Production of small cysteine-rich effector proteins in Escherichia coli for structural and functional studies

Zhang, Xiaoxiao, Nguyen, Neal, Breen, Susan, Outram, Megan A., Dodds, Peter N., Kobe, Bostjan, Solomon, Peter S. and Williams, Simon J. (2016) Production of small cysteine-rich effector proteins in Escherichia coli for structural and functional studies. Molecular Plant Pathology, 18 1: 141-151. doi:10.1111/mpp.12385


Author Zhang, Xiaoxiao
Nguyen, Neal
Breen, Susan
Outram, Megan A.
Dodds, Peter N.
Kobe, Bostjan
Solomon, Peter S.
Williams, Simon J.
Title Production of small cysteine-rich effector proteins in Escherichia coli for structural and functional studies
Formatted title
Production of small cysteine-rich effector proteins in Escherichia coli for structural and functional studies
Journal name Molecular Plant Pathology   Check publisher's open access policy
ISSN 1364-3703
1464-6722
Publication date 2016-02-24
Year available 2017
Sub-type Article (original research)
DOI 10.1111/mpp.12385
Open Access Status Not Open Access
Volume 18
Issue 1
Start page 141
End page 151
Total pages 11
Place of publication Chichester, West Sussex, United Kingdom
Publisher Wiley-Blackwell Publishing
Language eng
Subject 1312 Molecular Biology
1102 Agronomy and Crop Science
1111 Soil Science
1110 Plant Science
Abstract Although the lifestyles and infection strategies of plant pathogens are diverse, a prevailing feature is the use of an arsenal of secreted proteins, known as effectors, which aid in microbial infection. In the case of eukaryotic filamentous pathogens, such as fungi and oomycetes, effector proteins are typically dissimilar, at the protein sequence level, to known protein families and functional domains. Consequently, we currently have a limited understanding of how fungal and oomycete effectors promote disease. Protein biochemistry and structural biology are two methods that can contribute greatly to the understanding of protein function. Both techniques are dependent on obtaining proteins that are pure and functional, and generally require the use of heterologous recombinant protein expression systems. Here, we present a general scheme and methodology for the production and characterization of small cysteine-rich (SCR) effectors utilizing Escherichia coli expression systems. Using this approach, we successfully produced cysteine-rich effectors derived from the biotrophic fungal pathogen Melampsora lini and the necrotrophic fungal pathogen Parastagonospora nodorum. Access to functional recombinant proteins facilitated crystallization and functional experiments. These results are discussed in the context of a general workflow that may serve as a template for others interested in understanding the function of SCR effector(s) from their plant pathogen(s) of interest.
Formatted abstract
Although the lifestyles and infection strategies of plant pathogens are diverse, a prevailing feature is the use of an arsenal of secreted proteins, known as effectors, which aid in microbial infection. In the case of eukaryotic filamentous pathogens, such as fungi and oomycetes, effector proteins are typically dissimilar, at the protein sequence level, to known protein families and functional domains. Consequently, we currently have a limited understanding of how fungal and oomycete effectors promote disease. Protein biochemistry and structural biology are two methods that can contribute greatly to the understanding of protein function. Both techniques are dependent on obtaining proteins that are pure and functional, and generally require the use of heterologous recombinant protein expression systems. Here, we present a general scheme and methodology for the production and characterization of small cysteine-rich (SCR) effectors utilizing Escherichia coli expression systems. Using this approach, we successfully produced cysteine-rich effectors derived from the biotrophic fungal pathogen Melampsora lini and the necrotrophic fungal pathogen Parastagonospora nodorum. Access to functional recombinant proteins facilitated crystallization and functional experiments. These results are discussed in the context of a general workflow that may serve as a template for others interested in understanding the function of SCR effector(s) from their plant pathogen(s) of interest.
Keyword Effector proteins
Plant disease
Protein biochemistry
Protein expression
Small cysteine rich proteins
Structural biology
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID DP120100685
1003325
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
School of Chemistry and Molecular Biosciences
 
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Created: Sat, 12 Mar 2016, 00:26:51 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences