Efficient production of antifungal proteins in plants using a new transient expression vector derived from tobacco mosaic virus.

Xiaoqing Shi,Sandra Garrigues,María Coca,Jose F. Marcos,Teresa Cordero,José‐Antonio Daròs

doi:10.1111/pbi.13038

Abstract

SummaryFungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy‐to‐use, open access viral vector based on Tobacco mosaic virus (TMV). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFPs in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFPs were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFPs fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market.

Highlights

Disease-causing fungi that infect plants, animals and humans pose a serious threat to human and animal health, food security and ecosystem resilience (Fisher et al, 2016, 2012)
As a first step toward a new Tobacco mosaic virus (TMV)-derived vector system for plant biotechnology that uses the Gibson assembly reaction to insert the open reading frame (ORF) of interest, we constructed a TMV infectious clone that efficiently infects plants when delivered by agroinoculation
We transferred a fragment of the TMV cDNA containing the entire coat protein (CP) ORF to a minimal cloning vector

Summary

Introduction

Disease-causing fungi that infect plants, animals and humans pose a serious threat to human and animal health, food security and ecosystem resilience (Fisher et al, 2016, 2012). More people die every year from fungal infections than from malaria (Bongomin et al, 2017). Fungal infections can have fatal consequences for at-risk immunocompromised patients with HIV/AIDS, anti-cancer chemotherapies, corticosteroid therapies, and organ transplantation, among others (Campoy and Adrio, 2017). Fungi are a challenge to food security because they destroy major crops globally and contaminate food and feed with mycotoxins that are detrimental to animal and human health (Bebber and Gurr, 2015). There is an urgent need to develop novel antifungals, whose properties and mechanisms of action represent improvements on the existing ones, and which can be applied in diverse fields, including crop and postharvest protection, preservation in cosmetics, materials and food, and animal and human health

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Conclusion