Genomic Analyses and Transcriptional Profiles of the Glycoside Hydrolase Family 18 Genes of the Entomopathogenic Fungus <i>Metarhizium anisopliae</i>

<div><p>Fungal chitin metabolism involves diverse processes such as metabolically active cell wall maintenance, basic nutrition, and different aspects of virulence. Chitinases are enzymes belonging to the glycoside hydrolase family 18 (GH18) and 19 (GH19) and are responsible for the hydrolysis of β-1,4-linkages in chitin. This linear homopolymer of N-acetyl-β-D-glucosamine is an essential constituent of fungal cell walls and arthropod exoskeletons. Several chitinases have been directly implicated in structural, morphogenetic, autolytic and nutritional activities of fungal cells. In the entomopathogen <i>Metarhizium anisopliae,</i> chitinases are also involved in virulence. Filamentous fungi genomes exhibit a higher number of chitinase-coding genes than bacteria or yeasts. The survey performed in the <i>M. anisopliae</i> genome has successfully identified 24 genes belonging to glycoside hydrolase family 18, including three previously experimentally determined chitinase-coding genes named <i>chit1</i>, <i>chi2</i> and <i>chi3</i>. These putative chitinases were classified based on domain organization and phylogenetic analysis into the previously described A, B and C chitinase subgroups, and into a new subgroup D. Moreover, three GH18 proteins could be classified as putative endo-<i>N</i>-acetyl-β-D-glucosaminidases, enzymes that are associated with deglycosylation and were therefore assigned to a new subgroup E. The transcriptional profile of the GH18 genes was evaluated by qPCR with RNA extracted from eight culture conditions, representing different stages of development or different nutritional states. The transcripts from the GH18 genes were detected in at least one of the different <i>M. anisopliae</i> developmental stages, thus validating the proposed genes. Moreover, not all members from the same chitinase subgroup presented equal patterns of transcript expression under the eight distinct conditions studied. The determination of <i>M. anisopliae</i> chitinases and ENGases and a more detailed study concerning the enzymes’ roles in morphological or nutritional functions will allow comprehensive insights into the chitinolytic potential of this highly infective entomopathogenic fungus.</p></div

Phylogenetic tree of GH18 subgroup A chitinases.

<p>Chitinase amino acid sequences from <i>M. anisopliae</i> and other fungi were from the NCBI and BROAD Institute databases. The Neighbor-Joining (1000 bootstraps) phylogenetic tree was constructed using Mega 6 after alignment obtained from ClustalW. <i>M. anisopliae</i> chitinases are indicated as ▴ with their corresponding ID numbers.</p

Modular domain structure and transcripts from <i>M. anisopliae</i> GH18 genes.

<p><b>A)</b> GH18 genes exhibit characteristic conserved domains with different compositions. Coding exonic sequences are depicted as boxes (color codes are indicated) and introns as thin lines. Domains were identified using Conserved Database Domain (at NCBI), dbCAN and InterProScan. Signal peptide sequences were predicted using SignalP 4.1. Blank protein regions indicate the absence of characterized domains. Horizontal semi-arrows indicate oligonucleotide positions and directions used for transcript validation. <b>B)</b> The validation of GH18 genes was achieved by transcript detection (+ or – on V column) with RNA extracted from seven different culture conditions in RT-PCR reactions using specific primers shown by the semi-arrows on panel A. The length of PCR products is displayed as bp and compared to C+ (genomic DNA as template) and C− (no template added). RNA extracted from: <b>M1 -</b><b>mycelium</b> grown in complete medium (MCc); <b>M2</b> - mycelium grown in <b>induction GlcNAc</b> 0.25% medium; <b>CO -</b><b>conidia</b> suspension; <b>M3 -</b> mycelium under <b>autolysis</b>; <b>AP -</b> from cultures under <b>appressorium</b> induction; <b>GT -</b> from cultures under <b>germ tube</b> formation induction; <b>BL -</b> from cultures in <b>blastospore</b> induction medium. #indicates alternative transcript forms detected.</p

Phylogenetic relationships among GH18 domain-containing proteins and subgroup divisions in three <i>Metarhizium</i> strains and <i>Cordyceps militaris</i>.

<p>Amino acid sequences corresponding to GH18 domains from <i>M. anisopliae</i> chitinases and ENGases (marked as ▴), <i>M. acridum</i> (MAC), <i>M. robertsii</i> (MAA) and <i>C. militaris</i> (CCM) were obtained from the NCBI databases. The Neighbor-Joining (1000 bootstraps) phylogenetic tree was constructed using Mega 6 after ClustalW alignment. The scale bar indicates the genetic distance, which is proportional to the number of amino acid substitutions.</p

Relative chitinase and ENGase transcript profiles in <i>M. anisopliae</i>.

<p>Transcriptional profiles from 21 chitinase and 2 ENGase genes in eight different conditions using actin as the reference gene are displayed. The different cell types and culture conditions analyzed were mycelium grown on glucose, chitin 1%, GlcNAc 0.25% or autolysis conditions; and also induced blastospores, conidia, induced appressoria and 24 h fungal growth over tick cuticles. Data are shown as the mean ± SD from three experimental replicates of three biological replicates. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Phylogenetic tree of GH18 subgroup C chitinases.

<p>Chitinase amino acid sequences from <i>M. anisopliae</i> and other fungi corresponding to the GH18 domain were from the NCBI and BROAD Institute databases. The Neighbor-Joining (1000 bootstraps) phylogenetic tree was constructed using Mega 6 after alignment obtained from ClustalW. <i>M. anisopliae</i> chitinases are marked as ▴ with their corresponding ID numbers.</p

Phylogenetic tree of the GH18 subgroup D (chitinase) and subgroup E (ENGases).

<p>Amino acid sequences from <i>M. anisopliae</i> and other fungi were from the NCBI and BROAD Institute databases. The Neighbor-Joining (1000 bootstraps) phylogenetic tree was constructed using Mega 6 after alignment obtained from ClustalW. <i>M. anisopliae</i> chitinases/ENGases are marked as ▴ with their corresponding ID numbers.</p

Properties of the 24 <i>M. anisopliae</i> glycoside hydrolase family 18 genes (GH18 genes).

<p><i>M. anisopliae</i> chitinases and ENGases identification, subgroup classification, molecular mass and accession numbers are given. The presence of predicted signals and carbohydrate-binding motifs (CBM) detected on the 24 GH18 proteins are marked as positive (+). These include presence of signal peptides (SignalP), GPI-anchors (GPI) and regions recognized by non-classically secretion pathways (NCS). Proteins marked with *were previously studied in <i>M. anisopliae</i> E6, as shown in references 25, 26, 31–33. At mature protein kDa column, the experimentally observed molecular mass is indicated between parentheses.</p><p>Properties of the 24 <i>M. anisopliae</i> glycoside hydrolase family 18 genes (GH18 genes).</p

HeatMap and hierarchical clustering of the eight analyzed conditions by similar expression profiles.

<p>The HeatMap and hierarchical clustering were analyzed using GenePattern databases at BROAD Institute. The hierarchical clustering was used to group conditions (and not genes) by similar expression profiles. The highest transcript values are displayed as the reddest (hot), the lowest values are displayed as the bluest (cool), and intermediate values are a lighter color of either blue or red (2^−ΔCt) for chitinase/ENGase in different conditions. Conditions are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107864#pone-0107864-g007" target="_blank">Figure 7</a>.</p

Numbers of ORFs identified in <i>A. deanei</i> and <i>S. culicis</i> and their symbionts, according to the mechanisms of DNA replication and repair, signal transduction, transcription and translation.

<p>Numbers of ORFs identified in <i>A. deanei</i> and <i>S. culicis</i> and their symbionts, according to the mechanisms of DNA replication and repair, signal transduction, transcription and translation.</p