Cochliobolus lunatus (Nelson and Hassis) a member of Dothideomycetes predominantly produces four-celled conidia primarily disseminated by air. C. lunatus causes several diseases in human [1, 2] as well as in food crops such as rice (Oryza sativa L.), wheat (Triticum aestivum), potato (Solanum tuberosum L.), sorghum (Sorghum bicolor), cassava (Manihot esculenta) and maize (Zea mays) [3–8]. Proteomics analysis of virulence variations in C. lunatus strains revealed that melanin synthesis-related proteins and heat stress-related proteins (HSP70) are the basic virulence-growth factors during invasion in maize [7, 8]. Although only intracellular protein from mycelia was used in these studies [7, 8], the data indicated that a large repertoire of functional proteins of C. lunatus are unknown.

‘Secretome’ refers to a set of secreted proteins at a given physiologic condition; which plays a key role in cell signaling, intracellular trafficking and migration of invasive weaponries (i.e. candidate effectors) in pathogenic interactions. C. lunatus has attracted the interest of many workers on various aspects viz., induce-virulence variation, virulence differentiation and heat-dependent aggressiveness [7–12]. Experimentally, extracellular weaponries secreted by pathogens are crucial for increased virulence and disease development in the context of plant-pathogen interaction sensu stricto. Candidate effector molecules are believed to manipulate host cell structure and function, thereby facilitating infection and suppression of the host immune responses [13, 14]. Once candidate effectors are deployed, they act either in the exhaustorial matrix, the extracellular space or within the host cell cytoplasm to promote invasion and pathogenicity [13–15]. In conditions where candidate effectors are recognized by the host disease resistance (R) proteins, hallmark resistance occurs via programmed cell death. In this case candidate effectors are considered to have an avirulence activity. Often, fungi discharge their candidate effectors into their surroundings via a non-classical pathway which does not require an N-terminal signal peptide [15]. On the contrary in classical secretory pathway, candidate effectors are directed by the N-terminal peptide signal via the endoplasmic reticulum and Golgi systems to their extracellular locations [13–16].

Frequently, pathogens differentially produce enzymes based on the environmental conditions [16]. Fluctuations of temperature in most cases play a decisive role in the development of disease, since the physiology of either the host or pathogen can change and significantly modulate the interaction dynamics. Interestingly, C. lunatus virulence increases with ambient temperature upto 38°C [9–12]. Nevertheless, whether C. lunatus discharges secretome weaponries under heat stress conditions on putative host is not known. Thus, examining C. lunatus temperature-dependent secretome on a putative host is important and can permit the discovery of candidate effectors that govern its virulence and thermo-pathogenicity. To date, the secretome architecture of C. lunatus is not explored and could be of value for designing a suitable control measure in the context of the current rise in global temperature. In this study, microscopic analysis was performed first to decipher the nature of potato leaf invasion in the liquid phase. Subsequently, we used 2-D, MS-technology and in silico tools to analyze C. lunatus secretome discharged during interaction with potato leaf in the liquid phase. Our work provided first analysis of C. lunatus temperature-dependent secretome weaponries deployed during invasion in potato crop.

A great challenge in secretomics is how to trigger a pathogen to secrete proteins as starting material for downstream analysis. Studies based on genome scanning for secretome through in silico approach is less cumbersome and generates large data [15, 25–28]. Nevertheless, the likelihood that predicted proteins expressed are during host colonization is very low. Moreover, the physiological conditions for secretion cannot be predicted in silico.

During matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) we found that several peptide spots showed incongruities between their experimental and predicted molecular mass (Mr) and pI. These inconsistencies in experimental and theoretical values of pI and molecular masses have been observed in several proteomics studies [31–34]. While predicted values are based on whole protein sequence, experimental values are a function of protein subunits only detected on gels. Ecological niche can cause dramatic changes in experimental pI of proteins from acidic pI to basic pI [32]. Interestingly, it is reported that in vivo or in vitro protein degradation can result in experimental Mr significantly lower than predicted Mr [34]. This might hold true for this study given C. lunatus secreted proteolytic proteins (Table 1). Furthermore, post-translational modifications such as ubiquitination, sumoylation, glycosylation, alternative splicing and endoproteolytic cleavage can cause discrepancies in predicted and expected pI and Mr values [33, 35].

We found that C. lunatus modified the ambient pH. Some fungi have developed complicated regulatory mechanism to sense and respond to ambient pH dependent signals during prolonged interaction with the environment [36]. In addition, a few fungi secrete different set of proteins at different ambient pH [37]. For a successful invasion of a host, pathogens require coordinated secretion of chemical weaponries at the onset of interaction. For instance, an important hemibiotroph such as Botrytis cinerea differentially secretes enzymes in various plant tissues during invasion [16]. Cooperation of various extracellular proteins, though a few may be ‘real’ virulence factors [37] is decisive for the successful suppression of the host defense response. The changes in biomass and pH observed in this study at different temperature regimes demonstrated that C. lunatus successfully thrived on potato leaf by finely adjusting its microenvironment (Figure 1A,B).

The secretome studies of B. cinerea revealed a dark colored culture medium at pH 4 and pH 6 [37]. This dark color was due to secondary metabolites produced during the fungus growth [37]. In the present secretome analysis, the intense yellowish- and milky-colored culture medium (Figure 1D-G) and melanized colonizing hyphae (Figure 2) indicated that C. lunatus secretome comprises of colored secondary metabolites along with their candidate effectors. Previous study revealed that C. lunatus strains which produced high level of melanin are more virulent than those producing low level melanin [38]. Melanin and related secondary metabolites are virulence factors in C. lunatus pathogenicity [7, 8, 38]. A closer inspection of the colonizing hyphae (Figure 2D) suggested that the dark-brown pigments might be an adaptive feature required to sustain C. lunatus virulence at higher temperature. This assumption is based on previous findings that proteins related to melanin synthesis and heat-stress responses are up-regulated in C. lunatus [7, 8] during host colonization.

In this study, we found that C. lunatus invaded potato leaf in CDB medium. This suggested that CDB may not have negatively affected the interaction dynamics. Higher temperature-virulence phenomenon has been observed in Cryptococcus neoformans and Aspergillus fumigatus [39, 40]. In these cross-kingdom fungi, loss of genes required for higher temperature growth resulted to attenuated virulence and at times death [39, 40]. In our study, we found that conidia as well as hyphae at 28°C and 38°C were highly pigmented (Figure 2). Additionally, more necrotic zones were formed around the melaninated penetrating hyphae at 38°C (Figure 3). On the other hand, conidia were present and actively germinating, an important prerequisite for persistent virulence [9, 12]. Put together, we speculated that C. lunatus concomitantly sporulates, discharges toxic-colored metabolites (Figure 1G) and produces diverse peptides during colonization of potato leaf (Figures 4, 5 and 6). Elsewhere, melaninated appressorial was reported in secretome analysis of Colletotrichum higginisianum, an important plant-pathogenic hemibiotroph [41]. Secreted secondary metabolites and candidate effectors can change the phenotypic characteristics of a host during interaction. Thus, this possibly explains why C. lunatus produces variable symptoms as earlier observed on different hosts [3, 4, 8–12].

In 2-D analysis, some of the spots were unique at 20°C with potato leaf and did not match with spots detected at 38°C with potato leaf where negligible change in pH was observed (Figures 5, 6 and 1B). Based on the changes in the secretome set (Figure 7), it is tempting to suggest that acidic microenvironment triggers C. lunatus to release more proteins. Also, we could not abandon the possibility that leaf protein underwent proteolysis during interaction. However, we failed to precipitate protein in treatments containing leaf without C. lunatus and also failed to find GPI-anchors on identified proteins. Based on these observations, we assumed that identified peptide spots were products of interaction secreted uniquely by C. lunatus. Nonetheless, the highest homology ranked peptide was CCO (spot# 38) secreted via classical pathway (Additional file 7: Figure S4 and Additional file 8: Figure S5). Adhesins are primary virulence factors used by pathogens to adhere to host prior to invasion [26, 27] and only 10 peptide spots were found with adhesins motifs (Additional file 6: Table S2A, B). We concluded that majority of C. lunatus secreted proteins are highly soluble, positively charged (Table 1), discharged via the non-classical pathway and lack anchors to the cell surface.

Most phytopathogenic fungi progressively adapt to the genetic background of their host plants, thus creating a new type of virulent or physiological race [42]. This progressive process largely depends on the intensity of the pathogen-host specific interaction [43]. Nevertheless, during interaction, pathogenic fungi survived host counter attacks through coordinated secretion of their secretome weaponries primarily to harness nutrients and proliferate. Notably, the large number of up-regulated nucleic acid metabolic-related proteins, such as protein kinases (Figure 8: Table 1) indicated that under heat-stress or biological stress (i.e. with leaf), C. lunatus prioritized its proliferation. This is in agreement with the concept that protein profile of microorganisms change under biological or abiotic stress condition [44], which is also observed in this study (Figure 4). Intuitively, the abundant secretion of nucleic acid metabolic-related proteins enhances virulence as proliferation directly leads to large fungal biomass production. The secretome changes demonstrated that C. lunatus secretes varied repertoire of proteins depending on the ambient temperature (Figures 4 and 8). This is supported by unique unaligned peptide (spots# 27, 32, 34, 35, 36, 37 and 38) expressed only at 38°C (Figure 6: Additional file 6: Table S2A, B). This possibly could explain the survival and successful lifestyle of C. lunatus and related species at higher temperature [9–12]. Cautiously, the unique peptide spots could represent signature proteins required to sustain C. lunatus at elevated temperature only, not necessarily involved in pathogenicity sensu stricto.

Besides the peptide spots with known putative functions, abundantly expressed peptide spots with no catalytic domain and no predictable subcellular target got our notice. C. lunatus is a successful cross-kingdom pathogen and would cogently not over-secrete peptide spots# 6, 19 and 21 at 20°C with potato leaf (Figure 5: Table 1) and spot# 27 (Figure 6) at 38°C with potato leaf without a possible role in pathogenicity. As the normalization plot depicts (Additional file 5: Figure S3), C. lunatus secreted discrete extracellular weaponries at different temperature. Assuming that secreted proteins operates in a cooperative manner [37], it is reasonable to suggest that C. lunatus candidate effectors with no assignable functions exert an unforeseen synergistic role in temperature-dependent virulence. Secreted proteins with unknown functions is a common phenomenon in fungal-plant interactions [14, 31, 37, 41]. In recent years, progress has been made in effectoromics [36, 45–51]. Nonetheless, effectors are still ill-defined, but widely accepted that they manipulate host cell processes for the pathogen benefit [13–16].

Candidate effectors are understood to perform key functions prior to host colonization such as the provision of nutrients, mask recognition by the host defense system, cell-to-cell communication, detoxification of the environment, suppression of host-cell programmed cell death and killing of potential competitors [13, 45–48]. Therefore, fungal secretome analysis is important. For instance, mining protein effectors that matches specific resistance-proteins can assist the deployment of engineered resistance-genes in crops and can also permit scientist to survey crops when a particular pathotype overcome the resistance-genes [47, 48]. Importantly, effectors can be used as molecular probes to dissect focal immune responses, unravel the diversity of secretory vesicles and the cargo they transport [46]. We found that the secretome of C. lunatus on potato leaf shared motif- CLxxxxLHM (Figure 9). Cysteine is important and maintains effector stability in the extracellular space [36]. In addition, we observed a significant occurrence rate of CLxxxxLHM motif in other GenBank® fungal genome peptides (Figure 10). This shows that cross-kingdom pathogens share some common pathogenicity signatures in their secretome. For instance, in powdery mildew and rust fungi, motif Y/F/WxC [49] and Oomycetes effector motif RxLR-dEER [45, 50] shared no positional conserved amino acid residues. Noteworthy, these pathogens colonize only plant hosts. Intriguingly, C. lunatus colonizes plant hosts [4–8] and animal hosts [1, 2]. Critically, motif-CLxxxxLHM share features to motif-Y/F/WxC and motif-RxLR with conserved residue of cysteine (C) and leucine (L), respectively. In addition, histidine (H)-residue like arginine (R) is a basic amino acid residue and is rare in effector motif, but reported elsewhere [51]. Motifs with positional constraint amino acids such as M, L, H, N, Y, F, W and C have been reported in other fungi candidate effectors [49–52]. A closer inspection suggests histidine in motif-CLxxxxLHM is a unique signature in C. lunatus secretome on potato crop during invasion.

Pioneering effectoromics study indicated that fungal candidate effectors are most likely to be soluble and secreted into the extracellular matrix but do not form cross-link with fungal cell wall and show no homology to proteins with known functions [41]. Based on these criteria, we concluded that spots# 3 (C. lunatus genome peptide m118 v.2 accession no: 118437|e_gw1.13.184.1/ClEfc1), spot# 6 (19257|fgenesh1_pg.7_#_229/ClEfc2), spot# 19 (46252|estExt_Genemark1.C_210087/ClEfc3), spot# 21 (64448|estExt_fgenesh1_pg.C_160063/ClEfc4) and spot# 27 (132047|estExt_Genewise1.C_4_t40098/ClEfc5) (Table 1: Figure 11) with no homology to known proteins are candidate effectors of C. lunatus on potato crop. We found candidate effectors ClEfc1, ClEfc2, ClEfc3, ClEfc4 and ClEfc5 were secreted via non-classical pathway. As such, the candidate effectors did not have N-terminal signal peptide in agreement with conditions for non-conventional secretions [15, 53]. Sophistication in C. lunatus invasion strategies is further enhanced by its ability to use the classical and non-classical secretory pathways to explore its microenvironment. Since only spot# 38 (Additional file 8: Figure S5) was identified with a TM-networking, this agrees with previous finding that many fungal effectors are soluble and do not often cross-linked with the fungal cell wall after secretion [41]. Our findings demonstrate that C. lunatus secretes majority of its effectors via non-classical pathway.

At 20°C with potato leaf, known virulence related proteins such as HSP70-related protein [7, 41], tetratricopeptide (TPR)-related protein [54] and pleckstrin-like proteins were up-regulated. Based on the observation that the expression fold of HSP70-related proteins was greater than the cut-off limit, we concluded that it plays a decisive role in C. lunatus virulence on potato as previously reported in maize [7]. TPR-like proteins have a versatile helix associated with protein-protein interaction and intercellular trafficking [54] and a virulence domain [55]. Pleckstrin-like proteins controls diverse cellular trafficking interrelated with cell growth, survival, proliferation and metabolism [56]. In Candida albicans, pleckstrin-like proteins are understood to be involved in virulence and morphological switching [57]. Other putative proteins including protein kinases (spot# 1, 5 and 28), oxido-reductase (OXR, spot# 4) and cytochrome c oxidase (CCO, spot#38) were up-regulated. Although similar up-regulation was observed at the transcript level, their role in C. lunatus-increased temperature dependent-virulence is not clear (Figure 11A). Also, C. lunatus discharged an array of extracellular proteins that are often related with carbohydrate metabolism and proteolysis. Proteases play an important role in pathogenicity and maintenance of life cycle of fungi [37, 46, 58] and in this study, subtilisin protease (Pte) was constitutively expressed (Figure 11A). Toxin production in C. lunatus is believed to be positively correlated with up-regulation of NTR [7, 8]. Furthermore, SCD, SDR and NTR are important genes for melanin biosynthetic pathway [7, 8, 41]. It was showed that up-regulation of SCD and NTR increased melanin pigments [7, 8] and also improved the penetration strength of melanized appressorial [41]. Therefore, up-regulation of SCD, SDR and NTR might play important role in C. lunatus temperature-dependent virulence (Figure 11A). However, an evident up-regulation of inter-trafficking transcripts such as WD40, Grlp and ALP was observed (Figure 11A) which requires detailed investigation to decipher their possible role. Meanwhile, we suggest that WD40, Grlp and ALP may synergistically cooperate with candidate effectors ClEfc1, ClEfc2, ClEfc3, ClEfc4 and ClEfc5 to cause disease on potato.

Classical 2-D is a powerful approach for direct analysis of secretome [16, 37] and for the discovery of virulence factors in plant-pathogen interactions [7, 8, 58–61]. In this study, we have successfully used 2-D, MS-technology, RT-PCR and in silico tools to identify a set of 5 soluble candidate effectors of unknown function from C. lunatus secreted against potato crop at different temperature regimes. The findings throw light on the increased temperature-dependent virulence of C. lunatus in accordance with recent findings related to the release of pigments on maize and overexpression of stress related proteins [7]. Importantly, the discrete set of secretome provided new insight that can explain why C. lunatus can successfully thrives on a broad-spectrum of hosts under heat-stress conditions. The results showed that the sophisticated infection process of potato crop by C. lunatus comprises of: 1) intimate penetration of the leaf, 2) modulation of the ambient microenvironment pH, 3) release of colored secondary metabolites and 4) secretion of effectors candidate with diverse functions. In future, it will be interesting to integrate the analysis of secondary metabolites and the current secretome data to confirm the hypothesis generated in this study on C. lunatus thermo-pathogenicity.

BL conceived the experiment, run 2-D electrophoresis, performed in silico analysis, analyze gels and wrote the manuscript. SDW performed the fungal-potato interaction and assist to optimize the culture conditions. PR had the main responsibility for studies involving electrophoresis and interpretation of MS spectral, microscopy and proofreading of the manuscript. PKB assisted in gene expression studies. MWS assisted in statistical analyses. SG assisted in harvesting the extracellular proteins. CKS assisted in microscopy studies. NCT supplied all the reagents and assisted in microscopy analysis. All authors read and approved the final manuscript.