Team:Ecuador/Proof Of Concept
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Results
Proof Of Concept
Contribution
Proof of Concept
Introduction
We conceive, AgroBactory 593 as a modular bacterial platform that produces low-cost biopesticides to fight plant diseases. By producing and delivering dsRNA molecules to silence the expression of specific targets in the pathogen. To demonstrate the efficacy of the platform, we developed a biopesticide against Foc R1 in vitro, with genes that can also be used against FOC TR4.
To carry out our project we have divided it into three modules:
Module 1.-
Production and design of dsRNA, where homology and specificity are taken into account.
Module 2.-
Release and delivery of dsRNA molecules through a cell lysis protein, which is activated by the Quorum sensing mechanism that triggers cell lysis.
Module 3.-
dsRNA silencing of the chosen target gene and description of how dsRNA and RNA interference molecules can inhibit its gene expression, and eventually control Fusarium wilt.
Validating our project
Module 1.- Production and design of dsRNA
A key challenge and crucial step for RNAi-based crop protection strategies is the identification of effective target genes. We began a review of the literature to find genes involved in the pathogenicity of Foc TR4. The genes SGE1, ERG11, Velvet, and SIX1 were chosen for their role in the development of diseases such as conidia, virulence, synthesis of secondary metabolites, and wilt [1]. We confirm the presence of Velvet (450 pb), ERG11 (400 pb), SGE1(1089 pb) and Six1 (246 pb), in a sample of Foc R1, so we continued with the design and assembly of dsRNAs which will be assembled (using restriction enzymes) with the widely used plasmid L4440.
In order to improve the production of dsRNA in E. coli DH5α and HT115 strains, we enhance the conventional plasmid L4440, bidirectional sequences of the T7 promoter, and two terminators were introduced into the plasmid backbone using 3A assembly. We successfully obtained bacterial colonies with the proposed assemblies, verified by colony PCR and the fluorescence of the transformed bacteria in LB medium characteristic of the GFP indicator.
Module 2.- Release and delivery of dsRNA molecules
Module 2 was designed for delivering and realeasing dsRNA molecules of the velvet gene produced by Module 1. However, we replaced Module 1 by a fluorescent reporter to test the behaviour of the genetic device and the effectiveness of cell lysis. Thus, LuxR-AHL transcription factor activates simoultanously expression of GFP and the lysis protein PhiX174E, when sufficient amounts of cells in the population produce AHL.
We designed a temporal experiment to assess and quantify (if possible) cell lysate. We collected absorbance and fluorescence data from the gene circuit in vivo. One of the assays is shown in the Figure below.
Figures A and B depict how cells death when the lysis protein is activated by the lux promoter K2656003.
The total GFP fluorescence expressed by the population and a single-cell is shown in Figures C and D, respectively. At the beginning of the experiment, the number of cells producing GFP is very low (OD 600=3.05e-6). But after lysis, GFP molecules are released to the medium and some cells are still producing GFP. With module 2 we demonstrate the feasibility of Agrobactory's second release approach, as native plant bacteria release dsRNA through an oscillating lysis system, also controlling the bacterial population.
Module 3.- dsRNA silencing
The ultimate objective was to test our dsRNA-based biopesticide and identify the most effective gene segments to test in RNAi-inducing constructs to provide resistance to Panama disease. We plan to test the antifungal activity of gene-specific dsRNAs, produced in E coli and previously purified, by reduction in colony number following administration by imbibition into spores of Foc R1 [2]. We planned to carry out experiments to determine the optimal test concentration of dsRNAs to use in the spore germination inhibition bioassays and then we conducted the final assay.
Due to difficulties associated with the COVID-19 pandemic, we were unable to execute complete the Module 3 trials, however based on the results of Mumbanza et al. [2] we will expect that after optimization of the test parameters in preliminary experiments, the most effective test concentration to use in the spore germination inhibition bioassays will be determinate at a close value of 0.6 μg/mL of dsRNAs. Then, among the treatments, we would expect to observe a significant reduction in the number of colonies (between 80 – 90 % [2, 3, 4]) that will establish on the plates and identify the most effective target gene to inhibit Foc spore germination.
Conclusion
With the backup from previous researches and our results in modeling and experiments, we validated the feasibility of our modular bacterial platform that produces low-cost biopesticides to fight plant diseases. Agrobactory 593 has the potential to offer a preventive treatment for Panama disease (caused by the fungus Foc TR4), and any other disease affecting plants. Through our development scheme, which starts with the in silico design of dsRNAs for pathogen-specific genes, the optimized production of dsRNAs in E coli strains, with a structure that presents greater stability. Our oscillator as a release system based on quorum sensing and finally the development of a simple inhibition assay, Agrobactory demonstrates its potential as an innovative idea to ultimately reduce the use of pesticides in agriculture, within the framework of Sustainable Development Goals.
References
[1] V. Gurdaswani, S. B. Ghag, y T. R. Ganapathi, “FocSge1 in Fusarium oxysporum f. sp. cubense race 1 is essential for full virulence”, BMC Microbiology, vol. 20, núm. 1, p. 255, ago. 2020, doi: 10.1186/s12866-020-01936-y
[2] F. M. Mumbanza, A. Kiggundu, G. Tusiime, W. K. Tushemereirwe, C. Niblett, and A. Bailey, “In vitro antifungal activity of synthetic dsRNA molecules against two pathogens of banana, Fusarium oxysporum f. sp. cubense and Mycosphaerella fijiensis,” Pest Manag. Sci., vol. 69, no. 10, pp. 1155–1162, Oct. 2013, doi: 10.1002/ps.3480.
[3] X. Hou, B. An, Q. Wang, Y. Guo, H. Luo, and C. He, “SGE1 is involved in conidiation and pathogenicity of Fusarium oxysporum f. sp. cubense,” Can. J. Microbiol., vol. 64, no. 5, pp. 349–357, May 2018, doi: 10.1139/cjm-2017-0638.
[4] M. S. López-Berges et al., “The velvet complex governs mycotoxin production and virulence of Fusarium oxysporum on plant and mammalian hosts,” Mol. Microbiol., vol. 87, no. 1, pp. 49–65, Jan. 2013, doi: 10.1111/mmi.12082.
