we previously reported ( see ref . 15 ) a prediction of the tertiary protein structure of a c. graminicola cpls ( locus i d glrg_05578 ) using crystallized protein structures of subtilisins with sequence similarity to cplss . the most similar one was the sbt3 of solanum lycopersicum ( pdb i d 3i6s ) . recently , the crystal structure of the subtilisin cucumisin of cucumis melo was published . with this new structure available , we recalculated the 3-d model of glrg_05578 ( the cpls of colletotrichum graminicola ) using the phyre 2 server . the new structural model has only 57 residues predicted ab initio ( highly unreliable sites ) instead of 73 as reported in armijos jaramillo et al . the new glrg_05578 structural model was aligned to the structural models of sbt3 and cucumisin to determine the similarities and the differences with the previous model . the structural alignment of the previous model of glrg_05578 and sbt3 has a maximum rmsd ( root  mean  square  deviation ) of 11.68 and a minimum of 0.02 . the new model has a highest rmsd value of 12.65 and the lowest value of 0.03 . these results show that the new model is less similar to sbt3 than the one reported previously . however , the new model is more similar to the sbt3 subtilisin than to the cucumisin ( fig .  1 ) ( maximum rmsd of 17.59 and minimum rmsd of 3.09 with the old model and maximum rmsd of 15.12 and minimum rmsd of 1.84 with the new model ) . the new model reveals differences in the tertiary protein structures of the pa domain , -hairpin arrangement and one of the putative ca replacement sites between cpls and tomato subtilisin . these sites are important in dimerization and stabilization of tomato sbt3 and the differences with cpls could imply differences in the quaternary structure ( conformation of dimers ) and/or the specificity of interaction with other proteins . structural alignment of the new model predicted for the tertiary conformation of cpls glrg_05578 of colletotrichum graminicola and one monomer of the crystallized subtilisin cucumisin ( 3vta ) of cucumis melo . the color spectrum represents the values of pairwise rmsd , with blue specifying minimum values and red indicating maximum values . labels indicate the relevant domains and sites of the proteins conserved in subtilisin sbt3 of tomato in ottman et al . although the protein targets of the cplss are unknown , analysis of the most similar plant proteins could reveal potential clues . a blastp search in the uniprot / swissprot database shows that the most similar proteins to cplss are 3 subtilisins of arabidopsis thaliana ( o65351 , o64495 , q9lll8 ) and the cucumisin of cucumis melo ( q39547 ) . information about the interaction of the arabidopsis subtilisins is available in the string database , but it is not available for cucumisin . this database shows that o64495 is co - expressed with several proteins involved in the development of stomata , including at1g63700 , at5g62230 , at1g80080 , at5g53210 , at3g06120 , and others . the protein o65351 ( ara12 ) is predicted to interact with at1g22300 and at4g09000 , 2 general regulatory factors . gene q9lll8 is co - expressed with at4g35350 and at1g20850 , which have peptidase activity and cysteine - type peptidase activity , respectively . the interaction and co - expression of these proteins could help to explain the role of cplss in the host and help to plan future studies . however , as we commented in armijos jaramillo et al . , the subtilisins in plants have a variety of metabolic functions . plant subtilisins with high percentage of sequence similarity to cplss are involved in varied , and sometimes unrelated biological process . ( ara12 ) is essential for mucilage release from seed coats , whereas o64495 is involved in stomatal development and distribution , and q9lll8 is involved in negative regulation of catalytic activity . the functions of the cplss are not clear yet , but they could be involved in the mis - regulation or mimicry of any of those processes . alternatively , novel functions might also be expected for cpls . our hypothesis is that the cpls hgt event took place in an ancestor of the genus colletotrichum . therefore , cplss should be present in all of the extant species of colletotrichum and absent in species outside the genus . we previously showed that the cplss are present in the genome sequences of all 3 species of colletotrichum that were available at that time but absent in all other fungi . , reported the presence of subtilisins , likely resulting from an hgt event , in the genomes of c. orbiculare ( cob 12233 , cob 06327 ) and c. gloeosporioides ( cggc5 2662 ) , adding further support to our hypothesis . in addition , genome sequences for several more species of colletotrichum have recently become available , enabling us to further validate our hypothesis . a blast search of the genbank nr database revealed the presence of cpls homologs in the genome of c. gloeosporioides nara gc5 ( genbank accession numbers : ela37268 and ela38265 ) . we also performed blast searches against the proteomes of 2 additional species of colletotrichum ( c. gloeosporioides 23 and c. fiorinae mh 18 , http://www.jgi.doe.gov ) and found that both species have cpls homologs . a phylogenetic analysis ( data not shown ) shows that all of the putative cpls homologs share common ancestry with the cplss identified in our previous report . this idea is consistent with our hypothesis of a plant protein that was transferred to an ancestor of the genus colletotrichum . hgt events among eukaryotes are presumed to be rare , and we assume that those that we observe in populations , such as the cplss , have been fixed due to strong selective pressure . in the case of plant pathogenic fungi , such as colletotrichum , horizontally transferred genes may have some role in pathogenicity , enabling the fungus to adapt to new hosts or enhancing its ability to colonize its host . research is already underway which is aimed at determining whether cplss have a role in pathogenicity in colletotrichum .
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 .