The features of the proteases secreted by phytopathogenic fungi and their role in virulence factors.

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Abstract

Phytopathogenic fungi are the causal agents of many of the world’s most serious plant diseases that can result in significant yield loss in a large-scale commercial agriculture production. They produce an array of various hydrolytic enzymes for nutrition and/or pathogenicity during penetration. Among hydrolases, proteases from phytopathogenic fungi are required for survival and growth of phytopathogenic fungi. Extracellular and/or secretary proteases are the subject of this mini-review. Several extracellular proteases have been identified with the role in fungal growth, the formation of infection structures, cell wall degradation, proteolytic processing of pathogenesis-related proteins and act as an elicitors. In addition, there is a positive correlation between the protease secretion and disease aggressiveness and/or necrosis. Recently, much attention has been focused on proteases that are required for pathogenicity. The involvement of various secretary fungal proteases in diverse pathological mechanisms makes them potential targets of protease inhibitors designing and may provide an improved way to combat plant diseases and reduce dependence on fungicides in near future research. Introduction

Phytopathogenic fungi are among the most serious pathogens inducing plant diseases that contribute substantially to the overall loss in yield. According to Kubicek et al., (2014) and Horbach et al., (2011), ~10% of all known fungal species can cause disease in more than 10,000 plant species. When pathogenic fungi, encounter a host, that produces various cell wall digesting enzymes such as glycanases and proteases to fragment the plant cell wall polymers, thus facilitating the penetration of the host cells (Knogge, 1998; Lebeda et al., 2001; Kubicek et al., 2014). It has been porposed that, apart from the role as a cell wall degrading enzyme, proteases synthesized by the plant pathogenic fungi may function as virulence factors during host-pathogen interactions. Moreover, plant cell walls possess several proteins, glycoproteins, and structural proteins and therefore the proteolytic enzymes have been suggested to play an important role in pathogenesis (Carpita and Gibeaut, 1993). However, most of the saprophytic fungi produce proteolytic enzymes for their nutritional requirements. Therefore, the type and level of protease production determine the saprophytic or pathogenic characteristics of fungi. The role (if any) of these enzymes in the process of pathogenesis has been the subject of intensive research. This review describes the different features of the proteases secreted by phytopathogenic fungi and their role in virulence factors.

Classifications of Proteases

Proteases are one of the most important industrial enzymes, which account for nearly 65% of the total worldwide enzyme sales (Rao et al., 1998; Kasana et al., 2011; Jisha et al., 2013). Protease (E.C 3.4) is a group of enzymes hydrolyzing peptide bonds [Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB)]. The classification of proteases is based on their process of hydrolysis (endopeptidases and exopeptidases) or pH (acidic, alkaline and neutral proteases) or functional group at the active site (aspartic, cysteine, glutamic, metallo, serine, threonine, and unknown (NC-IUBMB). Proteolytic enzymes are also grouped according to the MEROPS peptidase database into 12 clans and 35 families on the basis of amino acid sequence similarity and catalytic mechanism (Rawlings et al., 2011).

Role of proteases in phytopathogenic fungi

In general, phytopathogenic fungi secrete a broad spectrum of proteolytic enzymes during penetration and colonization of the host tissues (Table 1). Proteases from phytopathogenic fungi play an essential role in post-secretion protein processing, aerial mycelium formation, nutrition, adaptation to different environmental condition, and appressorium formation (Archer and Peberdy, 1997; St. Leger et al., 1997; Tucker and Talbot, 2001; Chandrasekaran and Sathiyabama, 2014).

Fungal proteases are thought to be important in different aspects of infection process, including adhesion to host cells, initial penetration of the plant cell wall and colonization (Movahedi and Heale, 1990; Paris and Lamattina, 1999; Carlile et al., 2000; Olivieri et al., 2004). Olivieri et al. (2002) demonstrated that in potato tubers, the pathogenic fungi Fusarium solani secretes 30 kDa size serine protease which has the ability to degrade pathogenesis related (PR) proteins, and cell wall protein. It has also been reported that (poly)peptides released upon the action of proteases secreated from the phytopathogenic fungi may act as elicitors, and thereby initiating plant protection reactions (Ryan and Pearce, 2003). Metalloprotease from the rice blast fungus Magnaporthe grisea has been shown to function as a gene-for-gene-specific (Pt-a gene against AV-Pt) nonvirulence factor by directly binding the plant resistance gene product and stimulates a signaling cascade which in turn leads to resistance (Jia et al., 2000). Genetic studies on a protease-deficient, a non-pathogenic mutant of the plant pathogen Pyrenopeziza brassicae revealed that genes conferring extracellular cysteine protease production and pathogenicity were identical or closely linked (Ball et al. 1991). In Cochliobolus carbonum disruption of the ALP1 gene, which encodes trypsin-like protease (TLP) activities, had no distinct effect on virulence (Murphy and Walton 1996). Proteolytic enzyme produced by phytopathogenic fungi in culture media may be involved with its phytopathogenicity. In addition, proteases increases the permeability of the plant plasma membrane which suggest that they may have role in plant pathogenesis (Tseng and Mount, 1974).

Protease production and phytopathogenenicity

Many species of phytopathogenic fungi produce different proteases in a variety of culture media and host tissues (Morita et al., 1994; Griffen et al., 1997). According to Zaferanloo et al. (2014), the optimum condition for protease production of Alternaria alternata was 37 ï‚°C and pH 7.0 with soybean as substrate, while 48 °C, pH 9.1 was ideal when casein used as substrate as suggested by Dunaevsky et al. (1996). The optimum condition for protease production in A. solani was 27 ï‚°C and pH 8.5 with casein as substrate has been previously reported (Chandrasekaran and Sathiyabama, 2014). Addition of heat stable potato tuber proteins to the culure medium is vital for the secretion of exoprotease from Rhizoctonia solani and F. culmorum (Valueva et al. 2011). Recently, Mandujano-Gonzalez et al. (2013) identified a 41 kDa size extracellular aspartyl protease, Eap1 from Sporisorium reilianum both in solid and liquid culture media. These studies suggest that different nutritional sources can be crucial for the differential production of proteases.

A correlation between proteolytic activity in culture medium and in infected plant has been shown in several phytopathogenic fungi during their pathogenicity (Brown et al., 2001; Pekkarinen et al., 2002; Bindschedler et al., 2003). According to Movahedi and Heale, (1990), 38-39 kDa aspartyl acid proteases observed in both culture medium and in Botrytis cinerea infected carrot tissue but it was not detected in uninfected carrot tissue. Further, cell death was observed in infected carrot tissue suggesting that aspartic proteinase may be responsible for the initial stage of pathogenecity. A significant extracellular proteolytic activity is accumulated in Spunta tubers during the infection with F. eumartii (Olivieri et al., 1998). Moreover, at the time of infection process, the genes such as acp1 and asps were expressed in Sclerotinia sclerotiorum, which are responsible for the production of acid protease and aspartyl protease (Poussereau et al. 2001). Simillaly, in Ustilago maydis non-aspartyl acid extracellular protease pumAe plays a crucial role during infection of plant tissue (Mercado-Flores et al. 2003). Rolland et al. (2009) identified BcACP1, a member of the G1 family of acidic proteases, produced by B. cinerea during infection. It has been reported that extracellular proteinase is a significant pathogenic and virulent factor of Colletorichum coccodes, and its removal by mutagenesis converts a virulent pathogen into a non-pathogenic endophyte (Redman and Rodrigues, 2002). Furthermore, the necessity of extracellular proteasesin phytopathogenecity was confiremed by mutational studies in several fungal species (Dickman and Patil, 1986; Ball et al., 1991). Molecular studies also indicate a role of extracellular protease in pathogenesis (Rogers et al., 2000). An array of various types of proteases such as aspartic proteases, cysteine proteases, and serine protease was produced in Colletotrichum acutatum (soft rot causative fungi in fruits) (Gregori et al., 2010). ten Have et al., 2010 demonstrated that the involvement of aspartic proteases (APs) in pathogenicity induced by B. cinerea.

Transient expression of serine protease inhibitor gene, NmIMSP in a susceptible tobacco in turn shows higher/enhanced resistance to Peronospora hyoscyami f. sp. tabacina and Phytopthora parasitica var. nicotianae (Silva et al. 2013). Recently, PLPKI (serine protease inhibitor) inhibited the activity of proteases secreted by the two pathogens of potato, Phytopthora infestans and Rhizoctonia solani, however, it was inactive against proteases secreted into the culture medium by a non-pathogenic fungi, Rhizoctonia N2 in potato. These results suggest that the species specific protease is essential for phytopathogenicity (Feldman et al., 2014). The importance of proteases in pathogenecity is also suggested by the fact that plants have evolved mechanisms to counter pathogen-secreted enzymes by producing protease inhibitors (Ryan, 1990).

Proteases as markers of phytopathogenicity

Positive correlations between protease activity and aggressiveness have been reported in several plant pathogenic fungi. Staples and Mayer (1995) showed than an increase in the level of aspartic protease activity in B. cinerea led to an increase in pathogenicity. Brown et al., (2001) conducted a comparative study of virulent and non-virulent strains of Aspergillus flavus in cotton boll infection. The results showed that the virulent strain displayed higher protease activities than the non-virulent strain, suggesting some correlation between the enzyme activity and virulence of A. flavus. Redman and Rodriguez (2002) reported a direct relationship between the extracellular serine protease activity of C. coccodes and the induction of disease symptoms in tomato fruit. Oliveri et al. (2004), demonstrated a correlation between proteolytic activity detected in intercellular washings with size of lesions caused by F. eumartii in potato tubers of susceptible one, but not in the resistant one. During infection, Phytopthora infestans secrete trypsin-, subtilisin-like serine proteases (SLPs), and metalloproteases (Gvozdeva et al., 2004).

Extracellular fungal proteolytic enzymes are represented largely by serine proteases. There are two major families of serine proteases such as chymotrypsin (S1) and subtilisin (S8) in fungi (Rawlings et al., 2011). The interaction between exoproteases of the phytopathogenic fungus R. solani and plant inhibitors indicate that plant infection involves TLPs secreted by the fungus (Gvozdeva et al., 2004). Dunaevsky et al. (2001, 2006) reported that the production of TLPs is characteristic of plant pathogens, whereas extracellular endoproteolytic activity in the saprotrophs is mainly due to subtilisin-like proteases. A comparative study of extracellular proteases of six species of mycelial fungi, including three phytopathogens (A. alternata, Botrytis cinerea, and Ulocladium botrytis) and three saprophytes (Penicillium chrysogenum, P. terlikowskii and Trichoderma harzianum) showed that serine like proteases are present in all fungal species (Dunaevsky et al., 2007). More pathogenic F. sporotichoides exhibited a greater extracellular TLPs activity than the less pathogenic F. heterosporum (Dunaevsky et al., 2008). Some fungi such as T. harzianum and other saprotrophic fungi secrete solely subtilisin-like proteases. However, A. alternata and other phytopathogens was produces TLPs as well. By using SLPs, pathogenic fungus affects the physiological integrity of host during penetration and colonization (Li et al., 2010). These data indicate that the production of TLPs is a distinct feature of phytopathogenic fungi, whereas the production of SLPs is a specific feature of saprotrophic fungi (Jia et al., 2000; Mosolov and Valueva, 2006; Dubovenko et al., 2010). Thus, the presence of appreciable extracellular TLP activity in phytopathogenic fungi may serve as a marker of their phytopathogenicity.

Conclusion and future perspectives

It is clear from recent studies on phytopathogenic fungi that the importance of proteases in the disease process may depend upon the specific host-pathogen interaction. It is assumed that energy requirements of phytopathogens are met by degrading the plant cell wall proteins. Relatively little attention has been paid to the importance of proteases in the virulence of phytopathogens. Identification and characterization of fungal proteases will be a key aspect of the generation of molecular markers for phytopathogenicity. For better, understand the mechanism of interactions between plant and pathogenic fungi it is necessary to intensify the search for targets of inhibitors in the pathogen. Progress in this area, along with the available information from this review, will allow plant pathologist to design more efficient strategies to generate pathogen resistance plants based on mechanisms such as protease inhibitors.

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