Throughout evolution, plants and pathogenic fungi have been in a constant battle where
fungi have developed new mechanisms to infect plants while plants have co-evolved
to combat the infection. The early stages of plant-pathogen interactions occur in
the intercellular spaces of the plant tissue and thus involve a myriad of secreted
factors. Traditionally, all proteins released into the extracellular space were thought
to be transported via the ER-Golgi dependent classical secretory pathway. However,
non-classical secretion of proteins/RNA through extracellular vesicles (EVs) has recently
been reported to contribute to the milieu of extracellular molecules that mediate
plant-fungal interactions (Rodrigues et al., 2007; Meyer et al., 2009). EVs can be
broadly classified into exosomes and ectosomes (Keerthikumar et al., 2015). Exosomes
are secreted microvesicles (30–150 nm in diameter) of endocytic origin that are released
by multiple cell types and are conserved across various species (Lotvall et al., 2014;
Gangoda et al., 2015). In contrast, ectosomes or shedding microvesicles are larger
(100–1000 nm in diameter) and bud off directly from the plasma membrane (Keerthikumar
et al., 2015). For clarity, we will collectively refer to both types of membranous
vesicles as EVs in this article.
Recent studies on mammalian systems have highlighted the role of EVs in cell-cell
communication and the intercellular transport of cargo (proteins, nucleic acids, and
carbohydrates) (Batista et al., 2011; Cossetti et al., 2014). Whilst the role of EVs
in plant-fungal interactions is still poorly defined, this non-canonical secretory
pathway has been proposed as an alternative route for the secretion of virulence and
defense molecules by fungi and plants, respectively (Robatzek, 2007; Rodrigues et
al., 2011). The basic requirement for successful host colonization is the establishment
of a parasitic relationship between the fungal pathogen and the host. This requires
the induction of specific defense mechanisms in the fungus for protection against
the plant innate immune system (Hayes et al., 2013). Evasion or suppression of the
plant defense response is thought to be regulated by virulence factors that are secreted
from the fungus and act at the plasma membrane or in the cytoplasm of the plant cell
(Rodrigues et al., 2008a). Interestingly, recent studies allude to the EV-mediated
transport of virulence factors from the fungus into the host cell as a more efficacious
delivery mechanism than simple diffusion (Rodrigues et al., 2008a; Silverman and Reiner,
2011). Similarly, in plants, when the integrity of the cell wall is threatened by
a fungal pathogen, a response is mediated, at least in part, by multivesicular bodies
(MVBs) (An et al., 2006b). In mammalian cells, it is well documented that fusion of
MVBs with the plasma membrane results in the secretion of exosomes (Boukouris and
Mathivanan, 2015; Gangoda et al., 2015). Though the production of MVBs may not always
result in the secretion of EVs, the observation that plants produce MVBs in response
to a fungal infection leads to the speculation that EVs may play a critical role in
plant-fungal interactions. Here, we will discuss the current knowledge on EVs in the
context of human-fungal interactions and their potential roles in plant-fungal interactions.
Role of EVs in human-fungal interactions
Fungal EVs were first isolated from the human fungal pathogen Cryptococcus neoformans
(Rodrigues et al., 2007). These EVs contained well known virulence factors such as
the capsular polysaccharide glucuronoxylomannan (GXM) and the virulence regulator,
glucosylceramide (Rodrigues et al., 2008a). Rodrigues and colleagues also reported
the presence of several other pathogenicity-associated components that are delivered
into the host via EVs. Furthermore, the isolated EVs were biologically active as they
could invigorate phagocytes in the host and enhance their antimicrobial activity (Oliveira
et al., 2010a). Other mammalian fungal pathogens including Histoplasma capsulatum,
Candida parapsilosis, Sporothrix schenckii, and Candida albicans also deliver a variety
of effector molecules in a similar manner (Albuquerque et al., 2008; Vargas et al.,
2015; Gil-Bona et al., 2015b). Interestingly, the serum from patients with H. capsulatum
infections contains antibodies to proteins that are present in the EVs produced by
the pathogen indicating involvement of EVs in the host-pathogen interaction. Moreover,
characterization of EVs from the human pathogens C. neoformans, H. capsulatum and
Malassezia sympodialis has implicated them in the modulation of the host immune system
and regulation of the host-pathogen interaction in favor of the fungus (Rodrigues
et al., 2008b; Gehrmann et al., 2011).
Role of EVs in plant-fungal interactions
A major component of plant-fungal interactions is the secretion of small proteins
by both organisms. Plants produce pathogenesis related (PR) proteins, many of which
inhibit fungal growth or directly kill fungal cells (Sels et al., 2008). Fungi secrete
virulence factors encoded by the avirulence (AVR) genes (Stergiopoulos and De Wit,
2009; Rodrigues et al., 2014; Gil-Bona et al., 2015a). However, in spite of decades
of research, it is still unclear as to how these proteins cross the plasma membranes
and cell walls of both species. The AVR genes AVRa10 and AVRk1of the fungal pathogen
Blumeria graminis f. sp. hordei encode proteins that lack signal peptides. Despite
the lack of classical secretion signal, they still enter the cells in a susceptible
host plant and are required for the pathogenicity of the fungus (Ridout et al., 2006).
In mammalian systems, it is well established that certain proteins with and without
signal peptides are transported via EVs (Kalra et al., 2012; Simpson et al., 2012).
Hence, EVs could potentially mediate the transfer of these fungal virulence factors
into plant hosts (Rodrigues et al., 2008b). However, further studies are required
to understand this highly complex phenomenon.
The ability of a plant to mount a rapid defense response against potential pathogens
is vital to its survival. Intercellular organelle rearrangements and structural modulation
of the cytoskeleton with increased focal secretion of compounds lead to the formation
of a physical barrier at the attack site that might prevent successful infection (Frey
and Robatzek, 2009). These modifications may involve rapid and targeted delivery of
molecules via EVs. Fungal infection enhances the formation of both intracellular MVBs
and paramural vesicles between the plasma membrane and cell wall in the plant cells
indicating the critical role of this secretory pathway in plant innate immune response
(An et al., 2006a; Wang et al., 2014). For example, vesicular structures were associated
with the accumulation of phenolic compounds and H2O2 that prevented pathogenic establishment
of the powdery mildew fungus B. graminis in barley (Hordeum vulgare) leaves. Though
the detection of plant MVBs at the site of infection provides indirect evidence of
their role in plant defense (An et al., 2006a), more work is needed to define their
molecular composition and whether they do indeed transport innate immunity proteins
to the site of infection and/or into the fungal cell.
MVBs were first reported in the appressoria and haustoria of the powdery mildew fungus
B. graminis (Hippe, 1985; Hippe-Sanwald et al., 1992). More recently, microscopic
examination confirmed the presence of membrane bound vesicles at the biotrophic interface
between B. graminis and the host plant (Micali et al., 2011). Furthermore, the haustorial
complexes produced by Golovinomyces orontii in infected Arabidopsis leaves have vesicles
and MVBs in the haustorial body (cytoplasm), paramural space, and extrahaustorial
matrix. In addition, vesicle budding and fusion of MVB-like structures with the fungal
plasma membrane has been observed in this interaction, although the microscopic evidence
did not reveal whether the vesicles were derived from the plant or the fungus (Micali
et al., 2011). In other studies, EVs from fungal pathogen Paracoccidioides brasiliensis
and H. capsulatum were reported to transport antioxidants (superoxide dismutase and
catalase B) and heat shock proteins (Hsp60 and Hsp70) which may have an essential
role in the fungal defense mechanism (Albuquerque et al., 2008; Vallejo et al., 2012).
Only recently, Hsp60 was also reported in the proteome of EVs from the fungus Alternaria
infectoria. Several species of this genus Alternaria are also considered major plant
pathogens (Silva et al., 2014).
Biochemical analyses of EVs from various human fungal pathogens has revealed the presence
of a variety of lipids, proteins and RNA (Peres Da Silva et al., 2015). Although observed
by microscopy, EVs have not been isolated and characterized from a plant pathogenic
fungus. Nevertheless, upon uptake by the plant cell, it is possible that the contents
could modulate the plant response to the invading fungal pathogen by attenuating the
immune response. Similarly, plant exosomes are poorly characterized and their composition
is largely unknown. However, it is plausible that some plant exosomes, particularly
those produced in response to fungal threat, might contain small molecules and proteins
that are toxic to the fungus.
Finally, cell wall remodeling is a key process on both sides of the plant-fungus interaction
(Bellincampi et al., 2014). Proteomics studies highlighted the presence of various
enzymes (Endochitinase 1 precursor, Beta-glucosidase 4, Beta-1,3-glucanosyltransferase
3, and Chitin synthase B) in EVs secreted by H. capsulatum. Similarly, S. cerevisiae
secreted more than 20 proteins implicated in cell wall assembly including glucanases
and glucanosyl transferases (Oliveira et al., 2010b). These enzymes have the capacity
to regulate synthesis and hydrolysis of cell wall components highlighting the potential
role of EVs in cell wall remodeling (Albuquerque et al., 2008). Fungal cell wall synthesis
is also known to be mediated by chitosomes, small vesicles containing chitin microfibrils,
in Neurospora crassa (Riquelme et al., 2007). Chitosomes follow an unconventional
secretory pathway to transport various components of the chitin synthase family required
for fungal cell wall synthesis. Whilst the differences between chitosomes and EVs
are not clearly understood, it can be speculated that EVs can also play a pivotal
role in fungal cell wall remodeling. In plants, reinforcement of the cell wall is
one of the major strategies of the host to restrain further invasion by the pathogen
(Lionetti and Métraux, 2014). The delivery of the cell wall carbohydrates to the extending
chains of insoluble polysaccharides that make up majority of the cell wall is relatively
poorly understood. The role of EVs in cell wall remodeling in both the fungus and
plant is understudied and needs further research.
Conclusion
Recent findings pertaining to the role of EVs in the interaction between fungal pathogens
and humans have led us to ask whether EVs also have a major role in plant pathogen
interactions. It is still unknown how effectors and defense molecules are packaged
and transported across the plasma membranes and cell walls of the plant and fungal
cells. We propose that proteins lacking secretion signals could be packaged into EVs
for passage through the plasma membrane and the cell wall (Figure 1). Alternatively,
proteins containing a secretion signal could be secreted into the matrix of the cell
wall and then bind to EVs via a lipid binding motif. The protein then transits the
cell wall as a passenger on the outer leaflet of the vesicle. Based on the discovery
that EVs aid disease progression (Boukouris and Mathivanan, 2015; Gangoda et al.,
2015), we propose that EVs can mediate/aid in fungal infection. This could be achieved
via the transfer of effectors via EVs and/or by modulating the host cells response
in favor of the fungal pathogen. Similarly, we propose that plant EVs can aid in the
protection against pathogenic infections. Upon infection, defense molecules can be
packaged and delivered to the site of infection to protect against the invading pathogen.
Further to this, we propose that the molecular cargo present in EVs is specific to
the type of insult or infection. For instance, molecular cargo present in EVs of plants
during stress can be significantly different to that produced during fungal infection.
Thus the molecular cargo contained within EVs of plant or fungus can serve as indicators
of health, stress, and disease. Investigation of the role of EVs in plant-fungal interactions
is likely to uncover a new mechanism for delivery and identification of molecules
required for a productive infection and/or defense response. This knowledge will enhance
our ability to protect agricultural crops against the damaging effects of fungal pathogens
and securing our food sources for generations to come.
Figure 1
Schematic representation of putative crosstalk via EVs at the plant-fungal interface.
Exosome biogenesis and secretion in the fungal side: MVBs are formed from the early
endosomes. Within the MVBs, invagination of the limiting membrane results in the formation
of intraluminal vesicles which are packaged with protein and RNA cargo from the cell.
The MVBs either fuse with the plasma membrane or with the lysosome for degradation.
When the MVBs fuse with the plasma membrane, the intraluminal vesicles are released
as exosomes. The exosomes are considered to contain various molecules including effectors
that are required for the establishment of the pathogen and/or infection. Exosome
biogenesis and secretion in the plant side: Similarly, on the plant side, vesicles
from the MVBs may contain innate immunity proteins and defense molecules that can
impede fungal growth or lead to alterations in the fungal cell wall. Thus, the plant
and its fungal counterpart could utilize the exosomes as one of the many strategies
in their mutual struggle for survival.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.