High-efficiency green management of potato late blight by a self-assembled multicomponent nano-bioprotectant

Late blight, caused by the oomycete Phytophthora infestans, remains the most economically important potato disease globally, historically causing the Irish Famine and currently leading to annual losses exceeding $6 billion. Control largely relies on frequent applications of synthetic pesticides, raising environmental, health, and safety concerns. RNA interference (RNAi), mediated by double‑stranded RNA (dsRNA), enables sequence‑specific gene silencing and has been explored for spray‑induced gene silencing (SIGS) to manage plant diseases. However, P. infestans has low uptake efficiency of exogenous dsRNA, creating a delivery bottleneck that limits SIGS efficacy. Nanotechnology offers biocompatible vehicles to promote translocation of biomolecules. Building on prior work with a star polycation (SPc) nanocarrier exhibiting high intracellular delivery efficiency and biocompatibility, this study aims to overcome dsRNA delivery barriers in P. infestans and to develop a self‑assembled multicomponent nano‑bioprotectant that combines dsRNA with a plant elicitor to achieve synergistic protection via pathogen inhibition and enhanced plant defenses, suitable for greenhouse and field applications.

Background literature underscores: (i) the economic and historical impact of potato late blight and reliance on chemical control; (ii) RNAi/SIGS as a promising, target‑specific, eco‑friendly approach, with successful uptake in some fungi (e.g., Botrytis cinerea) but limited uptake in P. infestans; (iii) the need for efficient delivery systems for oomycetes; and (iv) nanocarriers that enhance cellular uptake and endocytosis in biomedical and agricultural contexts. Prior studies introduced SPc as a facile‑synthesized, biodegradable, low‑cost star polycation nanocarrier capable of binding nucleic acids via electrostatic interactions and loading hydrophobic actives, improving delivery of dsRNA and amplifying plant defenses with elicitors. These works motivated co‑delivery strategies for gene/drug combinations to address short RNA lifetimes and delivery bottlenecks.

• SPc synthesis: A star polycation (SPc) was synthesized using a pentaerythritol‑based star initiator (Pt‑Br) polymerized with DMAEMA under nitrogen. THF solvent was removed and reused; product purified by repeated dialysis in water and freeze‑dried.
• dsRNA design and synthesis: Targeted P. infestans infection genes PiHmp1 (haustorial membrane protein 1) and PiCut3 (cutinase) were amplified from cDNA (strain T30‑40) and used to produce dsRNAs in vitro (T7 RiboMAX). Fluorescent dseGFP was synthesized for uptake studies; dstdTomato (205 bp) was prepared for RNAi tests in a tdTomato‑expressing transformant.
• Bacterial large‑scale dsRNA production: PiHmp1 and PiCut3 were tandemly cloned into pET28a and co‑expressed in E. coli BL21(DE3) RNase III−. Cultures were induced with IPTG; dsRNA was released by heat/ethanol or lysozyme plus heat, purified, quantified, and mixed with SPc at defined ratios.
• Complex formation: dsRNA was complexed with SPc (optimized by gel retardation). For the multicomponent system, cellobiose was first loaded onto SPc (drug loading content determined), then dsRNA was assembled to form cellobiose/SPc/dsRNA nanoparticles. Component ratios were determined by agarose gel retardation and anthrone‑sulfuric acid colorimetry.
• Stability assays: RNase A degradation assays compared naked dsRNA vs SPc‑complexed dsRNA and multicomponent complexes. Persistence on plant leaves was evaluated over time.
• Uptake and delivery assays: Confocal microscopy assessed uptake of fluorescent dseGFP into P. infestans sporangia/hyphae and into potato and Nicotiana benthamiana tissues, comparing naked vs SPc‑loaded dsRNA. RNAi efficacy was tested by fluorescence reduction and qRT‑PCR (tdTomato transformant), normalizing to actin using 2^−ΔΔCT.
• Elicitor loading and plant defense assays: Cellobiose was loaded into SPc to form cellobiose/SPc complexes. Potato leaves were treated with cellobiose or cellobiose/SPc; qRT‑PCR quantified endocytosis‑related genes (StEPSIN, StVPS36, StVAMP1‑2, StVAMP3‑1, StRab) and defense genes (StPRI, StWRKY1, StPPO, StPT15), plus phytoalexin pathway genes (StCoum, StEpic). Coumarin levels were quantified by LC‑MS/MS.
• Self‑assembly characterization: Isothermal titration calorimetry (ITC) examined SPc–cellobiose interactions and subsequent binding to dsRNA, yielding thermodynamic parameters and dissociation constants. Dynamic light scattering (DLS) and TEM characterized particle sizes and morphologies of SPc, cellobiose, cellobiose/SPc, and cellobiose/SPc/dsRNA complexes.
• Bioassays on detached leaves: Potato cv. Favorita leaves were sprayed with dsPiHmp1, dsPiCut3, their SPc complexes, or controls; inoculated with P. infestans sporangia. Lesion areas were measured at 2, 5, and 15 dpi.
• Greenhouse and field trials: Whole plants were sprayed with water, SPc, mancozeb, dsRNA, dsRNA/SPc, cellobiose/SPc, or multicomponent cellobiose/SPc/dsRNA and challenged with P. infestans. Disease index, relative biomass, protection effect at 29 days, and AUDPC were assessed. Field plots (Hebei, China) used susceptible cv. Wotu 5; mancozeb applied at 5.6 kg/ha. Statistical analyses used t‑tests or one‑way ANOVA with Tukey’s HSD (p < 0.05).

• Delivery and stability: SPc protected dsRNA from RNase A degradation and enhanced uptake into P. infestans sporangia and hyphae. On potato leaves, SPc‑loaded dseGFP showed 3.65‑fold higher fluorescence intensity than naked dsRNA; SPc/dsRNA persisted on plants up to 12 days and was 1.5‑fold more abundant than naked dsRNA.
• Gene silencing: In a tdTomato‑expressing P. infestans transformant, dstdTomato/SPc reduced red fluorescence and decreased tdTomato expression by 2.7‑fold versus dstdTomato alone (p < 0.001).
• Disease suppression on leaves: SPc‑loaded dsPiHmp1 or dsPiCut3 provided strong protection on detached leaves when applied 24 h before inoculation, with no disease symptoms up to 15 dpi; naked dsRNAs were ineffective under the same conditions.
• Dual‑target dsRNA: Tandem dsPiHmp1+PiCut3 without SPc showed silencing of both targets but conferred protection only when delivered via SPc. A bacterial pET28‑BL21(DE3) RNase III− system produced dsRNA at 3‑fold higher yield than L4440‑HT115(DE3), enabling low‑cost, large‑scale production. SPc‑loaded dsPiHmp1+PiCut3 from the bacterial system matched in vitro dsRNA efficacy.
• Elicitor effects and defense amplification: Cellobiose loaded into SPc (DLC 12.70 ± 1.07%) up‑regulated potato endocytosis genes by 1.4–2.9‑fold versus cellobiose alone. Defense genes were further amplified; notably StWRKY1 increased by 920‑fold. Phytoalexin‑related genes increased by 2.5‑ and 24‑fold, and coumarin content rose by 2.1‑fold. SPc alone did not up‑regulate immune genes.
• Self‑assembly and composition: ITC indicated strong SPc–cellobiose interaction (Kd = 4.075 × 10^−4 M) driven by non‑covalent interactions; further binding to dsRNA involved hydrogen bonding, van der Waals, and electrostatic forces. Particle sizes: SPc ~100 nm; cellobiose ~219 nm; cellobiose/SPc ~35 nm; cellobiose/SPc/dsRNA ~88 nm. Component mass ratio in the multicomponent complex was 53 (cellobiose):1000 (SPc):1000 (dsRNA).
• Greenhouse efficacy: dsRNA/SPc, cellobiose/SPc, and the multicomponent nano‑bioprotectant all reduced disease indices and P. infestans biomass compared with controls, without growth defects.
• Field efficacy: Under natural epidemic conditions, the multicomponent nano‑bioprotectant achieved the lowest disease index (20) and 68% protective effect at 29 dpi, outperforming mancozeb (53% protection). AUDPC values were lowest for the multicomponent treatment, reflecting superior season‑long control.

The study addresses the central barrier to SIGS against P. infestans: poor pathogen uptake of dsRNA. By employing an SPc nanocarrier, dsRNA stability against nucleases and efficient intracellular delivery into both oomycete and plant cells were achieved, translating to robust gene silencing of key infection determinants (PiHmp1 and PiCut3) and prolonged protection on leaves. Incorporating a plant elicitor (cellobiose) into the same nanoplatform further enhanced systemic defense through elevated endocytosis and strong up‑regulation of defense genes and phytoalexin pathways. This dual mechanism—pathogen gene suppression and host defense amplification—yielded additive protection in greenhouse assays and superior control under field conditions compared to a widely used synthetic fungicide. The bacterial co‑expression system for dsRNA provided a practical, scalable, and cost‑effective production route, supporting field deployment. Overall, the results validate a nano‑enabled, multicomponent co‑delivery strategy as a green, effective alternative for managing late blight and potentially other plant diseases.

This work introduces a self‑assembled multicomponent nano‑bioprotectant that co‑delivers dsRNAs targeting P. infestans infection genes with a plant elicitor via an SPc nanocarrier. The platform overcomes dsRNA delivery and stability limitations, extends the RNAi protective window, and amplifies plant immunity, achieving greenhouse and field efficacy that surpasses a commercial fungicide. It also demonstrates a scalable, low‑cost bacterial system for dual‑target dsRNA production. The strategy offers a generalizable framework for eco‑friendly pesticide/drug design and integrated management of plant diseases and pests. Future research could expand target gene combinations, evaluate longevity and reapplication schedules under diverse agroecological conditions, assess non‑target and environmental safety profiles, and adapt the platform to other oomycetes and pathogens.