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Redox proteomics of tomato in response to Pseudomonas syringae infection.

Balmant KM, Parker J, Yoo MJ, Zhu N, Dufresne C, Chen S - Hortic Res (2015)

Bottom Line: In addition, the results of the redox changes were compared and corrected with the protein level changes.A total of 90 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, biosynthesis of cysteine, sucrose and brassinosteroid, cell wall biogenesis, polysaccharide/starch biosynthesis, cuticle development, lipid metabolism, proteolysis, tricarboxylic acid cycle, protein targeting to vacuole, and oxidation-reduction.This inventory of previously unknown protein redox switches in tomato pathogen defense lays a foundation for future research toward understanding the biological significance of protein redox modifications in plant defense responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Genetics Institute, University of Florida , Gainesville, FL, USA ; Plant Molecular and Cellular Biology Program, University of Florida , Gainesville, FL, USA.

ABSTRACT
Unlike mammals with adaptive immunity, plants rely on their innate immunity based on pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) for pathogen defense. Reactive oxygen species, known to play crucial roles in PTI and ETI, can perturb cellular redox homeostasis and lead to changes of redox-sensitive proteins through modification of cysteine sulfhydryl groups. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, little is known about redox proteins and how they function in PTI and ETI. In this study, cysTMT proteomics technology was used to identify similarities and differences of protein redox modifications in tomato resistant (PtoR) and susceptible (prf3) genotypes in response to Pseudomonas syringae pv tomato (Pst) infection. In addition, the results of the redox changes were compared and corrected with the protein level changes. A total of 90 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, biosynthesis of cysteine, sucrose and brassinosteroid, cell wall biogenesis, polysaccharide/starch biosynthesis, cuticle development, lipid metabolism, proteolysis, tricarboxylic acid cycle, protein targeting to vacuole, and oxidation-reduction. This inventory of previously unknown protein redox switches in tomato pathogen defense lays a foundation for future research toward understanding the biological significance of protein redox modifications in plant defense responses.

No MeSH data available.


Related in: MedlinePlus

Comparison of redox proteomes and identified redox proteins in different tomato genotypes. (a) Venn diagram showing the number of shared proteins among three different biological replicates. (b) The number of proteins underwent reduction (gray) or oxidation (dark) in the susceptible prf3 and resistant PtoR genotypes at early and late stage of Pst infection. PtoR clearly showed more oxidized proteins at both 4 hai and 24 hai, compared to prf3.
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fig2: Comparison of redox proteomes and identified redox proteins in different tomato genotypes. (a) Venn diagram showing the number of shared proteins among three different biological replicates. (b) The number of proteins underwent reduction (gray) or oxidation (dark) in the susceptible prf3 and resistant PtoR genotypes at early and late stage of Pst infection. PtoR clearly showed more oxidized proteins at both 4 hai and 24 hai, compared to prf3.

Mentions: Identification of redox-responsive cysteines, peptides, and proteins Redox proteomics approaches are based on differential labeling of redox-modified cysteines in proteins and have shown utility in unraveling important biological mechanisms. Here we chose to use the TCA method to precipitate proteins because TCA not only lyses the cells, but also protonates all thiolates to prevent thiol-disulfide exchange reactions.29 In addition, a reverse-labeling procedure was performed, in which IAM was used to block free thiol groups. After alkylation of the free thiols, reduction of reversibly oxidized cysteine residues and labeling with cysTMT reagents were conducted. This reverse-labeling procedure maintains the initial redox state of the proteins and prevents artificial oxidation during sample preparation. Therefore, the increases of the cysTMT signals from specific cysteine peptides derived from treated samples compared to control samples indicate the presence of oxidation responsive/sensitive cysteines (Figure 1). The use of this powerful methodology enabled identification of proteins with cysteines that underwent redox modifications in the resistant PtoR and susceptible prf3 genotypes of tomato at early and late time points after infection. In total, 4348 proteins were confidently identified among three biological replicates with a FDR of 0.05. A total of 3258 cysteine-containing peptides and 1580-associate proteins were observed. Of the cysteine-containing peptides, 2749 peptides (84.3%) were labeled with cysTMT tags and correlated to 1413 proteins. A comparison of the three biological replicates showed that 217 proteins were present in all the replicates, and 529 were present in at least two replicates (Figure 2). It should be noted that the three replicates represent independent biological replicates using the dip-inoculation method, which is close to natural infection, but known to generate more variations than the infiltration method.30 A peptide abundance greater than 1.2 or less than 0.8 with a p < 0.05 was considered to be redox-sensitive (Supplementary Figure S1). As previously addressed, the identification of redox proteins may be complicated due to protein turnover. This issue may be solved by comparing the redox proteomics data with protein level change data31,32 (Supplementary Figure S2). In this report, the total protein level data generated by Parker et al.26 was used to correct the redox changes. It is important to highlight that the same set of samples were used in the proteomics study23 and in our redox proteomics study in order to avoid sample variation. For example, a dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex-like protein was identified as a potential redox-regulated protein in the prf3 susceptible genotype with a fold change of 1.46 at 4 hai (Table 1). However, the iTRAQ analysis revealed a significant fold change of 2.06. Therefore, the redox-fold change may be due to the protein level change rather than a cysteine redox response. Cleary, our redox proteomics method showed utility in the identification of redox-responsive cysteines, peptides, and proteins.


Redox proteomics of tomato in response to Pseudomonas syringae infection.

Balmant KM, Parker J, Yoo MJ, Zhu N, Dufresne C, Chen S - Hortic Res (2015)

Comparison of redox proteomes and identified redox proteins in different tomato genotypes. (a) Venn diagram showing the number of shared proteins among three different biological replicates. (b) The number of proteins underwent reduction (gray) or oxidation (dark) in the susceptible prf3 and resistant PtoR genotypes at early and late stage of Pst infection. PtoR clearly showed more oxidized proteins at both 4 hai and 24 hai, compared to prf3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591677&req=5

fig2: Comparison of redox proteomes and identified redox proteins in different tomato genotypes. (a) Venn diagram showing the number of shared proteins among three different biological replicates. (b) The number of proteins underwent reduction (gray) or oxidation (dark) in the susceptible prf3 and resistant PtoR genotypes at early and late stage of Pst infection. PtoR clearly showed more oxidized proteins at both 4 hai and 24 hai, compared to prf3.
Mentions: Identification of redox-responsive cysteines, peptides, and proteins Redox proteomics approaches are based on differential labeling of redox-modified cysteines in proteins and have shown utility in unraveling important biological mechanisms. Here we chose to use the TCA method to precipitate proteins because TCA not only lyses the cells, but also protonates all thiolates to prevent thiol-disulfide exchange reactions.29 In addition, a reverse-labeling procedure was performed, in which IAM was used to block free thiol groups. After alkylation of the free thiols, reduction of reversibly oxidized cysteine residues and labeling with cysTMT reagents were conducted. This reverse-labeling procedure maintains the initial redox state of the proteins and prevents artificial oxidation during sample preparation. Therefore, the increases of the cysTMT signals from specific cysteine peptides derived from treated samples compared to control samples indicate the presence of oxidation responsive/sensitive cysteines (Figure 1). The use of this powerful methodology enabled identification of proteins with cysteines that underwent redox modifications in the resistant PtoR and susceptible prf3 genotypes of tomato at early and late time points after infection. In total, 4348 proteins were confidently identified among three biological replicates with a FDR of 0.05. A total of 3258 cysteine-containing peptides and 1580-associate proteins were observed. Of the cysteine-containing peptides, 2749 peptides (84.3%) were labeled with cysTMT tags and correlated to 1413 proteins. A comparison of the three biological replicates showed that 217 proteins were present in all the replicates, and 529 were present in at least two replicates (Figure 2). It should be noted that the three replicates represent independent biological replicates using the dip-inoculation method, which is close to natural infection, but known to generate more variations than the infiltration method.30 A peptide abundance greater than 1.2 or less than 0.8 with a p < 0.05 was considered to be redox-sensitive (Supplementary Figure S1). As previously addressed, the identification of redox proteins may be complicated due to protein turnover. This issue may be solved by comparing the redox proteomics data with protein level change data31,32 (Supplementary Figure S2). In this report, the total protein level data generated by Parker et al.26 was used to correct the redox changes. It is important to highlight that the same set of samples were used in the proteomics study23 and in our redox proteomics study in order to avoid sample variation. For example, a dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex-like protein was identified as a potential redox-regulated protein in the prf3 susceptible genotype with a fold change of 1.46 at 4 hai (Table 1). However, the iTRAQ analysis revealed a significant fold change of 2.06. Therefore, the redox-fold change may be due to the protein level change rather than a cysteine redox response. Cleary, our redox proteomics method showed utility in the identification of redox-responsive cysteines, peptides, and proteins.

Bottom Line: In addition, the results of the redox changes were compared and corrected with the protein level changes.A total of 90 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, biosynthesis of cysteine, sucrose and brassinosteroid, cell wall biogenesis, polysaccharide/starch biosynthesis, cuticle development, lipid metabolism, proteolysis, tricarboxylic acid cycle, protein targeting to vacuole, and oxidation-reduction.This inventory of previously unknown protein redox switches in tomato pathogen defense lays a foundation for future research toward understanding the biological significance of protein redox modifications in plant defense responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Genetics Institute, University of Florida , Gainesville, FL, USA ; Plant Molecular and Cellular Biology Program, University of Florida , Gainesville, FL, USA.

ABSTRACT
Unlike mammals with adaptive immunity, plants rely on their innate immunity based on pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) for pathogen defense. Reactive oxygen species, known to play crucial roles in PTI and ETI, can perturb cellular redox homeostasis and lead to changes of redox-sensitive proteins through modification of cysteine sulfhydryl groups. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, little is known about redox proteins and how they function in PTI and ETI. In this study, cysTMT proteomics technology was used to identify similarities and differences of protein redox modifications in tomato resistant (PtoR) and susceptible (prf3) genotypes in response to Pseudomonas syringae pv tomato (Pst) infection. In addition, the results of the redox changes were compared and corrected with the protein level changes. A total of 90 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, biosynthesis of cysteine, sucrose and brassinosteroid, cell wall biogenesis, polysaccharide/starch biosynthesis, cuticle development, lipid metabolism, proteolysis, tricarboxylic acid cycle, protein targeting to vacuole, and oxidation-reduction. This inventory of previously unknown protein redox switches in tomato pathogen defense lays a foundation for future research toward understanding the biological significance of protein redox modifications in plant defense responses.

No MeSH data available.


Related in: MedlinePlus