The Fungal Pathogen <em>Moniliophthora perniciosa</em> Has Genes Similar to Plant PR-1 That Are Highly Expressed during Its Interaction with Cacao

<div><p>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 <em>PR-1</em> genes, designated as <em>MpPR-1</em>, which were identified in the genome of <em>Moniliophthora perniciosa</em>, 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 <em>MpPR-1</em> genes were assessed by qPCR in different stages throughout the fungal life cycle. A specific expression pattern was verified for each member of the <em>MpPR-1</em> 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 <em>MpPR-1</em> family include neutralization of plant defenses, antimicrobial activity to avoid competitors and fruiting body physiology. This study provides strong evidence on the importance of <em>PR-1-like</em> genes for fungal virulence on plants.</p> </div

Homology modeling of MpPR-1 proteins.

<p>(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.</p

Transcriptional profile of <i>MpPR-1</i> family members throughout the <i>M. perniciosa</i> life cycle.

<p>Each <i>MpPR-1</i> gene has a distinct expression profile during fungal development. “Monokaryotic” and “Dikaryotic” hyphae represent the two mycelial stages (biotrophic and necrotrophic) grown under <i>in vitro</i> conditions. “Green broom” and “dry broom” correspond to the biotrophic and necrotrophic stages of <i>M. perniciosa</i>, respectively, during its interaction with cacao. Analyses were performed by qPCR and the <i>M. perniciosa β-actin</i> 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.</p

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

<p>(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 <i>Candida albicans</i> (GenBank AAG09789); Tex31, SCP/TAPS from the mollusk <i>Conus textile</i> (GenBank CAD36507); Na-ASP-2, <i>Necator americanus</i> secreted protein (GenBank AAP41952); GliPR-1, human glioma PR-1 protein (GenBank P48060); SC7, SCP/TAPS from the basidiomycete <i>Schizophyllum commune</i> (GenBank P35794).</p

Genomic organization and transcriptional profile of the <i>MpPR-1</i> gene cluster found in <i>M. perniciosa</i>.

<p>The <i>MpPR-1c</i>, <i>MpPR-1d</i> and <i>MpPR-1j</i> genes are arranged <i>in tandem</i> over a region of approximately 5 kbp. Analysis of the WBD RNA-seq Atlas shows the expression profile of these <i>MpPR-1</i> genes in different conditions (green broom – <i>in planta</i> development of the biotrophic monokaryotic hyphae; monokaryotic mycelium; dikaryotic mycelium; basidiomata and basidiospores). Data were visualized using the Integrative Genomics Viewer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045929#pone.0045929-Robinson1" target="_blank">[62]</a>. 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.</p

Domains identified in the MpPR-1g protein.

<p>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.</p

Genes most differentially expressed between the 2 stages of fiber development.

*<p>Brill, et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048855#pone.0048855-Brill1" target="_blank">[72]</a> reported 4 isoforms, A–D, ‘new ‘C isoform with highest level at SCW synthesis stage, which corresponded (best blast hits) to 2 of the contigs, Contig_19300_bb and Contig_56175_bb, which had the highest differential 22>10, with 67 and 103-fold.</p

Changes in expression of fiber genes for all possible pair-wise comparisons.

<p>Comparisons are contrasted between: A- fiber development dates (10 vs. 22 dpa), B-genotypes (<i>G. hirsutum</i> and <i>G. barbadense</i>, <i>Gh</i> vs. <i>Gb</i>), and C-sub-genomic origin of reads (A sub-genome vs. D sub-genome). Gene expression was assessed digitally according to the origins of reads within contigs. In each pair-wise comparison bar length is proportional to the number of contigs showing differential over-expression (number of contigs also indicated in parentheses). Symbol ‘*’ indicates a statistical difference (P0.05) for each pair-wise comparison using Fisher’s exact test. For example, within the A panel (all comparisons involving 10 vs. 22 dpa), the comparison Gh10_A vs. Gh22_A (164 vs. 102, significantly different) indicates that within <i>G. hirsutum</i>, there are 164 contigs for which the A-tagged reads are more abundant (≥2-fold) at 10 dpa than at 22 dpa; conversely 102 contigs have more abundant representation of A-tagged reads at 22 dpa than at 10 dpa.</p

Number of contigs in the different cell wall-related gene categories.

<p>The gene categories definitions refer to the 2^nd or 3^rd organization levels in Cell Wall Navigator, <a href="http://bioweb.ucr.edu/Cellwall/" target="_blank">http://bioweb.ucr.edu/Cellwall/</a>). Number of reads are indicated in square brackets.</p

Schematic partitioning of the 39,099 variant positions between intra- and inter-genotypic SNPs.

<p>Variant positions are counted among contigs covered by the 2 genotypes and with at least 6 reads of both. The blue and brown bars symbolize the A_T and D_T sub-genome co-assembled homoeo-copies. Genotypes are symbolized in plain (<i>G. hirsutum</i>) and dotted (<i>G. barbadense</i>) lines.</p