Biotechnological production of antifungal proteins for crop protection

Abstract

Plant diseases caused by pathogenic fungi are responsible of important crop losses endangering food security and safety. Antimicrobial peptides (AMPs), exhibiting potent and durable lytic activity specifically against microorganisms, have a great potential as novel natural fungicides for the control of pathogenic fungi. However, viable exploitation of AMPs requires fast, cost-efficient, and safe production systems. The main goal of this work was to develop a sustainable platform for the production of bioactive AMPs, and to characterize them in the control of fungal infections in plants to advance in their application in agriculture. Antifungal proteins (AFPs) secreted by filamentous fungi are a group of highly stable cysteine-rich AMPs that specifically target fungal cells. In this study, we demonstrate that Nicotiana benthamiana plants are an excellent biofactory for producing AFPs through transient expression using a new vector derived from the tobacco mosaic virus. Using this plant-based production system we efficiently produced two different bioactive AFPs, the Penicillium expansum AfpA and Penicillium digitatum AfpB. We found that the subcellular compartment where AFPs are accumulated has an important impact on protein yield, probably avoiding toxicity towards plant cells. The highest yields were achieved when targeting AFPs to vacuoles, reaching up to 0.170 mg/g of fresh leaves of the highly active AfpA and eight times more of AfpB (1.2 mg/g of leaf). We also show that plant crude extracts containing AFPs are fully active against plant pathogens without requiring further protein purification, thus reducing significantly downstream processing. Therefore, the developed system is efficient for the production of AFPs, and also it is economic and safe since it is based on plants. We also developed an alternative system for the production of the linear PAF102 antifungal peptide that was recalcitrant to be produced in biological systems. This system is based on targeting the peptide to lipid droplets (LDs) through the fusion to a plant oleosin protein. Using this oleosin fusion technology, we produced PAF102 in rice seed LDs, reaching moderate yields of about 20 μg of peptide per gram of grain. Production on rice seeds is long process in order to speed the process, we successfully transferred the plant oleosin fusion technology to the Pichia pastoris system. We produced commercially relevant yields of PAF102 in these yeast LDs, reaching values of 180 mg/l of culture in only 4 days. The accumulation of PAF102 in the LDs of rice seeds and yeast facilitated its downstream extraction and recovery by simple flotation on dense solutions, with the recovered PAF102 being biologically active against pathogenic fungi. Finally, we demonstrate that in planta produced AfpA and AfpB, either purified protein or protein extracts enriched with these two proteins, are efficient in controlling important fungal diseases on economically relevant crops, including Botrytis gray mold disease in tomato leaves and fruits, blast disease in rice plants and Fusarium proliferatum infection in rice seeds. Our results provide a sustainable production system of AFPs, and evidence their efficacy on protecting plants from fungal infection, strongly supporting the use of AFPs as environmental friendly and effective “green fungicides” in crop and postharvest protection.