The fungal pathogen Moniliophthora perniciosa has genes similar to plant PR-1 that are highly expressed during its interaction with cacao

Abstract

The widespread SCP/TAPS superfamily (SCP/Tpx-1/Ag5/PR-1/Sc7) has multiple biological functions, including roles in the immune response of plants and animals, development of male reproductive tract in mammals, venom activity in insects and reptiles and host invasion by parasitic worms. Plant Pathogenesis Related 1 (PR-1) proteins belong to this superfamily and have been characterized as markers of induced defense against pathogens. This work presents the characterization of eleven genes homologous to plant PR-1 genes, designated as MpPR-1, which were identified in the genome of Moniliophthora perniciosa, a basidiomycete fungus responsible for causing the devastating witches' broom disease in cacao. We describe gene structure, protein alignment and modeling analyses of the MpPR-1 family. Additionally, the expression profiles of MpPR-1 genes were assessed by qPCR in different stages throughout the fungal life cycle. A specific expression pattern was verified for each member of the MpPR-1 family in the conditions analyzed. Interestingly, some of them were highly and specifically expressed during the interaction of the fungus with cacao, suggesting a role for the MpPR-1 proteins in the infective process of this pathogen. Hypothetical functions assigned to members of the MpPR-1 family include neutralization of plant defenses, antimicrobial activity to avoid competitors and fruiting body physiology. This study provides strong evidence on the importance of PR-1-like genes for fungal virulence on plants.

Figure 1. Comparison of MpPR-1 and SCP/TAPS proteins of representative organisms.

(A) Domain arrangement of SCP/TAPS proteins. Hydrophobic signal peptides are shown in black and SCP/TAPS domains are represented in blue. The numbers on the right show the size of each protein. Large N-terminal and C-terminal expansions are observed in MpPR-1b and MpPR-1g, respectively. (B) Alignment of the conserved domain of SCP/TAPS proteins. In general, the SCP/TAPS superfamily members show similarities only over the SCP/TAPS domain. Conserved residues (100% of identity) are shown in blue and semi-conserved residues (at least 60% of identity) in green. Putative active site residues are highlighted in red and cysteines in yellow. Secondary structure elements are shown above the alignment (arrow: β-sheets; helix: α-helixes). P14, tomato PR-1 (GenBank P04284); RBT4, repressed by TUP1 from Candida albicans (GenBank AAG09789); Tex31, SCP/TAPS from the mollusk Conus textile (GenBank CAD36507); Na-ASP-2, Necator americanus secreted protein (GenBank AAP41952); GliPR-1, human glioma PR-1 protein (GenBank P48060); SC7, SCP/TAPS from the basidiomycete Schizophyllum commune (GenBank P35794).

Figure 2. Domains identified in the MpPR-1g protein.

In addition to the SCP/TAPS domain, this protein has a KEKE motif in its C-terminal extension. This motif is known to mediate the interaction with other proteins or ions.

Figure 3. Homology modeling of MpPR-1 proteins.

(A) Ribbon stick representation showing the folding of eleven MpPR-1 proteins and three SCP/TAPS proteins used to obtain these models. The putative residues forming the catalytic site are highlighted in dark blue (histidines) and light blue (glutamic acids). Note the presence of an additional protein module in MpPR-1b and MpPR-1g. These modules respectively correspond to the N-terminal and C-terminal extensions observed in these proteins. (B) MpPR-1b, MpPR-1c, MpPR-1d, MpPR-1e, MpPR-1h and MpPR-1j have the four putative active site residues of the SCP/TAPS domain. (C) These residues are partially or completely absent in MpPR-1a, MpPR-1f, MpPR-1g, MpPR-1i and MpPR-1k.

Figure 4. Transcriptional profile of MpPR-1 family members throughout the M. perniciosa life cycle.

Each MpPR-1 gene has a distinct expression profile during fungal development. “Monokaryotic” and “Dikaryotic” hyphae represent the two mycelial stages (biotrophic and necrotrophic) grown under in vitro conditions. “Green broom” and “dry broom” correspond to the biotrophic and necrotrophic stages of M. perniciosa, respectively, during its interaction with cacao. Analyses were performed by qPCR and the M. perniciosa β-actin gene was used as endogenous control to normalize data. Error bars represent standard deviations determined with two biological replicates. Representative drawings of the conditions analyzed are shown on the top.

Figure 5. Genomic organization and transcriptional profile of the MpPR-1 gene cluster found in M. perniciosa.

The MpPR-1c, MpPR-1d and MpPR-1j genes are arranged in tandem over a region of approximately 5 kbp. Analysis of the WBD RNA-seq Atlas shows the expression profile of these MpPR-1 genes in different conditions (green broom – in planta development of the biotrophic monokaryotic hyphae; monokaryotic mycelium; dikaryotic mycelium; basidiomata and basidiospores). Data were visualized using the Integrative Genomics Viewer . The black coverage plot shows cumulative RNA-seq read coverage along the transcripts in all different conditions. Note that these genes were named according to the order they were identified in the fungal genome, and the nomenclature does not necessarily reflect their relative localization in the genome.