Abstract
Activation of the disease resistance response in a host plant frequently requires the interaction of a plant resistance gene product with a corresponding, pathogenderived signal encoded by an avirulence gene. The products of resistance genes from diverse plant species show remarkable structural similarity. However, due to the general paucity of information on pathogen avirulence genes the recognition process remains in most cases poorly understood. NIP1, a small protein secreted by the fungal barley pathogen Rhynchosporium secalis, is one of only a few fungal avirulence proteins identified and characterized to date. The defense-activating activity of NIP1 is mediated by barley resistance gene Rrs1. In addition, a role of the protein in fungal virulence is suggested by its nonspecific toxicity in leaf tissues of host and non-host cereals as well as its resistance gene-independent stimulatory effect on the plant plasma membrane H+-ATPase. Four naturally occurring NIP1 isoforms are characterized by single amino acid alterations that affect the different activities in a similar way. As a step toward unraveling the signal perception/transduction mechanism, the solution structure of NIP1 was determined. The protein structure is characterized by a novel fold. It consists of two parts containing beta-sheets of two and three anti-parallel strands, respectively. Five intramolecular disulfide bonds, comprising a novel disulfide bond pattern, stabilize these parts and their position with respect to each other. A comparative analysis of the protein structure with the properties of the NIP1 isoforms suggests two loop regions to be crucial for the resistance-triggering activity of NIP1.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
In contrast to results reported previously [19, 33], the disulfide bonds did not appear to be accessible to TCEP in 6 M guanidine HCl (GuCl)
NMR Results and Structure Determination—The quality of the data obtained in the NMR experiment is exemplified in Fig. 2, which presents the 15N,1H HSQC spectrum of 2.0 mM NIP1 recorded at pH 6 and 298 K
Summary
Sample Preparation—For 15N labeling of the protein, Escherichia coli strain BL21 carrying the pQE30-NIP1 vector [13] was grown in 5 liters of minimal medium [18] consisting of M9 salts, 20% glucose, Fe2ϩ (5 ng literϪ1) and thiamine (500 ng literϪ1). 15NH4Cl (ARL, Groningen, The Netherlands) was used as the sole nitrogen source. Partial reduction was performed with a 5 molar excess of tris(2-carboxyethyl)phosphine (TCEP), a reagent that has proven to be an excellent reducing agent for disulfides at acidic pH [19] This reaction mixture was incubated at various temperatures for 15 min, directly followed by the addition of 4 –5 l of 0.1 M 1-cyano-4-. 2 The abbreviations used are: MALDI-TOF MS, matrix-assisted laser desorption-ionization time-of-flight mass spectroscopy; COSY, correlation spectroscopy; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum coherence; GHSQC, gradientselected HSQC; NIP1, necrosis-inducing protein 1; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser and exchange spectroscopy; TCEP, tris(2-carboxyethyl)phosphine; TOCSY, total correlation spectroscopy; GuCl, guanidine HCl; HPLC, high performance liquid chromatography. Exchanging amide protons were determined from the NH resonances from the fingerprint region cross peaks present in a TOCSY, COSY, and NOESY, recorded at 298 K of a NIP1 sample that was fully protonated and lyophilized. Coordinates have been deposited in the Protein Data Bank (accession code 1KG1)
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