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Genome-wide identification of HrpL-regulated genes in the necrotrophic phytopathogen Dickeya dadantii 3937.

Yang S, Peng Q, Zhang Q, Zou L, Li Y, Robert C, Pritchard L, Liu H, Hovey R, Wang Q, Birch P, Toth IK, Yang CH - PLoS ONE (2010)

Bottom Line: In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence.CONCLUSION/SIGNIFICANCES: We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors.Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT

Background: Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression.

Methodology/principal findings: To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. CONCLUSION/SIGNIFICANCES: We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens.

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HrpL binds to hrp box specifically.hrpA or hrpN promoter regions containing the hrp box were amplified by PCR and labeled with digoxigenin (DIG) (Pierce, Rockford, IL). The core RNAP or His6-HrpL sigma factor alone were mixed with 200 fmol labeled DNA probe (1×) (Lane 1–2); or core RNAP and His6-HrpL sigma factor mixture together with 1× labeled DNA probe were mixed with same unlabelled DNA probe with various concentrations up to 4000 fmol (20×), or up to 3200 fmol (16×) of unlabeled internal fragment of the hrpA gene. A: Labeled and unlabeled hrpA promoter region containing hrp box and the control fragments for the hrpA ORF region without hrp box (hrpAORF) were used. B: Labeled and unlabelled hrpN promoter region containing hrp box and the control fragments for the hrpA ORF region without hrp box were used.
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pone-0013472-g001: HrpL binds to hrp box specifically.hrpA or hrpN promoter regions containing the hrp box were amplified by PCR and labeled with digoxigenin (DIG) (Pierce, Rockford, IL). The core RNAP or His6-HrpL sigma factor alone were mixed with 200 fmol labeled DNA probe (1×) (Lane 1–2); or core RNAP and His6-HrpL sigma factor mixture together with 1× labeled DNA probe were mixed with same unlabelled DNA probe with various concentrations up to 4000 fmol (20×), or up to 3200 fmol (16×) of unlabeled internal fragment of the hrpA gene. A: Labeled and unlabeled hrpA promoter region containing hrp box and the control fragments for the hrpA ORF region without hrp box (hrpAORF) were used. B: Labeled and unlabelled hrpN promoter region containing hrp box and the control fragments for the hrpA ORF region without hrp box were used.

Mentions: To study the interaction of the HrpL protein with the promoter regions of hrpA and hrpN in 3937, an electrophoretic mobility shift assay (EMSA) was employed. RNAP (Epicentre Technologies, Madison, WI) and the His6-tagged HrpL (His6-HrpL) were incubated together with digoxigenin (DIG) labeled DNA fragments of hrpA or hrpN promoter regions containing hrp box sequences. Neither the His6-HrpL nor RNAP alone showed detectable binding to the hrp box DNAs of hrpA and hrpN (Fig. 1). However, mobility of the DNA fragments was retarded when hrp box DNA of hrpA or hrpN was incubated with RNAP and His6-HrpL (Fig. 1), indicative of the requirement of RNAP in the binding of HrpL to these DNA fragments.


Genome-wide identification of HrpL-regulated genes in the necrotrophic phytopathogen Dickeya dadantii 3937.

Yang S, Peng Q, Zhang Q, Zou L, Li Y, Robert C, Pritchard L, Liu H, Hovey R, Wang Q, Birch P, Toth IK, Yang CH - PLoS ONE (2010)

HrpL binds to hrp box specifically.hrpA or hrpN promoter regions containing the hrp box were amplified by PCR and labeled with digoxigenin (DIG) (Pierce, Rockford, IL). The core RNAP or His6-HrpL sigma factor alone were mixed with 200 fmol labeled DNA probe (1×) (Lane 1–2); or core RNAP and His6-HrpL sigma factor mixture together with 1× labeled DNA probe were mixed with same unlabelled DNA probe with various concentrations up to 4000 fmol (20×), or up to 3200 fmol (16×) of unlabeled internal fragment of the hrpA gene. A: Labeled and unlabeled hrpA promoter region containing hrp box and the control fragments for the hrpA ORF region without hrp box (hrpAORF) were used. B: Labeled and unlabelled hrpN promoter region containing hrp box and the control fragments for the hrpA ORF region without hrp box were used.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2957411&req=5

pone-0013472-g001: HrpL binds to hrp box specifically.hrpA or hrpN promoter regions containing the hrp box were amplified by PCR and labeled with digoxigenin (DIG) (Pierce, Rockford, IL). The core RNAP or His6-HrpL sigma factor alone were mixed with 200 fmol labeled DNA probe (1×) (Lane 1–2); or core RNAP and His6-HrpL sigma factor mixture together with 1× labeled DNA probe were mixed with same unlabelled DNA probe with various concentrations up to 4000 fmol (20×), or up to 3200 fmol (16×) of unlabeled internal fragment of the hrpA gene. A: Labeled and unlabeled hrpA promoter region containing hrp box and the control fragments for the hrpA ORF region without hrp box (hrpAORF) were used. B: Labeled and unlabelled hrpN promoter region containing hrp box and the control fragments for the hrpA ORF region without hrp box were used.
Mentions: To study the interaction of the HrpL protein with the promoter regions of hrpA and hrpN in 3937, an electrophoretic mobility shift assay (EMSA) was employed. RNAP (Epicentre Technologies, Madison, WI) and the His6-tagged HrpL (His6-HrpL) were incubated together with digoxigenin (DIG) labeled DNA fragments of hrpA or hrpN promoter regions containing hrp box sequences. Neither the His6-HrpL nor RNAP alone showed detectable binding to the hrp box DNAs of hrpA and hrpN (Fig. 1). However, mobility of the DNA fragments was retarded when hrp box DNA of hrpA or hrpN was incubated with RNAP and His6-HrpL (Fig. 1), indicative of the requirement of RNAP in the binding of HrpL to these DNA fragments.

Bottom Line: In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence.CONCLUSION/SIGNIFICANCES: We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors.Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT

Background: Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression.

Methodology/principal findings: To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. CONCLUSION/SIGNIFICANCES: We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens.

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