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Common protein sequence signatures associate with Sclerotinia borealis lifestyle and secretion in fungal pathogens of the Sclerotiniaceae.

Badet T, Peyraud R, Raffaele S - Front Plant Sci (2015)

Bottom Line: To spread successfully, S. borealis must synthesize proteins adapted to function in its specific environment.We found that enrichment in Thr, depletion in Glu and Lys, and low disorder frequency in hot loops are significantly associated with S. borealis proteins.High index proteins were also enriched in function associated with plant colonization in S. borealis, and in in planta-induced genes in S. sclerotiorum.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire des Interactions Plantes-Microorganismes, Institut National de la Recherche Agronomique, UMR441 Castanet-Tolosan, France ; Laboratoire des Interactions Plantes-Microorganismes, Centre National de la Recherche Scientifique, UMR2594 Castanet-Tolosan, France.

ABSTRACT
Fungal plant pathogens produce secreted proteins adapted to function outside fungal cells to facilitate colonization of their hosts. In many cases such as for fungi from the Sclerotiniaceae family the repertoire and function of secreted proteins remains elusive. In the Sclerotiniaceae, whereas Sclerotinia sclerotiorum and Botrytis cinerea are cosmopolitan broad host-range plant pathogens, Sclerotinia borealis has a psychrophilic lifestyle with a low optimal growth temperature, a narrow host range and geographic distribution. To spread successfully, S. borealis must synthesize proteins adapted to function in its specific environment. The search for signatures of adaptation to S. borealis lifestyle may therefore help revealing proteins critical for colonization of the environment by Sclerotiniaceae fungi. Here, we analyzed amino acids usage and intrinsic protein disorder in alignments of groups of orthologous proteins from the three Sclerotiniaceae species. We found that enrichment in Thr, depletion in Glu and Lys, and low disorder frequency in hot loops are significantly associated with S. borealis proteins. We designed an index to report bias in these properties and found that high index proteins were enriched among secreted proteins in the three Sclerotiniaceae fungi. High index proteins were also enriched in function associated with plant colonization in S. borealis, and in in planta-induced genes in S. sclerotiorum. We highlight a novel putative antifreeze protein and a novel putative lytic polysaccharide monooxygenase identified through our pipeline as candidate proteins involved in colonization of the environment. Our findings suggest that similar protein signatures associate with S. borealis lifestyle and with secretion in the Sclerotiniaceae. These signatures may be useful for identifying proteins of interest as targets for the management of plant diseases.

No MeSH data available.


Related in: MedlinePlus

Candidate proteins associated with colonization of the environment identified based on high sTEKhot values. (A) Multiple protein sequence alignment of B. cinerea BC1G_03854 (sTEKhot = 4.29), S. borealis SBOR_9046 (sTEKhot = 10.01), S. sclerotiorum SS1G_10836 (sTEKhot = 7.34) and the hyperactive Type I antifreeze protein “Maxi” from Pseudopleuronectes americanus (4KE2_A). (B) Superimposition of Maxi antifreeze protein structure (tan) and SS1G_10836 model structure (rainbow). (C) Surface hydrophobicity of SS1G_10836 model dimer. Dotted line corresponds to the position of the section shown on the right, illustrating the characteristic hydrophilic inner core of the dimer. (D) Multiple protein sequence alignment of B. cinerea BC1G_07573 (sTEKhot = 7.07), S. borealis SBOR_1255 (sTEKhot = 3.79), S. sclerotiorum SS1G_03146 (sTEKhot = 1.58) and the AA11 Lytic Polysaccharide Monooxygenase from Aspergillus oryzae (4MAH_A). (E) Superimposition of A. oryzae AA11 structure (tan) and SS1G_03146 model structure (rainbow). (F)SS1G_10836 and SS1G_03146 gene expression in vitro (PDB, Potato Dextrose Broth), during colonization of Arabidopsis thaliana (lesion periphery and lesion center) and in sclerotia. Error bars show standard error of the mean from two independent biological replicates.
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Figure 8: Candidate proteins associated with colonization of the environment identified based on high sTEKhot values. (A) Multiple protein sequence alignment of B. cinerea BC1G_03854 (sTEKhot = 4.29), S. borealis SBOR_9046 (sTEKhot = 10.01), S. sclerotiorum SS1G_10836 (sTEKhot = 7.34) and the hyperactive Type I antifreeze protein “Maxi” from Pseudopleuronectes americanus (4KE2_A). (B) Superimposition of Maxi antifreeze protein structure (tan) and SS1G_10836 model structure (rainbow). (C) Surface hydrophobicity of SS1G_10836 model dimer. Dotted line corresponds to the position of the section shown on the right, illustrating the characteristic hydrophilic inner core of the dimer. (D) Multiple protein sequence alignment of B. cinerea BC1G_07573 (sTEKhot = 7.07), S. borealis SBOR_1255 (sTEKhot = 3.79), S. sclerotiorum SS1G_03146 (sTEKhot = 1.58) and the AA11 Lytic Polysaccharide Monooxygenase from Aspergillus oryzae (4MAH_A). (E) Superimposition of A. oryzae AA11 structure (tan) and SS1G_03146 model structure (rainbow). (F)SS1G_10836 and SS1G_03146 gene expression in vitro (PDB, Potato Dextrose Broth), during colonization of Arabidopsis thaliana (lesion periphery and lesion center) and in sclerotia. Error bars show standard error of the mean from two independent biological replicates.

Mentions: To illustrate the value of the sTEKhot index for the exploration of the proteome of fungi from the Sclerotiniaceae, we analyzed in detail the sequence of two proteins with high sTEKhot but with no assigned function. Over the three proteomes analyzed, S. borealis SBOR_9046 had the highest sTEKhot (10.01). In S. sclerotiorum, its ortholog is SS1G_10836 which ranked as the 5th highest sTEKhot in S. sclerotiorum (7.34). In B. cinerea, its ortholog is BC1G_03854 which ranked as the 23rd highest sTEKhot in B. cinerea (4.29). No interproscan domain or GO terms were associated with these proteins of 171 amino acids (except SS1G_10836 which is 173 amino acids long). To get insights into their putative function, we performed protein structure modeling and fold recognition using the I-TASSER server (Zhang, 2008). The closest structural analog was the antifreeze protein Maxi from winter flounder (Pseudopleuronectes americanus) (Sun et al., 2014). Although sequence similarity with Maxi was limited (from 15.2% identity for SBOR_9046 to 16.2% identity for SS1G_10836), superimposition of SS1G_10836 predicted structure with Maxi structure showed a Root Mean Square Deviation < 2.3Å and a TM-score of 0.875, indicating structural similarity deviating significantly from random (Figures 8A,B). Analysis of SBOR_9046, SS1G_10836 and BC1G_03854 sequence by TargetFreeze (He et al., 2015) supported the prediction as antifreeze proteins. The Sclerotiniaceae proteins contain four Cys residues located in the kink of predicted structures that may stabilize folding like, although these residues were not predicted to form disulfide bonds by Disulfind (Ceroni et al., 2006). Antifreeze proteins have been reporting that rely on disulfide bonds for folding (Basu et al., 2015) whereas others do not (Kondo et al., 2012; Sun et al., 2014). Like other known fungal antifreeze proteins (Kondo et al., 2012), but unlike Maxi, SBOR_9046 and its orthologs are predicted to be secreted. A unique feature of Maxi among antifreeze proteins is the presence of ice-binding residues buried within the four-helix bundle instead of exposed on their surface (Sun et al., 2014). A prediction of SS1G_10836 dimer structure supports the existence of rather hydrophilic pockets buried within the four-helix bundle, suggesting that the mechanism of ice binding of Maxi could be conserved in SS1G_10836 and its orthologs (Figure 8C). To get insights into SS1G_10836 function, we analyzed the expression of the corresponding gene in mycelium grown in Potato Dextrose Broth (PDB), during the colonization of Arabidopsis plants and in sclerotia by quantitative RT-PCR. This revealed a 3.3-fold induction (log2 = 1.7) specific to sclerotia (Figure 8F). Since sclerotia overwinter in the soil, putative antifreeze proteins may contribute to survival of these structures both in arctic and temperate climates.


Common protein sequence signatures associate with Sclerotinia borealis lifestyle and secretion in fungal pathogens of the Sclerotiniaceae.

Badet T, Peyraud R, Raffaele S - Front Plant Sci (2015)

Candidate proteins associated with colonization of the environment identified based on high sTEKhot values. (A) Multiple protein sequence alignment of B. cinerea BC1G_03854 (sTEKhot = 4.29), S. borealis SBOR_9046 (sTEKhot = 10.01), S. sclerotiorum SS1G_10836 (sTEKhot = 7.34) and the hyperactive Type I antifreeze protein “Maxi” from Pseudopleuronectes americanus (4KE2_A). (B) Superimposition of Maxi antifreeze protein structure (tan) and SS1G_10836 model structure (rainbow). (C) Surface hydrophobicity of SS1G_10836 model dimer. Dotted line corresponds to the position of the section shown on the right, illustrating the characteristic hydrophilic inner core of the dimer. (D) Multiple protein sequence alignment of B. cinerea BC1G_07573 (sTEKhot = 7.07), S. borealis SBOR_1255 (sTEKhot = 3.79), S. sclerotiorum SS1G_03146 (sTEKhot = 1.58) and the AA11 Lytic Polysaccharide Monooxygenase from Aspergillus oryzae (4MAH_A). (E) Superimposition of A. oryzae AA11 structure (tan) and SS1G_03146 model structure (rainbow). (F)SS1G_10836 and SS1G_03146 gene expression in vitro (PDB, Potato Dextrose Broth), during colonization of Arabidopsis thaliana (lesion periphery and lesion center) and in sclerotia. Error bars show standard error of the mean from two independent biological replicates.
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Figure 8: Candidate proteins associated with colonization of the environment identified based on high sTEKhot values. (A) Multiple protein sequence alignment of B. cinerea BC1G_03854 (sTEKhot = 4.29), S. borealis SBOR_9046 (sTEKhot = 10.01), S. sclerotiorum SS1G_10836 (sTEKhot = 7.34) and the hyperactive Type I antifreeze protein “Maxi” from Pseudopleuronectes americanus (4KE2_A). (B) Superimposition of Maxi antifreeze protein structure (tan) and SS1G_10836 model structure (rainbow). (C) Surface hydrophobicity of SS1G_10836 model dimer. Dotted line corresponds to the position of the section shown on the right, illustrating the characteristic hydrophilic inner core of the dimer. (D) Multiple protein sequence alignment of B. cinerea BC1G_07573 (sTEKhot = 7.07), S. borealis SBOR_1255 (sTEKhot = 3.79), S. sclerotiorum SS1G_03146 (sTEKhot = 1.58) and the AA11 Lytic Polysaccharide Monooxygenase from Aspergillus oryzae (4MAH_A). (E) Superimposition of A. oryzae AA11 structure (tan) and SS1G_03146 model structure (rainbow). (F)SS1G_10836 and SS1G_03146 gene expression in vitro (PDB, Potato Dextrose Broth), during colonization of Arabidopsis thaliana (lesion periphery and lesion center) and in sclerotia. Error bars show standard error of the mean from two independent biological replicates.
Mentions: To illustrate the value of the sTEKhot index for the exploration of the proteome of fungi from the Sclerotiniaceae, we analyzed in detail the sequence of two proteins with high sTEKhot but with no assigned function. Over the three proteomes analyzed, S. borealis SBOR_9046 had the highest sTEKhot (10.01). In S. sclerotiorum, its ortholog is SS1G_10836 which ranked as the 5th highest sTEKhot in S. sclerotiorum (7.34). In B. cinerea, its ortholog is BC1G_03854 which ranked as the 23rd highest sTEKhot in B. cinerea (4.29). No interproscan domain or GO terms were associated with these proteins of 171 amino acids (except SS1G_10836 which is 173 amino acids long). To get insights into their putative function, we performed protein structure modeling and fold recognition using the I-TASSER server (Zhang, 2008). The closest structural analog was the antifreeze protein Maxi from winter flounder (Pseudopleuronectes americanus) (Sun et al., 2014). Although sequence similarity with Maxi was limited (from 15.2% identity for SBOR_9046 to 16.2% identity for SS1G_10836), superimposition of SS1G_10836 predicted structure with Maxi structure showed a Root Mean Square Deviation < 2.3Å and a TM-score of 0.875, indicating structural similarity deviating significantly from random (Figures 8A,B). Analysis of SBOR_9046, SS1G_10836 and BC1G_03854 sequence by TargetFreeze (He et al., 2015) supported the prediction as antifreeze proteins. The Sclerotiniaceae proteins contain four Cys residues located in the kink of predicted structures that may stabilize folding like, although these residues were not predicted to form disulfide bonds by Disulfind (Ceroni et al., 2006). Antifreeze proteins have been reporting that rely on disulfide bonds for folding (Basu et al., 2015) whereas others do not (Kondo et al., 2012; Sun et al., 2014). Like other known fungal antifreeze proteins (Kondo et al., 2012), but unlike Maxi, SBOR_9046 and its orthologs are predicted to be secreted. A unique feature of Maxi among antifreeze proteins is the presence of ice-binding residues buried within the four-helix bundle instead of exposed on their surface (Sun et al., 2014). A prediction of SS1G_10836 dimer structure supports the existence of rather hydrophilic pockets buried within the four-helix bundle, suggesting that the mechanism of ice binding of Maxi could be conserved in SS1G_10836 and its orthologs (Figure 8C). To get insights into SS1G_10836 function, we analyzed the expression of the corresponding gene in mycelium grown in Potato Dextrose Broth (PDB), during the colonization of Arabidopsis plants and in sclerotia by quantitative RT-PCR. This revealed a 3.3-fold induction (log2 = 1.7) specific to sclerotia (Figure 8F). Since sclerotia overwinter in the soil, putative antifreeze proteins may contribute to survival of these structures both in arctic and temperate climates.

Bottom Line: To spread successfully, S. borealis must synthesize proteins adapted to function in its specific environment.We found that enrichment in Thr, depletion in Glu and Lys, and low disorder frequency in hot loops are significantly associated with S. borealis proteins.High index proteins were also enriched in function associated with plant colonization in S. borealis, and in in planta-induced genes in S. sclerotiorum.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire des Interactions Plantes-Microorganismes, Institut National de la Recherche Agronomique, UMR441 Castanet-Tolosan, France ; Laboratoire des Interactions Plantes-Microorganismes, Centre National de la Recherche Scientifique, UMR2594 Castanet-Tolosan, France.

ABSTRACT
Fungal plant pathogens produce secreted proteins adapted to function outside fungal cells to facilitate colonization of their hosts. In many cases such as for fungi from the Sclerotiniaceae family the repertoire and function of secreted proteins remains elusive. In the Sclerotiniaceae, whereas Sclerotinia sclerotiorum and Botrytis cinerea are cosmopolitan broad host-range plant pathogens, Sclerotinia borealis has a psychrophilic lifestyle with a low optimal growth temperature, a narrow host range and geographic distribution. To spread successfully, S. borealis must synthesize proteins adapted to function in its specific environment. The search for signatures of adaptation to S. borealis lifestyle may therefore help revealing proteins critical for colonization of the environment by Sclerotiniaceae fungi. Here, we analyzed amino acids usage and intrinsic protein disorder in alignments of groups of orthologous proteins from the three Sclerotiniaceae species. We found that enrichment in Thr, depletion in Glu and Lys, and low disorder frequency in hot loops are significantly associated with S. borealis proteins. We designed an index to report bias in these properties and found that high index proteins were enriched among secreted proteins in the three Sclerotiniaceae fungi. High index proteins were also enriched in function associated with plant colonization in S. borealis, and in in planta-induced genes in S. sclerotiorum. We highlight a novel putative antifreeze protein and a novel putative lytic polysaccharide monooxygenase identified through our pipeline as candidate proteins involved in colonization of the environment. Our findings suggest that similar protein signatures associate with S. borealis lifestyle and with secretion in the Sclerotiniaceae. These signatures may be useful for identifying proteins of interest as targets for the management of plant diseases.

No MeSH data available.


Related in: MedlinePlus