Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins.
The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins.
Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, United Kingdom.
B B
BB
Wulff
C M
CM
Thomas
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Smoker
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Grant
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Jones
D000595:Amino Acid Sequence; D001483:Base Sequence; D002963:Cladosporium / Q000235:genetics / Q000472:pathogenicity*; D018744:DNA, Plant / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D018628:Gene Dosage; D005800:Genes, Fungal; D017343:Genes, Plant; D018551:Solanum lycopersicum / Q000235:genetics* / Q000382:microbiology* / Q000502:physiology; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D030821:Plants, Genetically Modified; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid; D014774:Virulence / Q000235:genetics
C085987:AVR4 protein, Cladosporium fulvum; C109609:Cf protein, Lycopersicon esculentum; D018744:DNA, Plant; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins