The pathogen Moniliophthora perniciosa promotes differential proteomic modulation of cacao genotypes with contrasting resistance to witches´ broom disease

Abstract

Background: Witches' broom disease (WBD) of cacao (Theobroma cacao L.), caused by Moniliophthora perniciosa, is the most important limiting factor for the cacao production in Brazil. Hence, the development of cacao genotypes with durable resistance is the key challenge for control the disease. Proteomic methods are often used to study the interactions between hosts and pathogens, therefore helping classical plant breeding projects on the development of resistant genotypes. The present study compared the proteomic alterations between two cacao genotypes standard for WBD resistance and susceptibility, in response to M. perniciosa infection at 72 h and 45 days post-inoculation; respectively the very early stages of the biotrophic and necrotrophic stages of the cacao x M. perniciosa interaction.

Results: A total of 554 proteins were identified, being 246 in the susceptible Catongo and 308 in the resistant TSH1188 genotypes. The identified proteins were involved mainly in metabolism, energy, defense and oxidative stress. The resistant genotype showed more expressed proteins with more variability associated with stress and defense, while the susceptible genotype exhibited more repressed proteins. Among these proteins, stand out pathogenesis related proteins (PRs), oxidative stress regulation related proteins, and trypsin inhibitors. Interaction networks were predicted, and a complex protein-protein interaction was observed. Some proteins showed a high number of interactions, suggesting that those proteins may function as cross-talkers between these biological functions.

Conclusions: We present the first study reporting the proteomic alterations of resistant and susceptible genotypes in the T. cacao x M. perniciosa pathosystem. The important altered proteins identified in the present study are related to key biologic functions in resistance, such as oxidative stress, especially in the resistant genotype TSH1188, that showed a strong mechanism of detoxification. Also, the positive regulation of defense and stress proteins were more evident in this genotype. Proteins with significant roles against fungal plant pathogens, such as chitinases, trypsin inhibitors and PR 5 were also identified, and they may be good resistance markers. Finally, important biological functions, such as stress and defense, photosynthesis, oxidative stress and carbohydrate metabolism were differentially impacted with M. perniciosa infection in each genotype.

Keywords: Disease resistance; Plant-pathogen interaction; Proteomics; Theobroma cacao.

Fig. 1

T. cacao seedlings inoculated and non-inoculated with M. perniciosa and protein yield. a Theobroma cacao seedlings of Catongo (left) and TSH1188 (right), inoculated and non-inoculated with basidiospores of Moniliophthora. perniciosa at 72HAI (hours after inoculation) and 45DAI (days after inoculation). Typical symptoms of WBD (stem swellings), characteristic of fungal biotrophic phase was observed in both genotypes at 45DAI. b Protein total yield from 0.2 g of plant tissue of Catongo and TSH1188 genotype, inoculated (72HAI and 45DAI) and non-inoculated (72HNI and 45DNI) with basidiospores of M. perniciosa

Fig. 2

Representative 2D gels of proteins extracted from shoot apexes of TSH1188. Inoculated and non-inoculated (control) cacao genotypes collected at 72HAI and 45DAI post-infection with M perniciosa. Total proteins extract (500 μg) were focused on IPG strips (13 cm), pH ranging from 3 to 10 NL, separated by SDS-PAGE (12.5%) and stained with CBB G-250. Circles indicate protein spots identified. Spots number corresponds to protein indicated at Table 1 and Additional file 4

Fig. 3

Representative 2D gels of proteins extracted from shoot apexes of Catongo. Inoculated and non-inoculated (control) cacao genotypes collected at 72HAI and 45DAI post-infection with M perniciosa. Total proteins extract (500 μg) were focused on IPG strips (13 cm), pH ranging from 3 to 10 NL, separated by SDS-PAGE (12.5%) and stained with CBB G-250. Circles indicates protein spots identified. Spots number corresponds to proteins indicated in the Table 2 and Additional file 5

Fig. 4

Spot detection and hierarchical clustering of gel replicates. a Total number of common spots detected in each treatment performed by Image Master 2D Platinum software 7.0 on 2D gels triplicates images. Spot detection was made by matching the experimental triplicates of each treatment from TSH1188 and Catongo in inoculated conditions (72HAI and 45DAI) and non-inoculated conditions (72HNI and 45DNI). b Hierarchical clustering indicating the similarity between experimental replicates based on spot intensity values. This analysis was performed using the NIA array analysis tool software

Fig. 5

Venn diagrams representing the total number of proteins identified by mass spectrometry in 2D gels from Catongo and TSH1188 cacao genotypes at two time points after inoculation with M. perniciosa. a 72 h after inoculation (7HAI) and b 45 days after inoculation (45DAI). Proteins are discriminated by their occurrence: Gray dashed circles represent non-inoculated treatments, black circles represent inoculated treatments and in the diagrams intersections, the number of significantly common spots altered with Fold change (FC) ≥ 1.5

Fig. 6

Number of identified proteins discriminated by functional characterization and regulation (up and down). We used the Blast2Go software to divide proteins into eight functional groups: oxidative stress, stress and defense, photosynthesis, metabolism and energy, signal transduction, nucleic acid metabolism, protein metabolism and unknown. Functional characterization of differentially expressed proteins in Catongo (a) and TSH1188 (b) at 72HAI, and in Catongo (c) and TSH1188 (d) at 45DAI

Fig. 7

Differentially expressed proteins of TSH1188 and Catongo during interaction with M. perniciosa subjected PPI analysis. Networks of up regulated (a) and down regulated (b) proteins in TSH1188 at 45DAI. Networks of up regulated (c) and down regulated (d) proteins in Catongo at 45DAI. Dark circles represent highly clustered proteins related to important biological functions. Network nodes represent proteins in which each node represents all the protein by a single, protein-coding gene locus. Small nodes indicate proteins of unknown 3D structure, large nodes indicate proteins which 3D structures are known or predict (can be visualized by close-up the nodes). Different line colors indicate the types of evidence for the associations. Query proteins not connected with network were removed for better visualization

Fig. 8

Response model of T. cacao genotypes during M. perniciosa infection through proteomic approaches. The response of the susceptible (Catongo) and resistant (TSH1188) genotypes to M. perniciosa infection vary mainly due the differential protein expression observed by 2D-PAGE-LC/MSMS approach applied in this study. Proteins expression patterns reflect biological functions such as metabolism and energy, oxidative stress, photosynthesis and stress and defense. In general, resistance genotype is mainly related to the early and intense activation of defense pathways/signaling. Nevertheless, the susceptible genotype not only present latter and less intense activation of the mentioned biological functions, but they may be carried out by different proteins from the same biological functions compared to resistant genotype, which can be strongly related to the differential response observed between the evaluated genotypes