Characterization and proteome analysis of the extracellular vesicles of Phytophthora capsici
Introduction
Extracellular vesicles (EVs) are a group of membranous structures that are released from the living cells and function in the extracellular space. The secretion of EVs is an evolutionarily conserved bioprocess present in the majority of prokaryotes and all eukaryotes [1]. Generally, EVs are classified into three types according to their biogenesis and size range: exosomes, microvesicles, and apoptotic bodies. Exosomes originate from the cell, and their size range is 30–100 nm. Microvesicles, whose size range is 100–1000 nm, are generated by shedding from the plasma membrane. Apoptotic bodies originate from the plasma membrane during the late stage of programmed cell death, and they are larger than 1000 nm [2]. EVs can carry many bioactive molecules, including enzymes, sterols, phospholipids, polysaccharide pigments [3], and nucleic acids [4]. They have important functions in intercellular communication and pathogenesis.
Studies of EVs inclusions have revealed that there are many virulence factors and immunity elicitors. For example, in bacteria, EVs are very important to their physiology and pathogenicity and can increase the resistance of bacteria to antibiotics and phages [5]. In Leishmania spp., EVs are an important means of protein delivery from parasite to the host cells, and EVs can elicit the immunity of the host cell by inducing interleukin-8 (IL-8) [6]. In the fungi Cryptococcus neoformans, EVs can transfer the important virulence factor urease, which can affect the invasion and pathogenesis of the infected host cell [7]. In previous studies, EVs are generally considered biomarkers of some diseases and are used for diagnosis [8]. Some studies have provided evidence that EVs play a role during host–pathogen interaction in activities including immunomodulator presentation [9], immune activation [10], infection promotion [11], and intercellular communication [12].
The study of the function of EVs in fungal and plant interactions is still in its infancy. The majority of fungus EV research is of pathogenic fungi in humans. For example, an investigation of the proteome of Paracoccidioides brasiliensis EVs identified a total of 205 proteins. The GO functional annotation analysis of EV proteins involved in diverse processes such as translation, metabolism, signaling transport, as well as stress response [13]. Research in Candida albicans EVs found 75 proteins, and the no singnalpeptied multifunction proteins that related exocytosis and endocytosis processes were specific carriers in the C. albicans EVs [14]. Studies on plant vesicle proteomics have found that it can transport immune-related proteins and participate in the process of plant and pathogen interaction. For example, in the proteome of Arabidopsis thaliana EVs, researchers found many stress response proteins and defensive related proteins such as syntaxin AtSYP121/PENETRATION1 (PEN1), which is responsible for producing papillary callose and prevent the invasion of phytopathogens [15]. Previous research also observed EVs surrounding the infection site of plants by using fluorescent marked tetraspanins genes TET8 and TET9 in A. thaliana [16], proving EVs play a vital role in the interaction of plants and pathogens.
The characteristics and biological function of the EVs from the oomycete pathogen Phytophthora capsici have not been thus far investigated. P. capsici is a kind of filamentous oomycete pathogen that can cause root, crown, foliar, and fruit rot on multiple important vegetables [17]. It has a broad host range and can infect over 50 species of cultivated plants and cause significant economic loss [18,19]. To successfully infect host tissue, P. capsici enables its survival and dissemination by releasing an array of effectors to modulate the host immune responses. EVs are an important alternative mechanism of protein delivery and have functions during pathogen–host interaction [20,21].
In the current study, we first time isolated the EVs from the oomycete pathogen P. capsici. Observing EVs through transmission electron microscopy (TEM) and scanning electron microscopy (SEM), EVs are round lipid bilayered structures of similar size. Results of the inoculation assay indicate that EVs are bioactive with some materials (not simply cell structure components) as mixing EVs with zoospores increased the pathogenicity of the pathogen. Detection by liquid chromatography in tandem with mass spectrometry (LC-MS/MS) technology, over 200 proteins were identified. GO analysis indicated the major functions of these proteins include metabolism, translation, oxidation/reduction, and transport. Many proteins are considered moonlighting proteins, which not only participate in metabolism but also could alter the morphology of the pathogen. Notably, three elicitors and one apoplastic effector were found in the EVs.
Section snippets
Strain and culture conditions
The sequencing strain of P. capsici LT1534 was first cultivated on V8 juice plates (10% V8 juice, 1.4 g CaCO3, 15 g agar, and adding deionized water to 1 L) and grown for 3 d at 25 °C. Then 15 plugs of 5 mm diameter of mycelium were cultured in 1 L 20% potato dextrose broth (40 g slices of potato, 18 g glucose, and adding deionized water to 1 L) culturing in a shaking incubator (121 rpm) at 25 °C for 3 d. Eight surface-sterilized chili leaves were added to the culture and incubated for another
Characterization of EVs purified from P. capsici culture supernatant
After the co-culturing of P. capsici and leaves of Capsicum annuum L. for four days, cell components were removed from the culture supernatant. EVs released from P. capsici were subsequently obtained through 100,000 g ultracentrifugation. TEM observation of the EVs revealed the morphology of EVs from P. capsici is similar to the EVs described in mammalian cells. They were spherical and possess a lipid bilayer and a cup-like shaped morphology (Fig. 1 A–B, arrows). A total of 298 EV diameters were
Discussion
Phytophthora capsici is one of the top ten plant-pathogenic oomycetes and a significant threat to crop production [22]. It is a hemibiotrophic pathogen that secretes many bioactive molecules during the infection process. Effector proteins are important plant–pathogen interaction materials that could promote infection through suppression of the immune response and manipulation of host processes. Revealing the secretion mechanism of these effectors is of critical importance for the study of
Conclusions
First extraction EVs from the important oomycete pathogen Phytophthora capsici. The morphology of EVs from P. capsici is similar to the EVs described in mammalian cells and other fungal pathogens, they were spherical and possess a lipid bilayer. Preliminary data showed EVs were able to enhance the pathogenicity of P. capsici. Proteomics results also revealed a variety of pathogenic-related proteins were exist in EVs, like oxidation/reduction-related proteins, stress response proteins as well as
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
This work was funded by the National Key Research and Development Program of China (2017YFD0200501) and National Science Foundation of China (31730075).
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