Proteome alteration observed in TSH1188 differs from Catongo and may be related to resistance.
Plants during biologic stress may allocate energy to defense response against pathogens in detriment of other normal functions [35], which is usually observed at the early 48HAI. Accumulation of H2O2 during the first 72 hours in infected shoot apexes [36] and high peroxidase activity in protein extracts from leaves of cacao seedlings [37] were observed in the present pathosystem. These alterations require a physiological cost to host organism that are reflected in the proteome alterations observed at that time, since it was observed that both genotypes showed less detected spots and protein identification at 72HAI (Additional file 3, figure A) [38, 39]. A similar pattern was observed in 2D-PAGE gels of the strawberry inoculated with Colletotrichum fragariae pathosystem [19].
Considering that TSH1188 showed more spots compared to Catongo at both times and the metabolic shift from an inhibitory metabolism at 72HAI to an inductive metabolism at 45DAI (Additional file 3, figure A and B), it can be inferred that these responses may be associated with disease resistance in this genotype. Also, it seems to be related with up regulation of metabolic framework compared to the overall repressor pattern observed in Catongo, which showed more repressed proteins in both times. These results differ from da Hora Junior and collaborators (2012) [40]. These authors found in this pathosystem, more differentially expressed genes in Catongo in a transcriptomic study of shoot apexes of cacao challenged with M. perniciosa. However, these findings cannot be properly compared to the results of the present study because the authors used different collection times from ours: a pool of samples to characterize early stage (24, 48 and 72 hours) and samples from 30 and 60 days. Nevertheless, proteomic and transcriptomic studies often have a weak correlation. This divergence can be explained mainly by post-translational modifications that proteins can undergo and directly influence the structure, location, degradation, metabolism, functions in addition to their stability. These modifications may also influence protein abundance, suggesting that the accumulation of proteins is partially determined by the accumulation and degradation of mRNAs [18,41,42]. These finds highlight the differences in proteomic response between genotypes and indicates an overall repressive metabolic pattern in Catongo.
Oxidative stress proteins production is differently controlled between genotypes during infection: TSH1188 shows a strong mechanism of detoxification.
Oxidative oxygen species (ROS) such as superoxide O2-, hydrogen peroxide (H2O2) and hydroxyl radical (OH), are known to be toxic for plants, so they are removed by antioxidative enzymes. Nevertheless, they participate in important signaling pathways, such as development, growth, cell death, and mainly in response to biotic and abiotic stress, acting directly against the pathogens [43]. Moreover, they may function as signaling molecules in subsequent defense response [44]. Furthermore, ROS are toxic for both host and pathogens, therefore, the balance between production and removal of ROS are important during stress response [43]. TSH1188 exhibited up regulation of stress oxidative proteins at 72HAI, among them, isoforms GAPDH. The gene coding this protein was predicted involved in this pathosystem, however, in silico confirmation was not achieved [13]. This protein has other important functions besides its participation in glycolytic pathway [45]. Its cysteine residues can be oxidized [46] and act like ROS signaling transducers as observed during abiotic stress in A. thaliana [47]. Hydrogen peroxide formation in cacao tissue infected with M. perniciosa increases significantly in the first 72HAI in TSH1188 compared to Catongo, which in turn did not vary [40]. It was verified the inhibition of peroxidase 3 and 4 at 72HAI in TSH1188. That fact may be associated with the need of ROS accumulation, which in cacao tissues, is similar to a hypersensitive response (HR) in early infection stage, therefore improving the resistance response and disease control [40].
At 45DAI, TSH1188 showed up regulation of oxidative stress proteins twice as large as Catongo, particularly in proteins related to ROS detoxification (Figure 6, Table 1 and Additional file 4). This change in pattern, may be associated with the fungus’ shift from biotrophic to saprophytic-like stage which has already started at 45DAI, since clamp connections (characteristic of saprophytic mycelium) have been observed in hyphae of M. perniciosa at 45DAI in this pathosystem [5]. Thereby, suggesting that this time point can be considered as a transitional stage. Such mycelium had a remarkable intracellular aggressive growth, leading to tissue death. The stress generated may influence the up regulation burst of oxidative stress proteins observed. Increases in H2O2 levels at 45DAI were also observed in Catongo [6] and TSH1188 [36], but the increase of H2O2 in susceptible genotype may be related to promotion of pathogen life cycle [36]. Additionally, our results showed that both genotypes expressed peroxidases. The consistent increase in quantity and diversity in proteins of oxidative stress observed in TSH1188, point out that, in the resistant genotype, this response may be related to a more efficient mechanism of detoxification. This efficiency is required once the burst of ROS in that genotype must be finely controlled to either limit the pathogen infection and minimize the host damage through expression of detoxifying proteins.
Modulation of carbohydrates metabolism and photosynthesis proteins are required to energy supply during infection in both genotypes.
During plant infection, the host may present a reduction on photosynthetic rates to mobilize energy to defense response [48]. This "metabolic cost" has been observed in several pathosystems [19,49]. The energy required to maintain the responses, results in a greater aid of assimilates, mainly in the form of carbohydrates, however this is a two-edged sword, since the pathogen may use these compounds to self-nutrition, increasing its demand [49]. The up regulation of proteins related to metabolism of carbohydrates observed in our pathosystem may indicate the increase of respiration required. This pattern is a common response and has been observed in the strawberry x Colletotrichum fragariae pathosystem [19], maize inoculated with sugarcane mosaic virus [50] and abiotic stress [51].
The levels of soluble sugar increases in the first days of interaction in our pathosystem [52], also, the starch storage levels decrease during early disease stage, being higher in Catongo compared to TSH1188 in the first 15 days, although, at 45DAI, the levels of starch were higher in TSH1188 compared to Catongo [5]. These findings corroborate our results, since we found more up regulated proteins related to metabolism of carbohydrates in TSH1188 at 45DAI, which may be related to more efficient process of hexoses production via starch metabolism to supply the energy requirement at this stage [52]. Notwithstanding, these molecules may be used by the fungus as well, and probably perform important function during the mycelium shift from biotrophic to saprophytic [53]
Both genotypes showed increase in accumulation of proteins related to photosynthesis at 72HAI. Photosynthesis activation can benefit cells through supplying of carbon skeleton and energy to subsequent defense response [54]. The same pattern was observed in the proteomic profile of Pinus monticola challenged with Cronartium ribicola in compatible and incompatible interaction [55]. Nevertheless, this expression pattern changed at 45DAI when both genotypes showed down regulation of photosynthesis related proteins (Figure 6). This may be related to the hexoses accumulation that can modulate negatively photosynthesis-associated genes during plant-pathogen interaction [49]. Also, this pattern was already observed in other pathosystem [19]. Moreover, the up accumulation of sugar metabolism proteins observed in our work and the sugar accumulation observed at 45DAI by Sena and colleagues (2014) [5] reinforce that possibility.
Positive regulation of defense and stress proteins are more robust in TSH1188 genotype during early and late response to infection.
Fungal matrix cell wall is composed mainly by chitin, although the host did not produce this molecule, they developed, through evolution, enzymes (e.g chitinases) that are capable to degrade the fungus cell wall during defense response [56]. In the TSH1188 these proteins were detected up regulated at both times and in Catongo, only at 45DAI, evidencing the importance of these proteins during plant pathogen interaction. Transgenic plants expressing chitinases increases its resistance against fungus and other pathogens, once chitin fragments are important pathogen-associated molecular pattern (PAMP), which recognition by hosts results in activation of defense signaling pathways [57]. However, recently Fiorin and colleagues (2018) [58], observed that M. perniciosa evolved an enzymatically inactive chitinase (MpChi) that binds with chitin immunogenic fragments, therefore prevents chitin-triggered immunity, evidencing a strategy of immune suppression of the host response by the pathogen. Moreover, PAMPs are expressed during biotrophic development and recent studies showed that Cerato-platanin, a PAMP from M. perniciosa, might bind chitin in a high affinity way, leading to an eliciting of plant immune system by fungal chitin released fragments [59, 60]. Furthermore, the ionic channels which trough the PAMPs are recognized [61], are up regulated in TSH1188 at both times and only at 45DAI in Catongo, indicating that in the resistant genotype this mechanism of recognition is activated earlier. This information highlights the complex molecular relation during plant-pathogen interactions.
The resistance response of TSH1188 was also highlighted by the expression of several PRs, mainly at 45DAI, that shows representatives of four families. PRs are a heterogeneous group of proteins with basal expression in plants that are induced mainly during pathogen infection [62, 63]. Gesteira and colleagues (2007) [13] found that PR4 proteins were more represented at the cDNA libraries of TSH1188 in our pathosystem. Moreover, it was also observed, in our present study, the exclusive expression of PR5 in TSH1188, an important protein which has antifungal activity in a large number of fungal species, such as inhibition of spores germination and hyphae growth [64, 65, 66], and enhances resistance against plant pathogens, e.g in transgenic banana x Fusarium oxysporum sp and transgenic potato x Macrophomina phaseolina and Phytophthora infestans [67, 68]. In addition, data of the present study indicates that Ankyrin repeat domain-containing protein 2 has opposite expression profile between genotypes. This protein is associated with regulation of PRs coding genes and positive regulation of PCD (programmed cell death) [69, 70] which can contribute to the shift of phase of the M. perniciosa (biotrophic to saprophytic) by releasing nutrients to fungal mycelium [32]. Furthermore, the trypsin inhibitors, that are natural plant defense proteins against herbivory and related to biotic and abiotic resistance [71, 72], were found isoforms in both genotypes, however, in the cDNA library it was found only in TSH1188 [13]. In addition, only in this genotype were found its up regulation at 45DAI. It is well known that M. perniciosa at the biotrophic phase release lytic proteins and proteases that contributes to the pathogenicity [73].
The serine protease inhibitors are widely distributed in living organisms like, fungi, plants, bacteria and humans. Further, it has been related to plant resistance [74]. In cacao, the accumulation of these serine protease inhibitors varies in different tissues and genotypes in response to several stress. It was highly represented in the RT library of the resistant interaction between T. cacao and M. perniciosa [13]. These inhibitor shows high abundance in proteomic profile of cacao seed [75], zygotic embryo during development [76] and cacao root submitted to flooding [77], and in cacao leaves also varies in response to heavy metal stress [78]. The most abundant proteinases in the genome of M. perniciosa are deuterolysins, a type of fungal metalloproteinases that are similar to bacterial thermolysin [10]. Nevertheless, although this serine protease inhibitor variation is not a specific response to the fungus M. perniciosa, we believe that it is an important plant defense response of cacao genotypes to stress, that in this case might act protecting the cacao cells against the fungal hydrolases.
PPI analysis reveals a global protein network involving important biological functions in response to M. perniciosa infection.
M perniciosa is one of the most important pathogens to cacao trees and to understand the biological processes underlying the proteomic mechanisms during infection is mandatory. Thus, a detailed protein-protein interaction network is highly demanded. Construction of predict PPI networks are challenging for non-model plants, [79, 80] especially when it comes to high-throughput proteomic data. In order to further investigate the resistance and susceptibility of cacao genotypes against M. perniciosa we have utilized homology-based prediction to identifying PPI among differentially expressed proteins identified in the pathosystem. It is important to emphasize that, some proteins that were identified as isoforms in the 2D-PAGE electrophoresis, were identified as the same protein in the course of the identification process, which diminish the total number of identifications in the PPI networks due to duplicity of the input.
Proteins are not solitary entities; rather, they function as components of a complex machinery, which functional connections are determinant to general metabolism. The effects of M. perniciosa infection on the metabolism of TSH1188 and Catongo are illustrated in the figure 7, showing different protein components interacting with their partners in different biological functions, such as stress and defense, oxidative stress, protein metabolism, photosynthesis and carbohydrate metabolism. Surely, these clusters are not separated objects, and they form a global protein network in response to M. perniciosa infection, which can help us better understand how these undelaying mechanisms are connected, enabling to predict new functional interactions. This is very important, once available information about PPI in non-model plants is scarce. Similar maps were constructed in other pathosystem, such as, soybean and Fusarium virguliforme [81] and may be useful to find out specific proteins that respond to infection [82]. A layer of complexity was added to our study, once we noticed that one or more proteins might be cross-talkers between these biological functions. Such connectivity suggests that there is important PPI related to functional regulation, and they are different between both genotypes during M. perniciosa infection. Besides, one of the correlations found between some of these proteins was co-expression. It is known that co-expressed genes are often functionally related, ‘guilt by association’ [83], and may acting in similar pathways. This could result in a set of regulated protein that responds to specific perturbations. Thus, the information generated from PPI analysis, may be helpful to identify new potential disease related proteins and regulation models, aiming the formulation of new hypotheses in order to elucidating the molecular basis of our pathosystem and to improve defense strategies.
These results provide hints about the molecular mechanisms of resistance and susceptibility in the pathosystem. Although these predicted interaction networks still need to be verified and further analyzed in following investigations, it is known that PPI are broadly conserved between orthologous species [84, 85], strengthening the results presented in this paper.