• CRAG researchers have developed a strategy to efficiently produce antifungal proteins on a large scale in plants.
• The key to this advance lies in directing the production of these proteins to the vacuole, a cell compartment that has shown to be ideal for their accumulation.
• These plant-produced antifungal proteins offer great potential as safe and environmentally friendly biofungicides to protect crops and harvests.

A new study resulting from the collaboration between researchers at CRAG and the Institute of Agrochemistry and Food Technology (IATA-CSIC) has revealed a highly effective strategy for producing antifungal proteins (AFPs) in plants. Published in the journal Plant Biotechnology Journal, this work opens new avenues for the development of more sustainable and safer biofungicides for agriculture.

An innovative strategy towards high yields

AFPs, such as PeAfpA from Penicillium expansum and PdAfpB from Penicillium digitatum, are molecules of great biotechnological interest due to their potent activity against a wide range of pathogenic fungi. They are small and remarkably stable, making them resistant to heat, proteases, and extreme pH conditions. Moreover, they are non-toxic to plant or mammalian cells.

Traditionally, protein production in plants has explored various compartmentalization strategies, including secretion into the apoplast (the extracellular space of the cell). However, this research has shown that an alternative for achieving extraordinarily high yields of these AFPs lies in targeting them to this specialized organelle of the plant cells: the vacuole.

“The main novelty lies in the use of the large vacuolar compartment, usually considered unsuitable for protein storage due to its lytic environment, where proteins are prone to degradation”, explains Juan José López-Moya, co-author of the study. However, the inherent resistance of AFPs to this proteolytic environment makes them ideal candidates for this innovative strategy. By incorporating a vacuolar targeting signal in addition to an apoplastic one into the expression vectors, the researchers achieved a significant increase in protein accumulation. Specifically, PeAfpA production increased more than 9-fold, and PdAfpB by 3.5-fold, compared to constructs targeted only to the apoplast.

The subcellular localization of the proteins was confirmed by transmission electron microscopy, showing that the AFPs were mainly stored in the vacuole. Importantly, the functionality of the proteins was not affected by the change in targeting, and its vacuolar localization does not require different purification steps.

Practical applications and future outlook

Plant extracts enriched with these AFPs, without further complex purification, showed antifungal activity comparable to that of purified fungal-derived proteins, in both in vitro and in vivo assays.

The plant protection assays were very promising: (i) Effective protection was observed in tomato plants and fruits against Botrytis cinerea, the fungus causing gray mold, which leads to significant economic losses worldwide. (ii) AFPs protected rice seeds from infections by Fusarium proliferatum, a fungus associated with Bakanae disease and mycotoxin contamination. (iii) Their efficacy was also demonstrated in rice leaves against Magnaporthe oryzae, the causal agent of rice blast disease, one of the most serious threats to this crop.

These results suggest that AFPs could be widely used as natural fungicides in crop protection, including both pre-harvest and post-harvest applications. The transient expression system in plants is fast, cost-effective, and scalable, making it an attractive platform for producing these “green biofungicides”. Although further field studies and ecological impact assessments will be needed, this breakthrough represents a crucial step towards a more sustainable agriculture.

*All co-authors from CRAG and IATA dedicate this final publication to the memory of our dear Maria Coca, who died in 2021, honouring her role in the project design, supervision of the experiments, and drafting of the initial version.

Reference Article

Bugeda A, Shi X, Castillo L, Marcos JF, Manzanares P, López-Moya JJ, Coca M. High yield production of the antifungal proteins PeAfpA and PdAfpB by vacuole targeting in a TMV-based expression vector. Plant Biotechnol Journal. 2025 May 3. https://doi.org/10.1111/pbi.70093

About the authors and funding of the study

The research was funded by multiple grants from MICIU/AEI/10.13039/501100011033 (RTI2018-101115B-C22 to MC, RTI2018-101115B-C21 and PID2021-125858OB-100 to JFM and PM), and through the Severo Ochoa Program for Centres of Excellence (CEX2019-000902-S to CRAG and CEX2021-001189-S to IATA). Additional funds were provided by the CERCA Programme of the Generalitat de Catalunya.