Leucine-rich repeat-mediated intramolecular interactions in nematode recognition and cell death signaling by the tomato resistance protein Mi.
The root-knot nematode resistance gene Mi from tomato encodes a nucleotide-binding/leucine-rich repeat (NB/LRR) protein with a novel amino-terminal domain compared to related disease-resistance genes. The closely linked paralog Mi-1.1, which does not confer nematode resistance, encodes a protein 91% identical to the functional copy, Mi-1.2. The chimeric construct Mi-DS3, which encodes the 161 amino-terminal residues from Mi-1.1 fused to the remainder of Mi-1.2, induces localized necrosis when transiently expressed in Nicotiana benthamiana leaves. We produced mutant constructs that exchanged sequences encoding each of the 40 amino acid differences from the Mi-1.1 LRR region into Mi-DS3 and into Mi-1.2. For 23 of the substitutions, necrosis was lost upon transient expression of the mutated Mi-DS3 in N. benthamiana, and nematode resistance was lost when the altered Mi-1.2 was expressed in the tomato roots. One substitution, R961D, failed to give Mi-DS3-induced necrosis, but produced a dominant lethal phenotype when introduced into Mi-1.2. This gain-of-function phenotype was suppressed by co-expression with the amino-terminal region of Mi-1.1, suggesting that residue 961 is critical for negative regulation by the corresponding N-terminal region. Substitutions of Mi-1.1 residues 984-986 retained the ability to cause necrosis in Mi-DS3, but resulted in loss-of-nematode resistance in Mi-1.2, suggesting that these residues are essential for nematode recognition. None of the loss-of-function mutations in Mi-1.2 had a dominant negative phenotype. These results indicate that the Mi-1.2 LRR is involved in regulation of the transmission of the resistance response as well as in recognition of the nematode.
Hwang
Chin-Feng
CF
Department of Nematology, Center for Engineering Plants for Resistance Against Pathogens, University of California, Davis, CA 95616, USA.
Williamson
Valerie M
VM
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D016923:Cell Death; D004198:Disease Susceptibility; D005799:Genes, Dominant / Q000235:genetics; D007930:Leucine / Q000378:metabolism*; D018551:Solanum lycopersicum / Q000166:cytology / Q000378:metabolism* / Q000469:parasitology*; D009154:Mutation; D009348:Nematoda / Q000502:physiology*; D010641:Phenotype; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000166:cytology / Q000378:metabolism / Q000469:parasitology; D015398:Signal Transduction
D010940:Plant Proteins; D007930:Leucine