The endlessly growing hyphae of filamentous fungi, are among the most polarized cells in nature. The continuous flow of secretory vesicles, from the hyphal cell body to the growing hyphal tip, is essential for CW and membrane extension (Fischer et al 2008). The molecular mechanisms underlying protein secretion pathways, including exocytosis, have been intensively studied in filamentous fungi because of their ability to secrete abundant proteins (Higuchi 2021). Many features of CW biosynthesis are unique to fungi, and are therefore considered excellent targets for the development of antifungal drugs (Nimrichter et al. 2016).
Most proteins are released from the cell into the vesicular compartments, that pass through the CW and reach the extracellular environment. In this respect, EVs act as molecular carriers, which are part of a conserved secretion mechanism shared by all domains of life. However, little is known about the assembly, biogenesis, and immunobiological functions of EVs (Rizzo et al 2020a). When compared to yeast, the EVs in filamentous fungi are poorly understood, although they have been described in several species, including Trichophyton interdigitale (a causative agent of superficial mycosis in humans), Rhizopus delemar (an agent of mucormycosis in immunocompromised individuals), Aspergillus fumigatus and Aspergillus flavus (toxin-producing agents and the most common causes of invasive aspergillosis) (Bleackley et al. 2020; Bitencourt et al., 2018; Liu et al. 2018; Silva et al. 2014). Regarding the genus Trichoderma, de Paula et al. (2019) first described the isolation and characterization of EVs from Trichoderma reesei, a fungus associated with lignocellulose degradation. Intriguingly, they demonstrated that, when compared to the repressive or neutral carbon sources, glucose or glycerol, the growth of T. reesei in the presence of cellulose generated a higher number of EVs. This is particularly interesting because regardless of the fungal lifestyle, specific elicitors may stimulate the flow of EVs and the transport of substances through the mycelium, mainly to the tip of the hyphae. In the present study, we have shown a similar behavior, where the presence of a potential prey, in this case, the phytopathogen S. slcerotiorum, led to an intense flow of EVs, accelerating and polarizing mycelial growth, simultaneously along with the production, structuring, and remodeling of CW in T. harzianum under conditions of direct confrontation with the host. These findings are consistent with previous descriptions of the functions of CP-type proteins abundant in the secretome during hyphal growth, mycelial mass formation, sporulation, and spore maturation. They are also crucial for fungal growth, development, recognition, adhesion, morphogenesis, remodeling, and cell wall enlargement during hyphal growth and chlamydospore formation (Gomes et al. 2015; Baccelli et al. 2014; Friscmann et al 2013; Yu et al. 2012; Pazzagli et al. 1999). Epl-1, in particular, has been previously described as a plant resistance elicitor (triggering the expression of defense-related genes) (Gomes et al. 2017), affecting the mycoparasitism process (e.g. defective host mycoparasitic coilling process and altered expression patterns of mycoparasitism genes) and self-CW protection and recognition (Gomes et al. 2015).
Changes in the environment, including nutrient availability, pH, and temperature, are associated with CW remodeling. Ene et al. demonstrated a substantial CW transformation after 30 s in response to hyperosmotic stress (Ene et al. 2015). Many enzymes target specific sites on the surface of fungal cells, thereby fine-tuning remodelling and preventing cellular damage. As these rearrangements imply high diversity in the molecular composition of the cell surface, they must have a direct impact on the recognition and/or interaction between fungal pathogens and their hosts (Higuchi 2021). Analysis of isolated CWs indicates that this structure is semi-permeable with a defined permeability. The porosity of fungal CWs may vary depending on the study and the method used. For instance, atomic force microscopy has suggested that the CW of Saccharomyces cerevisiae has pores of approximately 200 nm, which can increase to 400 nm under stress conditions. Electron microscopy analysis showed vesicle-like systems in the fungal CW, suggesting that structures may temporarily occupy CW spaces (Casadevall et al. 2009; Rodrigues et al. 2007; Pereira and Geibel 1999). In this regard, the present study has shown that after the hyphae appear to be fully formed, the flow of EVs in the periplasmic space decreases considerably to such an extent that it was no longer possible to visualize them using fluorescence optical microscopy. Simultaneously, fission lines appeared in the T. harzianum CW structure. As a result, the CW region between the cleavage lines collapses, sediments, reorganizes, remodels, and assumes its final structural configuration. Once CW sedimentation began, it extended along the entire length of the hyphae in an uncoordinated manner. Assuming that this event is a fact, we can speculate that because of the large amount of Epl-1 protein present in the CW region (evidenced by the high level of fluorescence in this specific region) and the fact that Epl-1 has a greater weight owing to the presence of GFP, the CW has consequently collapsed and reorganized itself in a manner closer to the plasma membrane. Previous analyses have shown that the presence of GFP does not effectively affect the function and lifestyle of the fungus, and T. harzianum RecEpl-1-GFP regained all of the characteristics observed in the wild-type strain (Gomes et al. 2017, 2015).
Detailed studies on the effects of EV traffic and the importance of correct delivery and distribution of their cargo are essential, as evidence has suggested that certain biomolecules can affect the interaction between fungi and their respective hosts. Therefore, in addition to packaging and loading cytosolic proteins, lipids, polysaccharides, toxins, allergens, and pigments, recent evidence has shown that messenger RNA (mRNA), small RNA (sRNA), non-coding RNA (ncRNA), and RNA-binding proteins are packaged in EVs, suggesting a critical role in cross-kingdom communication. EV-mediated RNA export has been discussed as a universal mechanism for inter-kingdom and intra-kingdom communication, and it is reasonable to suggest that RNAs transported by fungal EVs may play an important role in cell communication (Alves et al. 2019; de Paula et al. 2019; Bielska et al. 2018; Cai et al. 2018; Rodrigues and Casadevall 2018; Tsatsaronis et al. 2018). Several delivered sRNAs have been shown to reduce fungal virulence by targeting the genes normally involved in infection (Dallastella et al. 2023; Rizzo et al. 2020a; Cai et al. 2018; Rodrigues and Casadevall 2018; Zamith et al. 2018). Thus, these results support the idea that fungal cell walls are permissive for the internalization of intact bioactive EVs. EVs loaded with molecules capable of neutralizing virulence genes are expected to have great therapeutic potential. The revelation that fungal EVs can deliver sRNAs to reduce virulence by targeting specific genes presents a compelling parallel for clinical applications in human health. This mechanism could be harnessed to develop innovative therapies in which human-derived EVs are engineered to carry similar sRNAs or other therapeutic molecules, aiming to target and neutralize virulence genes in pathogenic microbes or even in malignant human cells. The ability of EVs to traverse complex cell walls and deliver intact bioactive molecules underscores their potential as precise and minimally invasive vehicles for drug delivery. This approach could revolutionize the treatment of a variety of diseases, offering a highly targeted and efficient method for mitigating pathogenicity and enhancing patient outcomes in clinical settings (Kim and Kim 2023; Lucena et al. 2023; Alves et al. 2019; Bielska et al. 2018; Polakovicova et al. 2018).
In the future, unraveling the complexities of EVs in filamentous fungi has promising implications for environmental and clinical biology. Understanding EV-mediated RNA export and its role in cell communication, will pave the way for innovative therapeutic strategies and potentially harness these mechanisms for disease control and drug delivery systems. The specificity of fungal EV cargoes and their pathways, opens a new frontier in biotechnology and medicine, offering exciting prospects for targeted treatments and a deeper understanding of fungal biology and its implications for health and disease. The environmental implications of fungal EVs, extend beyond their clinical applications, highlighting their significance in ecosystem dynamics and bioremediation. Fungal EVs play crucial roles in soil health, plant growth, and also organic matter decomposition by mediating cellular communication and nutrient exchanges. Their roles in cross-kingdom interactions, particularly in symbiotic and pathogenic relationships, influence biodiversity and ecosystem resilience. Harnessing the potential of EVs in environmental management could revolutionize sustainable agricultural practices, enhance biodegradation processes, and offer novel approaches to combat climate change by maintaining soil health and fostering resilient plant communities. The intricate relationship between fungi, their EVs, and the environment embodies a complex web of interactions, and is a promising fertile ground for future ecological studies and biotechnological innovations.