EVs and plant-host interactions – the fungal pathogen viewpoint
The first report of EVs in a plant fungal pathogen came from the powdery mildew fungus Blumeria graminis (Hippe, 1985; Hippe-Sanwald et al., 1992). However, their earliest description was in 2007 fromC. neoformans (Rodrigues et al., 2007; Samuel et al., 2015; Rodrigues et al., 2008a; Zhao et al., 2019). Nonetheless, the mechanism behind the production of EVs from the fungal cell wall is not well understood, although several mechanisms have been suggested (e.g., via passage channels, turgor pressure, cell-wall degrading enzymes, and viscoelasticity of the cell wall) (Wolf and Casadevall, 2014; Brown et al., 2015; Kuipers et al., 2018). For instance, a recent study onS. cerevisiae showed that EVs may contain enzymes and cell wall-related proteins believed to take part in cell wall remodelling, therefore, aiding their transition across the fungal cell wall (Zhao et al., 2019).
Whilst a shortfall of protocols and specific biomarkers for EV isolation continues to be a challenge in the field of EV biology, particularly for filamentous fungi, numerous studies on fungal EV production remain biased towards human fungal yeast pathogens (Albuquerque et al., 2008; Gehrmann et al., 2011; Vallejo et al., 2011; Vallejo et al., 2012; Vargas et al., 2015; Leone et al., 2017; Bielska et al., 2018; Ikeda et al., 2018; Peres da Silva et al., 2019; Lavrin et al., 2020). Consequently, very little is known about the release of EVs in plant pathogenic fungi, although a large number of studies have confirmed their presence in these organisms (Silva et al., 2014; Bitencourt et al., 2018; Liu et al., 2018; de Paula et al., 2019; Souza et al., 2019; Bleackley et al., 2020; Brauer et al., 2020; Rizzo et al., 2020; Costa et al., 2021; Garcia-Ceron et al., 2021). However, the production of EVs in fungi is considered unconventional because they mostly transport proteins lacking signal peptides (Samuel et al., 2015). For example,B. graminis secrete avirulence proteins (i.e., AVRa10 and AVRk1) that are ‘leaderless’ (Samuel et al., 2015; Ridout et al., 2006). Mammalian studies have, however, reported the delivery of proteins with or without signal peptides by EVs, suggesting that this unconventional secretory pathway is a highly complex phenomenon (Samuel et al., 2015).
Fungal virulence is associated with the release of EVs that are involved in modulating acceptor cells. Thus, EVs are required for fungal pathogenesis due to their ability to modulate receipt cells to promote virulence (Rodrigues et al., 2007; Vargas et al., 2015; Joffe et al., 2016). In addition to their phytotoxic activity as mentioned previously,F. oxysporum f. sp. vasifectum EVs were found to comprise of virulent polyketide synthases and proteases etc. (Garcia-Ceron et al., 2021). Also, EVs in C. neoformans were found to contain virulence factors such as glucuronoxylomannan (GXM) and glucosylceramide (Rodrigues et al., 2007; Rodrigues et al., 2008a).
Several studies on plant pathogenic fungi including M. oryzae , and the oomycete pathogen Phytophthora infestans , revealed that the ESCRT pathway is utilized to export virulence-associated proteins via vesicles (Giraldo et al., 2013; Wang et al., 2017). The citrus fungal pathogen, P. digitatum , is reported to release EVs of myriad cargos containing mycotoxins and alkaloids (tryptoquialanine A) during infection (Costa et al., 2021). Moreover, B. cinerea EVs carry sRNAs which promote pathogen infection by silencing the plant immunity genes, suggesting that EVs mediate the delivery of fungal virulent factors into host plants (Rodrigues et al., 2008b; Weiberg et al., 2013; Wang et al., 2016, 2018). EVs from fungal species such asHistoplasma capsulatum and C. parapsilosis , have also been shown to contain effector proteins, which may play a role in modulating their host immune responses during host-pathogen interaction (Albuquerque et al., 2008; Vargas et al., 2015; Gil-Bona et al., 2015). Taken together, these findings suggest that the role of EVs as carriers of virulence factors and immunomodulators is universal.