Comparison of domains and sites between SBT3 (PDB 3I6S) and the predicted tertiary structure of GLRG_05578.

*<p>Only relevant residues were named.</p

Horizontal Transfer of a Subtilisin Gene from Plants into an Ancestor of the Plant Pathogenic Fungal Genus <em>Colletotrichum</em>

<div><p>The genus <i>Colletotrichum</i> contains a large number of phytopathogenic fungi that produce enormous economic losses around the world. The effect of horizontal gene transfer (HGT) has not been studied yet in these organisms. Inter-Kingdom HGT into fungal genomes has been reported in the past but knowledge about the HGT between plants and fungi is particularly limited. We describe a gene in the genome of several species of the genus <i>Colletotrichum</i> with a strong resemblance to subtilisins typically found in plant genomes. Subtilisins are an important group of serine proteases, widely distributed in all of the kingdoms of life. Our hypothesis is that the gene was acquired by <i>Colletotrichum</i> spp. through (HGT) from plants to a <i>Colletotrichum</i> ancestor. We provide evidence to support this hypothesis in the form of phylogenetic analyses as well as a characterization of the similarity of the subtilisin at the primary, secondary and tertiary structural levels. The remarkable level of structural conservation of <i>Colletotrichum</i> plant-like subtilisin (CPLS) with plant subtilisins and the differences with the rest of <i>Colletotrichum</i> subtilisins suggests the possibility of molecular mimicry. Our phylogenetic analysis indicates that the HGT event would have occurred approximately 150–155 million years ago, after the divergence of the <i>Colletotrichum</i> lineage from other fungi. Gene expression analysis shows that the gene is modulated during the infection of maize by <i>C. graminicola</i> suggesting that it has a role in plant disease. Furthermore, the upregulation of the CPLS coincides with the downregulation of several plant genes encoding subtilisins. Based on the known roles of subtilisins in plant pathogenic fungi and the gene expression pattern that we observed, we postulate that the CPLSs have an important role in plant infection.</p> </div

Functions of subtilisin family members in plants and fungi.

*<p>Genome project accession number.</p

Gene expression during anthracnose development.

<p>Due to the low representation of fungal mRNA in the samples, semi-quantitative RT-PCR assays were conducted to test the expression of CPLS GLRG_05578 of <i>C. graminicola</i> and the selected maize putative subtilisins. The amount of total RNA used in each PCR reaction was adjusted to the amount needed to provide equal amplification levels of CgTub in all samples. PCR products were visualized after electrophoresis on 2% agarose gel and ethidium bromide staining. <b>a</b>) RT-PCR products for GLRG_05578 and CgTub. <b>b</b>), RT-PCR products of nine genes encoding putative subtilisins in maize. <i>ZmGAPc</i> was amplified as an internal loading control. The number of cycles in PCR reactions was optimized to be in the linear amplification range of each gene. These assays were repeated two times with similar results. In both panels, the numbers over the lanes indicate the time-point at which RNA samples were taken. M indicates RNA samples from mock-inoculated leaves and G indicates genomic DNA.</p

a) Schematic view of protein domains found in <i>C. graminicola</i> subtilisin GLRG_05578.

<p>b) 3d surface view of the protein GLRG_05578. The peptidase s8 domain is colored in yellow, PA domain in blue and Fn III-like domain in green. The ß-hairpin like domain is colored in orange, the residues of the catalytic site are colored in cyan and the putative sites of Ca⁺ replacement are in red. The signal peptide and I9 inhibitor are in pink and violet respectively. The gray residues have not been assigned any domain c) Alignment between mature forms of subtilisin SBT3 of tomato and GLRG_05578 of <i>C. graminicola</i>. The tomato subtilisin is in black and the <i>C. graminicola</i> subtilisin is colored as in (b).</p

Representative portion of a multiple sequence alignment of CPLSs and subtilisins from plants, bacteria and fungi.

<p>The three best BLAST hits to GLRG_05578 from each taxonomic group were used to create the alignment. Amino acid disagreements to GLRG_05578 are represented by dots. Gaps are represented with a dash symbol. The arrow over the alignment indicates the position of the conserved histidine residue of the catalytic site of subtilisins.</p

Phylogenetic tree of subtilisins of <i>Zea mays</i> (GRMZM2G or AC) colored in red and <i>Colletotrichum graminicola</i> (sequence IDs beginning with GLRG) and <i>C. higginsianum</i> (CH) colored in blue. Internal nodes are labeled with percentage of bootstrap support.

<p>Phylogenetic tree of subtilisins of <i>Zea mays</i> (GRMZM2G or AC) colored in red and <i>Colletotrichum graminicola</i> (sequence IDs beginning with GLRG) and <i>C. higginsianum</i> (CH) colored in blue. Internal nodes are labeled with percentage of bootstrap support.</p