of in vivo produced dsRNA with chitosan and alginate for enhanced plant protection against tobacco mosaic virus
Benjamin Moorlach, Ana R. Sede, Katharina Hermann, Alesia A. Levanova, Minna M. Poranen, Michael Westphal, Martin Wortmann, Elzbieta Stepula, Désirée Jakobs‐Schönwandt, Manfred Heinlein, Waldemar Keil, Anant Patel
Abstract
We developed a formulation of long double-stranded RNA (dsRNA) using interpolyelectrolyte complexes (IPECs) composed of the biopolymers chitosan and alginate, in order to protect the dsRNA from biotic and abiotic factors. Our primary objectives were to enhance stability of dsRNA against environmental nucleases and, secondarily, to mitigate the negative charge of the dsRNA, which may promote foliar uptake. Our approach relies on submicron particles with adjustable surface charge being either positive or negative. Following this approach, we obtained a high encapsulation efficiency of 94 %. Subsequently, we investigated the influence of the charge ratio and total polymer content on the size, size distribution and ζ-potential of the IPECs. We discovered that formulating at low polymer concentrations ≤0.05 g/L with charge ratios of ≤0.9 (+/−) and ≥ 1.25 (+/−), respectively, produced <100 nm particles. Furthermore, the IPEC formulation protected dsRNA from enzymatic degradation by RNase III and micrococcal nuclease. In addition, we observed outstanding protection of formulated dsRNA from heat degradation. Experiments on Nicotiana benthamiana plants showed that formulated dsRNA offered protection against tobacco mosaic virus. In essence, this formulation demonstrates versatility for the production of IPECs with customizable size, surface charge, and nucleic acid content. In this study, we developed a biopolymer-based formulation using chitosan and alginate to protect long dsRNA from biotic and abiotic degradation. This method produced ~100 nm particles with customizable size and charge, achieving a 94.3 % encapsulation efficiency. The IPEC formulation effectively protected dsRNA, demonstrating its potential for dsRNA-based crop protection against pathogens.