Phytotoxic Tryptoquialanines Produced In Vivo by Penicillium digitatum Are 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. IMPORTANCE During the postharvest period, citrus fruits can be affected by phytopathogens such as Penicillium digitatum, which causes green mold disease and is responsible for up to 90% of total citrus losses. Chemical fungicides are widely used to prevent 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.

Small Molecule Analysis of Extracellular Vesicles Produced by Cryptococcus gattii: Identification of a Tripeptide Controlling Cryptococcal Infection in an Invertebrate Host Model

“…Noteworthy, in the H. capsulatum study, the conditions used for small molecule identification differed from those used in our study. Under similar conditions, we identified a comparable number of molecules in the EVs produced by the plant pathogen P. digitatum (11) and in C. gattii (this study). Specifically, small peptides were found in the EVs of these two distant species, reinforcing the notion that this molecular class is present in different fungal EVs.…”

“…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.…”

“…Fungal toxins were also identified in P. digitatum, but this class of molecules is not normally produced by members the Cryptococcus genus. In 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. Two recent studies have 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).…”