Phytotoxic tryptoquialanines producedin vivobyPenicillium digitatumare exported in extracellular vesicles

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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“…So far, tryptoquialanine A is the only low mass component of fungal EVs with a reported function. In this model, the mycotoxin fungisporin was also detected (11), but its function in fungal EVs remains to be determined. Together, these findings illustrate the need for an improved knowledge of the composition and functions of EV metabolites in fungi.…”

“…In H. capsulatum , carbohydrate metabolites were abundantly detected in EVs, in addition to L-ornithine and ethanolamine, among other small molecules (10). In the plant pathogen P. digitatum , EVs were characterized as the carriers of tryptoquialanine A, a toxin that inhibited the germination of orange seeds (11). So far, tryptoquialanine A is the only low mass component of fungal EVs with a reported function.…”

“…Molecules of low molecular mass, however, have been overlooked. A recent study has characterized the small molecule composition of Histoplasma capsulatum (10) and Penicillium digitatum EVs (11), but the low molecular mass components of other fungal EVs are unknown. Considering the molecular diversity found in both H. capsulatum and P. digitatum , it is plausible to predict that many still unknown functions of EV components of low molecular mass remain to be characterized.…”

“…Considering the molecular diversity found in both H. capsulatum and P. digitatum , it is plausible to predict that many still unknown functions of EV components of low molecular mass remain to be characterized. In fact, the metabolome analysis of P. digitatum EVs revealed the presence of phytopathogenic molecules that inhibited the germination of the plant host’s seeds (11).…”

“…The copyright holder for this this version posted January 18, 2021. ; https://doi.org/10.1101/2021.01. 18.427068 doi: bioRxiv preprint the metabolome analysis of P. digitatum EVs revealed the presence of phytopathogenic molecules that inhibited the germination of the plant host's seeds (11).…”