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

Abstract

Summary Fungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins ( AFP s) 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 AFP s 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 AFP s 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 AFP s 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.

The Antifungal Protein AfpB Induces Regulated Cell Death in Its Parental Fungus Penicillium digitatum

“…AFPs exhibit potent antifungal activity in the micromolar range against important human and plant pathogens ( 5 , 6 , 8 , 9 , 12 , 15 – 18 ). Furthermore, exploitation of AFPs is feasible, since safe and efficient fungus- or plant-based biofactories have been developed for their production ( 5 , 19 , 20 ).…”

“…AfpB is a recently identified AFP in the genome of the citrus postharvest pathogen Penicillium digitatum (26). The protein could not be detected in natural P. digitatum cultures but has been biotechnologically produced in large amounts in the same fungus or in a plant-based system (5,20). This antifungal protein is very active against major phytopathogenic fungi, especially Penicillium species, including the parental P. digitatum (5).…”

“…This antifungal protein is very active against major phytopathogenic fungi, especially Penicillium species, including the parental P. digitatum (5). Moreover, AfpB has been shown to be effective in plant protection treatments against fungal infections, such as Botrytis cinerea, the causal agent of gray mold disease in more than 200 vegetable species (6,20). We show here that AfpB cytotoxicity is determined by interaction with the cell wall of the target fungus and by its active internalization into fungal cells, where it induces a regulated cell death program.…”

Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants

“…Infectious clones (or infectious transcripts) of plant viruses remain a basic research tool in plant pathology, concerning mostly studies on virus-derived pathogenicity determinants [ 23 ]. On the other hand, plant virus vectors were described as platforms for heterologous protein production in plants [ 24 ] (revised in [ 25 ]) or for silencing gene expression in hosts (most commonly tobacco rattle virus and potato X virus). The goal of this study was to develop stable ToTV-based constructs suitable for expressing a reporter protein, GFP, in plants.…”

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

“…The overexpression of recombinant proteins in E. coli is commonly employed for manufacturing proteins in significant amounts owing to advantages, such as the development using cheap sources of energy, rapid deposition of biomass, suitability for fermentation with high cell density, and fairly quick output [ 14 ]. However, according to the data published by the Center for Eukaryotic Structure Genomics (CESG) (http:/targetdb.pdb.org/statistics/sites/CESG.html), only approximately 30% of the cloned targets in E. coli , among the total of 8048 targets, were presented in a soluble form, while the rest were presented either in a deteriorated form or as insoluble aggregates, classified as inclusion bodies [ 147 ]. While inclusion bodies cannot be used specifically for experiments involving protein activity, their insolubility offers a simple source of fairly pure protein, provided only certain proteins can be transformed to their natural and active conformation.…”