New insights into the evolution and structure of Colletotrichum plant-like subtilisins (CPLSs)

The Colletotrichum plant-like subtilisins (CPLSs) are a family of proteins found only in species of the phytopathogenic fungus Colletotrichum. CPLSs have high similarity to plant subtilisins and our previous work has shown that they were acquired by an ancient horizontal gene transfer event from plants. The rapid growth of sequence data in public databases enabled us to reexamine the structure and evolution of the CPLSs. A new plant subtilisin structural model aided us in refining the tertiary structure of CPLSs. Also, new information about protein interactions of plant subtilisin has provided new insights into the putative function of CPLSs. The availability of new genome sequences of members of the genus Colletotrichum gave us the opportunity to further validate our hypothesis that the CPLSs are unique to the Colletotrichum lineage. Together, this information furthers our knowledge of the potential role of the CPLSs in pathogenicity and the role of HGT in the genome evolution of plant pathogenic fungi.Fil: Armijos Jaramillo, Vinicio. Universidad de Salamanca; EspañaFil: Vargas, Walter Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentina. Universidad de Salamanca; EspañaFil: Sukno, Serenella A.. Universidad de Salamanca; EspañaFil: Thon, Michael R.. Universidad de Salamanca; Españ

Canonical or noncanonical? Structural plasticity of serine protease-binding loops in Kunitz-STI protease inhibitors

The Kunitz-Soybean Trypsin Inhibitor (Kunitz-STI) family is a large family of proteins with most of its members being protease inhibitors. The versatility of the inhibitory profile and the structural plasticity of these proteins, make this family a promising scaffold for designing new multifunctional proteins. Historically, Kunitz-STI inhibitors have been classified as canonical serine protease inhibitors, but new inhibitors with novel inhibition mechanisms have been described in recent years. Different inhibition mechanisms could be the result of different evolutionary pathways. In the present work, we performed a structural analysis of all the crystallographic structures available for Kunitz-STI inhibitors to characterize serine protease-binding loop structural features and locations. Our study suggests a relationship between the conformation of serine protease-binding loops and the inhibition mechanism, their location in the β-trefoil fold, and the plant source of the inhibitors. The classical canonical inhibitors of this family are restricted to plants from the Fabales order and bind their targets via the β4–β5 loop, whereas serine protease-binding loops in inhibitors from other plants lie mainly in the β5–β6 and β9–β10 loops. In addition, we found that the β5–β6 loop is used to inhibit two different families of serine proteases through a steric blockade inhibition mechanism. This work will help to change the general perception that all Kunitz-STI inhibitors are canonical inhibitors and proteins with protease-binding loops adopting noncanonical conformations are exceptions. Additionally, our results will help in the identification of protease-binding loops in uncharacterized or newly discovered inhibitors, and in the design of multifunctional proteins

The dilemma of bacterial expansins evolution. The unusual case of Streptomyces acidiscabies and Kutzneria sp. 744

Expansins are a superfamily of proteins mainly present in plants that are also found in bacteria, fungi and amoebozoa. Expansin proteins bind the plant cells wall and relax the cellulose microfibrils without any enzymatic action. The evolution of this kind of proteins exposes a complex pattern of horizontal gene transferences that makes difficult to determine the precise origin of non-plant expansins. We performed a genome-wide search of inter-domain horizontal gene transfer events using Streptomyces species and found a plant-like expansin in the Streptomyces acidiscabies proteome. This finding leads us to study in deep the origin and the characteristics of this peculiar protein, also present in the species Kutzneria sp.744. Using phylogenetic analyses, we determine that indeed S. acidiscabies and Kutzneria sp.744 expansins are located inside the plants expansins A clade. Using secondary and tertiary structural information, we observed that the electrostatic potentials and the folding of expansins are similar, independently of the proteins’ origin. Using all this information, we conclude that S. acidiscabies and Kutzneria sp.744 expansins have a plant origin but differ from plant and bacterial canonical expansins. This finding suggests that the experimental research around this kind of expansins can be promissory in the future

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

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

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

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

Functions of subtilisin family members in plants and fungi.

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