Mutational analysis of the Arabidopsis nucleotide binding site-leucine-rich repeat resistance gene RPS2.
Disease resistance proteins containing a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region compose the largest class of disease resistance proteins. These so-called NBS-LRR proteins confer resistance against a wide variety of phytopathogens. To help elucidate the mechanism by which NBS-LRR proteins recognize and transmit pathogen-derived signals, we analyzed mutant versions of the Arabidopsis NBS-LRR protein RPS2. The RPS2 gene confers resistance against Pseudomonas syringae strains carrying the avirulence gene avrRpt2. The activity of RPS2 derivatives in response to AvrRpt2 was measured by using a functional transient expression assay or by expressing the mutant proteins in transgenic plants. Directed mutagenesis revealed that the NBS and an N-terminal leucine zipper (LZ) motif were critical for RPS2 function. Mutations near the N terminus, including an LZ mutation, resulted in proteins that exhibited a dominant negative effect on wild-type RPS2. Scanning the RPS2 molecule with a small in-frame internal deletion demonstrated that RPS2 does not have a large dispensable region. Overexpression of RPS2 in the transient assay in the absence of avrRpt2 also led to an apparent resistant response, presumably a consequence of a low basal activity of RPS2. The NBS and LZ were essential for this overdose effect, whereas the entire LRR was dispensable. RPS2 interaction with a 75-kD protein (p75) required an N-terminal portion of RPS2 that is smaller than the region required for the overdose effect. These findings illuminate the pathogen recognition mechanisms common among NBS-LRR proteins.
Tao
Y
Y
Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, Maryland 21250, USA.
Yuan
F
F
Leister
R T
RT
Ausubel
F M
FM
Katagiri
F
F
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D004252:DNA Mutational Analysis; D018076:DNA, Complementary; D007930:Leucine / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D010641:Phenotype; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D009711:Nucleotides; D010940:Plant Proteins; C089426:RPS2 protein, Arabidopsis; D007930:Leucine