Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein. Isolation and characterization of a rice Mlo homologue.
Transient influx of Ca(2+) constitutes an early event in the signaling cascades that trigger plant defense responses. However, the downstream components of defense-associated Ca(2+) signaling are largely unknown. Because Ca(2+) signals are mediated by Ca(2+)-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens should provide insights into the mechanism by which Ca(2+) regulates defense responses. In this study, we isolated a gene encoding rice Mlo (Oryza sativa Mlo; OsMlo) using a protein-protein interaction-based screening of a cDNA expression library constructed from pathogen-elicited rice suspension cells. OsMlo has a molecular mass of 62 kDa and shares 65% sequence identity and scaffold topology with barley Mlo, a heptahelical transmembrane protein known to function as a negative regulator of broad spectrum disease resistance and leaf cell death. By using gel overlay assays, we showed that OsMlo produced in Escherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca(2+)-dependent manner. We located a 20-amino acid CaM-binding domain (CaMBD) in the OsMlo C-terminal cytoplasmic tail that is necessary and sufficient for Ca(2+)-dependent CaM complex formation. Specific binding of the conserved CaMBD to CaM was corroborated by site-directed mutagenesis, a gel mobility shift assay, and a competition assay with a Ca(2+)/CaM-dependent enzyme. Expression of OsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules. We propose that binding of Ca(2+)-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.
Kim
Min Chul
MC
Division of Applied Life Science, Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju 660-701, Korea.
Lee
Sang Hyoung
SH
Kim
Jong Kyong
JK
Chun
Hyun Jin
HJ
Choi
Man Soo
MS
Chung
Woo Sik
WS
Moon
Byeong Cheol
BC
Kang
Chang Ho
CH
Park
Chan Young
CY
Yoo
Jae Hyuk
JH
Kang
Yun Hwan
YH
Koo
Seong Cheol
SC
Koo
Yoon Duck
YD
Jung
Jae Cheol
JC
Kim
Sun Tae
ST
Schulze-Lefert
Paul
P
Lee
Sang Yeol
SY
Cho
Moo Je
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D015152:Blotting, Northern; D015139:Blotting, Southern; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D017343:Genes, Plant; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000378:metabolism* / Q000502:physiology*; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors
D002147:Calmodulin; D018076:DNA, Complementary; C104922:MLO protein, Hordeum vulgare; D010455:Peptides; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D010727:Phosphoric Diester Hydrolases; D002118:Calcium