Chlorella EV-Embedded Small RNAs for Durable Phytopathogen Control
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Solution Overview
Problem
Current methods for producing vectorized small RNAs to protect plants from phytopathogens are not scalable or cost-effective for industrialization, and existing RNA-based biologicals lack durability due to pathogen escape mutations.
Innovation Solution
The method involves expressing exogenous RNA interference precursors in Chlorella cells to produce Extracellular Vesicle (EV)-embedded antimicrobial small RNAs, which are resistant to ribonuclease digestion and can be efficiently produced and stored, enabling their use in microalgae-based biologicals for broad-spectrum pathogen protection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods are used to produce vectorized small RNAs, then production is possible, but scalability and cost-effectiveness for industrialization are poor
Solution Approach 1:
Chlorella cells are engineered to autonomously produce and package small RNAs into extracellular vesicles through their own cellular machinery. The transgenic Chlorella system self-assembles the RNAi precursors, processes them through endogenous pathways, and secrettes EVs containing the active small RNAs, eliminating the need for complex external vectorization and purification steps
Solution Approach 2:
The Chlorella production system serves multiple functions simultaneously: it acts as a factory for small RNA synthesis, a packaging system for EV formation, a protection mechanism against degradation, and a delivery vehicle for plant application. This multi-functionality consolidates multiple separate processes into a single integrated biological system
2Reliability
If existing RNA-based biologicals are used, then pathogen protection is provided, but durability is reduced due to pathogen escape mutations
Solution Approach 1:
The small RNA population is segmented into multiple variants targeting different regions of the pathogen genome. This segmentation creates a diverse arsenal where if one target region mutates, other segments continue to provide protection, preventing complete pathogen escape
Solution Approach 2:
The system employs variable parameters in the small RNA sequences, targeting multiple different genomic regions and pathways of the pathogen. This parameter diversity includes targeting essential genes, virulence factors, and resistance genes simultaneously, creating multiple barriers to pathogen adaptation
3Ease of operation
If small RNAs are released into the environment, then pathogen targeting is achieved, but ribonuclease-mediated digestion degrades the small RNAs
Solution Approach 1:
Extracellular vesicles serve as an intermediary carrier that protects small RNAs from direct exposure to ribonucleases in the environment. The EV membrane acts as a physical barrier, allowing the small RNAs to remain intact during transport and application while still being able to interact with pathogen cells
Solution Approach 2:
The extracellular vesicle membrane provides a flexible protective shell around the small RNA cargo. This lipid bilayer shell is permeable enough to allow RNA delivery to pathogen cells while being sufficiently robust to resist enzymatic degradation by ribonucleases in the external environment
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for high-yield, cost-effective production of stable EV-embedded small RNAs that effectively target virulence factors and antimicrobial resistance genes, providing durable resistance against a wide range of phytopathogens and enhancing beneficial microbial effects on plants.
Implementation Method 1
the expression of exogenous RNA interference (RNAi) precursor(s) in Chlorella cells, which in turn express and release Extracellular Vesicle (EV)-embedded and/or-associated antimicrobial small RNAs
Implementation Method 2
Chlorella EVs protect small RNAs from ribonuclease-mediated digestion, indicating that these lipid-based particles not only act as natural vectors of small RNAs towards pathogenic cells, but also presumably limit their degradation in the environment
Data Source
AI summary
The invention relates to a novel method to produce small RNAs targeting virulence factors, essential genes and/or antimicrobial resistance genes of phytopathogens. More specifically, the invention involves the expression of exogenous RNA interference (RNAi) precursor(s) in Chlorella cells, which in turn express and release Extracellular Vesicle (EV)-embedded antimicrobial small RNAs. These EVs can be collected from the cell-free medium of Chlorella cultures, and further concentrated and purified for biocontrol applications. Importantly, Chlorella EVs protect small RNAs from ribonuclease-mediated digestion, indicating that these lipid-based particles not only act as natural vectors of small RNAs towards pathogenic cells, but also presumably limit their degradation in the environment. The invention can thus likely be used to reduce the pathogenicity and growth of a wide range of pathogens or, potentially, to enhance beneficial effects and growth of plant-associated symbiotic and commensal microbes. Furthermore, because the integrity of Chlorella EV-embedded antimicrobial siRNAs remains unaltered when produced in photobioreactors, and when stored frozen, this method has the potential to be further exploited for the industrialization and manufacturing of a novel generation of microalgae-based biologicals.