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bioRxiv · 10.1101/2020.12.03.411132

Phytotoxic tryptoquialanines produced in vivo by Penicillium digitatum are exported in extracellular vesicles

Costa, J. H.·Bazioli, J. M.·Barbosa, L. D.·dos Santos Junior, P. L. T.·Reis, F.·Klimeck, T.·Crnkovic, C. M.·Berlinck, R. G. S.·Sussulini, A.·Rodrigues, M. L.·Fill, T. P.
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Abstract

Penicillium digitatum is the most aggressive pathogen of citrus fruits. Tryptoquialanines are major indole alkaloids produced by P. digitatum. It is unknown if tryptoquialanines are involved in the damage of citrus fruits caused by P. digitatum. To investigate the pathogenic roles of tryptoquialanines, we initially asked if tryptoquialanines could affect the germination of Citrus sinensis seeds. Exposure of the citrus seeds to tryptoquialanine A resulted in a complete inhibition of germination and an altered metabolic response. Since this phytotoxic effect requires the extracellular export of tryptoquialanine A, we investigated the mechanisms of extracellular delivery of this alkaloid in P. digitatum. We detected extracellular vesicles (EVs) released by P. digitatum both in culture and during infection of citrus fruits. Compositional analysis of EVs produced during infection revealed the presence of a complex cargo, which included tryptoquialanines and the mycotoxin fungisporin. The EVs also presented phytotoxicity activity in vitro, and caused damage to the tissues of citrus seeds. Through molecular networking, it was observed that the metabolites present in the P. digitatum EVs are produced in all of its possible hosts. Our results reveal a novel phytopathogenic role of P. digitatum EVs and tryptoquialanine A, implying that this alkaloid is exported in EVs during plant infection. IMPORTANCEDuring the post-harvest period, citrus fruits can be affected by phytopathogens such as Penicillium digitatum, which causes the green mold disease and is responsible for up to 90 % of the total citrus losses. Chemical fungicides are widely used to prevent the green mold disease, leading to concerns about environmental and health risks. To develop safer alternatives to control phytopathogens, it is necessary to understand the molecular basis of infection during the host-pathogen interaction. In the P. digitatum model, the virulence strategies are poorly known. Here, we describe the production of phytotoxic extracellular vesicles (EVs) by P. digitatum during the infection of citrus fruits. We also characterized the secondary metabolites in the cargo of EVs and found in this set of molecules an inhibitor of seed germination. Since EVs and secondary metabolites have been related to virulence mechanisms in other host-pathogen interactions, our data are important for the comprehension of how P. digitatum causes damage to its primary hosts.

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Costa, J. H., Bazioli, J. M., Barbosa, L. D., dos Santos Junior, P. L. T., Reis, F., Klimeck, T., Crnkovic, C. M., Berlinck, R. G. S., Sussulini, A., Rodrigues, M. L., Fill, T. P.. 2020-12-06. Phytotoxic tryptoquialanines produced in vivo by Penicillium digitatum are exported in extracellular vesicles. https://doi.org/10.1101/2020.12.03.411132

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