Introduction
Extracellular vesicles (EVs) mediate cell-cell-dependent environmental responses (Rodrigues et al., 2015; Anand et al., 2019, Baldrich et al., 2019). Numerous studies have isolated and characterized fungal pathogen EVs and found that they produce phytotoxic effects, among other things, when inoculated on plant tissues such as leaves (Bleackley et al., 2020). This suggests that cargo within these vesicles can be disease-promoting. How this cargo is sorted is currently elusive in plant pathogenic fungi, which limits our understanding of virulence factors transmitted through non-canonical pathways. A repertoire of EV constituents, including nucleic acids, proteins, and secondary metabolites often reflect the pathophysiological state of the cell from which EVs are secreted. These cargos can be taken up by other cells naked or enclosed in the EV membrane to yield the different EV-mediated physiological states in the acceptor cells (Koch et al., 2020). As EVs carry various bioactive molecules, many of which facilitate cell-cell communication (Rodrigues et al., 2015; Anand et al., 2019), they have also been implicated in plant-fungal interaction and pathogenesis. For instance, EVs of several species of fungi have been shown to traffic virulence factors, such as cell wall degrading enzymes, protein effectors, and toxins with phytotoxic effects on their plant host tissues (Bleackley et al., 2020; Costa et al., 2021; Regente et al., 2017). However, there is still a lack of understanding about the regulatory mechanisms involved in cargo sorting, packaging and trafficking of the respective vesicles, especially in filamentous fungi.
The ESCRT (e ndosomal s orting c omplexr equired for t ransport) pathway is utilized by many eukaryotes including fungi whereby it is involved in the sorting and trafficking of molecules within the endosomal system. This system is involved in the formation and sorting of endosomal vesicles, which are part of the endocytic pathway responsible for internalizing molecules from the cell surface and delivering them to specific destinations within the cell. In filamentous fungi, the ESCRT pathway plays a role in the biogenesis of filamentous growth and pathogenesis (Zheng et al., 2018; Sun et al., 2022). Studies have shown that the ESCRT pathway is required for the conventional organization of the fungal cytoskeleton (An et al., 2006; Henne et al., 2013), which is essential for the formation of hyphae. The ESCRT pathway also plays a role in the splitting of daughter nuclei during cell division and the sorting of proteins destined for the plasma membrane (Henne et al., 2013). Additionally, the ESCRT pathway is involved in the formation of endosomal vesicles that are important for nutrient uptake and stress response (Fan et al., 2015; Wang et al., 2020). In pathogenic fungi, the ESCRT pathway also plays a role in the secretion of virulence factors and evasion of host immunity (Regente et al., 2017; Martínez-López et al., 2022).
The ESCRT pathway is vital for endocytosis whereby it allows for the packaging of extracellular materials and membrane proteins into EVs for trafficking into the cytoplasm (Fig 1(A) , Ahmed et al., 2019). Endocytosis is characterized by the presence of late endosomes or multivesicular bodies (MVBs), which harbour intraluminal vesicles (ILVs) that usually arise from the invagination and budding of the endosomal membrane (Haag et al., 2015; Ahmed et al., 2019). The process of endocytosis is fundamental to various cellular process ranging from signal transduction to morphogenesis (Schimid et al., 2014). In certain fungal species, endocytosis also plays a role during interaction with plants including in the apical growth of hyphae (Toshima et al., 2006; Bielska et al., 2014). In addition to growth and development, ESRCT-based regulation of cellular function also appears to be crucial for adaptation and response to both external and internal stimuli such as biotic and abiotic stress factors (Mosesso et al., 2019; Rosa et al., 2020). Consequently, many of the basic components of the ESCRT pathway are conserved across eukaryotes, albeit with notable lineage-specific adaptations in some taxa (Leung et al., 20008).
Most of the significant contributions to our understanding of ESCRT derive strongly from studies on model organisms (Thompson et al., 2005; Vaccari & Bilder, 2005; Herz et al., 2006; Spitzer et al., 2006). Adding to these are several studies highlighting the key roles of ESCRT proteins in mammals (Pornillos et al., 2002; Xu et al., 2003; Skibinski et al., 2005; Parkinson et al., 2006; Saksena and Emr, 2009; Stuffers et al., 2009; Hurley et al., 2015). The ESRCT pathway was originally discovered in Archaeal cytokinesis (Lindas et al., 2008; Samson et al., 2008) and in fungi it was first discovered in the model fungusSaccharomyces cerevisiae (Emr et al., 2001). In this yeast, the ESCRT pathway’s constituents were named based on their functions in sorting ubiquitinated membrane proteins into lysosome/vacuole lumens for degradation (Katzmann et al., 2001; Babst et al., 2002a; Xie et al., 2019b). The pathway represents a complex endomembrane system that consists of five complexes, namely ESCRT-0, -I, -II -III and Vps4 (v acuolar p rotein s orting 4 ). Together with various accessory proteins, the individual elements of the pathway act in concert to form MVBs during diverse processes including cytokinesis, membrane repair and autophagy (Roxrud et al., 2010; Henne et al., 2013; Hurley et al., 2015).
As in other eukaryotes, fungal MVBs are critical for transporting ubiquitinated membrane proteins to the vacuole with the aid of the ESCRT machine, which both recognizes and packages these ubiquitin-modified proteins into the ILVs contained inside MVBs (An et al., 2006; Hurley and Hanson, 2010; Henne et al., 2011). Studies in yeast also showed that ILVs form when MVB membranes evaginate and undergo fission (Ahmed et al., 2019; Anand et al., 2019). Others have reported that impairment in the ESCRT apparatus can lead to reduced formation of ILVs and aberrant endosomal compartments (Raymond et al., 1992; Xie et al, 2019a). In the filamentous fungal pathogen, Fusarium graminearum , such defects can significantly impact cellular processes like deoxynivalenol production, growth and pathogenicity, as well as sexual and asexual reproduction (Xie et al. 2019a). However, details regarding this pathway have been considered in only a few phytopathogens, most notablyMagnaporthe oryzae and F. graminearum (Oh et al., 2012; Xie et al., 2016; Cheng et al., 2018; Xie et al., 2019a, b; Que et al., 2019). Currently, most fungal work pertaining to the ESCRT pathway is focused on human pathogenic yeasts including Cryptococcus neoformans and Candida albicans (Godinho et al., 2014; Hu et al., 2015; Zhang et al., 2015; Park et al., 2020). The elucidation of the core functions of the ESCRT pathway in fungal plant pathogens represents a critical aspect given the functional importance of this pathway. Therefore, this review reflects on the recent ESCRT discoveries as they relate to filamentous fungi and touch on the relevance of released EVs, which are the end-products of ESCRT, in fungal biology and fungal-host interactions.