Overview

Fungal infections have detrimental impacts in agriculture by decreasing crop yields. Particularly, Fusarium wilt, a fungal disease caused by Fusarium oxysporum, affects a wide variety of foodstuffs. Current treatments for Fusarium wilt involve the cultivation of resistant crops; however, ever-evolving fungal pathogens can circumvent those resistances and induce blight within the resistant crop strains. As a result, an inherent component of all fungal cell walls, chitin, was targeted to combat fungal infections in plants. A diversely found glucose-derivative, chitin provides rigidity in all fungal species and also acts as a similar constituent of arthropod and insect exoskeletons and harder external tissues in various organisms. Our proposed mechanism for the antifungal treatment was to degrade chitin through the use of the enzyme chitinase. Various chitinase isozymes act upon the varying chitin structures in different organisms, so chitinase LbCHI31 was selected as the chitinase of choice for Fusarium oxysporum.

Procedural Outline

The goal of this project was to engineer a bacterial strain capable of secreting viable chitinase to attack the cellular stability and structures of Fusarium oxysporum, thereby inducing structural collapse of the fungal cell wall. A plasmid containing genes encoding for LbCHI31 production and secretion was used to transform bacteria using standard heatshock and recovery protocols.

Plasmid Design

The pBR322 plasmid was used as a base construct for the plasmid design. When designing the plasmid, the gene sequence for chitinase LbCHI31 was incorporated with a GFP tag and a linker into the plasmid. Since the GFP gene was linked to the LbCHI31, the presence of green fluorescence under UV exposure would indicate successful secretion of the GFP-LbCHI31 complex (i.e., since GFP is secreted, the linked the LbCHI31 product is consequently secreted). A signal peptide following the GFP tag was added and linked to the chitinase in order to signal the E. coli to begin type II secretion. The type II secretion system is generally utilized to secrete degradative enzymes such as chitinase. Additionally, the pBR322 contains inherent ampicillin resistance; as such, by using ampicillin as a selective agent, transformed e. coli was easily isolated and cultured. In order to break down the chitin in the cell wall of fungus, our team has chosen to utilize E. coli and have them secrete a chitinase that will break down the chitin. Chitinases essentially break down glycosidic bonds in chitin, suggesting that by getting E. coli to secrete chitinases, we can successfully combat fungal infections. The particular chitinase we chose was LbCHI31, an enzyme that is known to be able to degrade the chitin in Fusarium oxysporum. Through transformation, we will introduce a plasmid with the gene that encodes for LbCHI31 into E. coli.

PROTOCOLS

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SAFETY

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RESULTS

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Team:TP CC-SanDiego/test-project