Structure-function analysis of cf-9, a receptor-like protein with extracytoplasmic leucine-rich repeats.
The tomato (Lycopersicon pimpinellifolium) resistance protein Cf-9 belongs to a large class of plant proteins with extracytoplasmic Leu-rich repeats (eLRRs). eLRR proteins play key roles in plant defense and development, mainly as receptor-like proteins or receptor-like kinases, conferring recognition of various pathogen molecules and plant hormones. We report here a large-scale structure-function analysis of an eLRR protein. A total of 66 site-directed mutants of Cf-9 were analyzed for activity in Avr9 recognition and for protein stability and the results interpreted with the help of a homology model of the Cf-9 structure. Conserved Trp and Cys pairs in the N-terminal LRR-flanking domain appear to be important for Cf-9 activity and are probably exposed at the putative concave inner surface of the Cf-9 protein, where recognition specificity also resides. Removal of each of the 22 putative N-linked glycosylation sites (PGS) revealed that many PGSs contribute to Cf-9 activity and that the PGSs in the putative alpha-helices of the LRR modules are essential. Immunoblot analysis and mass spectrometry showed that all but one of the PGSs are N-glycosylated. Introduction of glycosylation at the putative concave beta-sheet surface blocks Cf-9 activity, in some cases probably by disturbing specific recognition, and in another case by steric hindrance with existing N-glycans. The glycosylation pattern and several other features are conserved in other eLRR proteins, where similar mutations show similar phenotypes.
van der Hoorn
Renier A L
RA
Wageningen University, Laboratory of Phytopathology, 6709 PD, Wageningen, The Netherlands.
Wulff
Brande B H
BB
Rivas
Susana
S
Durrant
Marcus C
MC
van der Ploeg
Anke
A
de Wit
Pierre J G M
PJ
Jones
Jonathan D G
JD
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D017343:Genes, Plant; D006031:Glycosylation; D007930:Leucine / Q000737:chemistry; D018551:Solanum lycopersicum / Q000235:genetics / Q000502:physiology; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D030821:Plants, Genetically Modified; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
C109609:Cf protein, Lycopersicon esculentum; D008562:Membrane Glycoproteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D007930:Leucine
Plant Cell. 1998 Mar;10(3):319-30
9501107
Cell. 2002 Jul 26;110(2):213-22
12150929
Plant J. 1992 May;2(3):367-73
1303801
Biochemistry. 2001 Jul 31;40(30):8861-7
11467947
Science. 1995 Dec 15;270(5243):1804-6
8525370
Curr Opin Plant Biol. 2003 Oct;6(5):507-16
12972053
Plant Cell. 2003 May;15(5):1198-211
12724544
Plant J. 2004 Feb;37(4):517-27
14756760
Mol Plant Microbe Interact. 2001 Apr;14(4):508-15
11310738
Proc Natl Acad Sci U S A. 2002 Jul 9;99(14):9585-90
12060717
Genes Dev. 2001 May 1;15(9):1128-39
11331608
Nature. 2000 Dec 14;408(6814):796-815
11130711
Plant Cell. 1999 Oct;11(10):1925-34
10521522
Cell. 1997 Sep 5;90(5):929-38
9298904
Cell. 2003 Jun 27;113(7):935-44
12837250
Science. 2002 May 24;296(5572):1470-2
12029134
Mol Cell. 2000 Jun;5(6):1003-11
10911994
Science. 2002 May 31;296(5573):1697-700
12040198
Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14440-5
8962070
Plant Cell. 2000 Jul;12(7):1179-201
10899983
Nature. 1970 Aug 15;227(5259):680-5
5432063
Plant J. 2000 Mar;21(6):529-36
10758503
Nature. 2002 Nov 28;420(6914):426-9
12442172
Plant Cell. 2001 Feb;13(2):273-85
11226185
Nature. 2002 Jun 27;417(6892):959-62
12087405
Plant Physiol. 2000 Aug;123(4):1247-56
10938344
Plant J. 2004 Aug;39(4):643-54
15272880
Cell. 1996 Feb 9;84(3):451-9
8608599
Proc Natl Acad Sci U S A. 2003 May 27;100(11):6860-5
12748390
Plant Cell. 2001 Feb;13(2):255-72
11226184
Biochemistry. 2001 Jan 16;40(2):569-76
11148052
EMBO J. 1999 May 4;18(9):2352-63
10228150
Proc Natl Acad Sci U S A. 2001 May 8;98(10):5916-21
11320207
Science. 1994 Nov 4;266(5186):789-93
7973631
Plant Cell. 2002 Sep;14(9):2277-87
12215520
Plant Cell. 2003 Jan;15(1):93-106
12509524
Nucleic Acids Res. 2000 Jan 1;28(1):235-42
10592235
Genetics. 2004 May;167(1):459-70
15166169
Proc Natl Acad Sci U S A. 2001 May 22;98(11):6511-5
11331751
Science. 2002 Aug 16;297(5584):1176-9
12183630
Cell. 2002 Jul 26;110(2):203-12
12150928
Plant Cell. 2000 Sep;12(9):1591-606
11006334
J Mol Biol. 1996 Dec 20;264(5):1028-43
9000628
Plant Cell. 2001 May;13(5):1155-63
11340188
Nature. 2002 Nov 28;420(6914):422-6
12442170
Cell. 1997 May 16;89(4):575-85
9160749
Plant Cell. 2004 Jun;16(6):1604-15
15155877
Plant J. 2003 Mar;33(5):949-56
12609035
Cell. 2000 Oct 13;103(2):211-25
11057895
Proc Natl Acad Sci U S A. 2004 Jan 20;101(3):886-90
14715897
Cell. 2002 Dec 13;111(6):825-36
12526809
Plant Cell. 1996 Apr;8(4):735-46
8624444
Genes Dev. 2002 Aug 1;16(15):2021-31
12154130
Methods Enzymol. 1997;277:505-24
18488322
Plant Cell. 1999 Mar;11(3):393-406
10072399
Nature. 1993 Dec 23-30;366(6457):751-6
8264799
Plant Cell. 2002 Mar;14(3):689-702
11910014
Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):10124-8
12904578
Plant J. 2003 Aug;35(3):305-15
12887582
Genes Dev. 2000 Jan 1;14(1):108-17
10640280
Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10763-8
11526204
Genes Dev. 2001 Oct 15;15(20):2755-66
11641280
Mol Gen Genet. 1990 Jan;220(2):245-50
2325623
Biochemistry. 1998 May 12;37(19):6833-7
9578569
Curr Biol. 2002 Oct 15;12(20):1718-27
12401166
J Immunol. 2001 Feb 1;166(3):1433-8
11160179
Plant Physiol. 2004 Jun;135(2):1100-12
15155873
J Biol Chem. 1980 Oct 10;255(19):9236-42
7410422
Plant J. 1993 Mar;3(3):451-6
8220453
Nature. 2001 Mar 15;410(6826):380-3
11268216
Mol Plant Microbe Interact. 2000 Apr;13(4):439-46
10755307
Plant Physiol. 2003 Mar;131(3):1313-26
12644681
Science. 1998 Oct 2;282(5386):115-7
9756474
Science. 2003 Jan 3;299(5603):109-12
12411574
Plant Cell. 2002 Nov;14(11):2707-22
12417696
Trends Biochem Sci. 2003 Apr;28(4):210-4
12713905
J Biol Chem. 1994 Jul 8;269(27):18062-71
8027066
J Mol Biol. 1998 Apr 3;277(3):519-27
9533877
Nature. 2002 Jun 27;417(6892):962-6
12087406
Cell. 1997 Dec 12;91(6):821-32
9413991
Acta Crystallogr D Biol Crystallogr. 1997 Sep 1;53(Pt 5):580-7
15299889
J Biol Chem. 1996 Mar 15;271(11):6363-6
8626433
Plant J. 2003 Nov;36(3):291-300
14617087