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.

Both plant and animal cells elevate their cytosolic free calcium level ([Ca 2ϩ ] cyt ) 1 in response to a variety of external stimuli, including pathogen attack (1)(2)(3)(4)(5). A rapid increase in [Ca 2ϩ ] cyt has been reported in a number of plant-pathogen interactions (6 -9). Ratio imaging of cowpea epidermal cells after challenge with the fungus Uromyces phaseoli demonstrated an increased [Ca 2ϩ ] cyt level in an incompatible interaction at the time when the pathogen was penetrating the host cell wall (6). A rapid and sustained rise in [Ca 2ϩ ] cyt was observed in whole leaves of Arabidopsis during race-specific resistance in response to the phytopathogenic bacterium Pseudomonas syringae (7). Treatment of suspension-cultured plant cells with non-race-specific fungal elicitors leads to Ca 2ϩ fluxes in the micromolar range (8,9). Because treatment of plant cells with Ca 2ϩ chelators or Ca 2ϩ channel blockers compromises the oxidative burst that is thought to drive defense responses at sites of attempted pathogen invasion, pathogen-triggered Ca 2ϩ fluxes are likely to play an important role in signaling during plant defense (8,9). However, little is known of how Ca 2ϩ signals impinge on plant defense pathways at the molecular level.
In plants, Ca 2ϩ either directly activates a group of enzymes called calcium-dependent protein kinases (10 -12) or acts indirectly through Ca 2ϩ -modulator proteins such as CaM (13,14). CaM is a ubiquitous Ca 2ϩ -binding protein in eukaryotes and the primary intracellular Ca 2ϩ sensor or adaptor. It transduces Ca 2ϩ signals by modulating the activity of numerous and diverse CaMBPs, thereby generating physiological responses to various stimuli (15). In plant cells, multiple CaM genes exist that code for numerous CaM isoforms in wheat (16), potato (17), Arabidopsis (18), and soybean (19). Expression of CaM isoform genes is differentially induced in response to physical and chemical stimuli, including pathogen invasion (20). CaM isoforms have been found to regulate differentially many Ca 2ϩ / CaM-dependent enzymes in vitro (21). Soybean CaM isoform-1 (SCaM-1) activates NAD kinase and calcineurin, whereas soy-bean CaM isoform-4 (SCaM-4) serves as a competitive antagonist of these activations (22). The opposite is true for mammalian nitric-oxide synthase (23,24). Furthermore, specific CaM isoform(s) mediate SA-independent plant defense signal transduction in vivo (20), although the exact defense signaling cascades mediated by the CaM isoforms remain to be elucidated. There is considerable interest in identifying and characterizing CaM-binding proteins because at least some are expected to have a direct role in mediating the outcome of plant-pathogen encounters.
Barley Mlo is the founder of a plant-restricted and sequencediversified class of seven-transmembrane proteins located in the plasma membrane (25). Homozygous mutations in barley Mlo confer broad spectrum resistance to the powdery mildew fungus and exhibit a developmentally controlled phenotype of spontaneous leaf cell death, indicating that the wild-type gene has a negative regulatory function in plant defense and leaf cell death (26,27). Although the subcellular location, topology, and sequence diversification of Mlo family members within a plant species are reminiscent of G-protein-coupled receptors (GPCR) in animals and fungi, direct experimental evidence for Mlo receptor function is lacking (25)(26)(27)(28).
Here we report the isolation from rice of a gene encoding an Mlo homologue (OsMlo) that shares 65% sequence identity with the barley gene, HvMlo. OsMlo expression was found to be responsive to pathogen and plant defense signaling molecules. We identified a 20-amino acid sequence in the C-terminal tail of OsMlo that is required for Ca 2ϩ -dependent complex formation with CaM. The conservation of the novel CaMBD among Mlo family members in diverse plant species suggests that activity modulation via CaM could be a common feature of Mlo proteins.

EXPERIMENTAL PROCEDURES
Screening of the Rice cDNA Expression Library-A rice cDNA expression library was constructed in a ZAPII vector (Stratagene) from rice (Oryza sativa L. Milyang 117) suspension cells treated with a fungal elicitor as described previously (29). SCaM-1 was conjugated to a maleimide-activated HRP using the EZ-Link maleimide-activated HRP conjugation kit (Pierce) as described in a previous report (30). We screened the rice cDNA expression library as described by Sambrook et al. (31), using the HRP-conjugated SCaM-1 (SCaM-1:HRP) as a probe. Approximately 4 ϫ 10 4 plaque-forming units were plated per 15-cm LB plate using Escherichia coli XL1-blue MRF (Stratagene) as the host strain. The plates were incubated at 42°C until the plaques appeared. The plates were then overlaid with nitrocellulose filters that had previously been soaked in 10 mM IPTG. The incubation was continued at 37°C for 6 -8 h, and the plates were cooled to 4°C. The filters were removed and rinsed twice in a large volume of TBS-T (TBS containing 0.1% (v/v) Tween 20). The filters were blocked by incubation in 7% (w/v) non-fat dry milk/TBS-T overnight, and the blocked filters were washed three times with TBS-T for 5 min and equilibrated in overlay buffer (50 mM imidazole HCl (pH 7.5), 150 mM NaCl) for 1 h. The membranes were blocked a second time by incubating the filters in overlay buffer containing 9% (v/v) gelatin (Sigma), 0.5% (v/v) Tween 20, and 1 mM CaCl 2 for 3.5 h. SCaM-1:HRP was added to the gelatin-containing buffer at a final concentration of 0.2 g/ml, and the filters were incubated for 1 h. Final washing was done in three steps, with each step consisting of five repeats of a 5-min wash: first, in TBS-T/50 mM imidazole HCl (pH 7.5) and 1 mM CaCl 2 ; second, in 20 mM Tris-HCl (pH 7.5), 0.5% Tween 20, 50 mM imidazole HCl, 0.5 M KCl, and 1 mM CaCl 2 ; and third, in 20 mM Tris-HCl (pH 7.5), 0.1% Tween 20, and 1 mM MgCl 2 . Bound CaM:HRP was visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences). A total of 5 ϫ 10 5 recombinants were screened, and 50 positive clones were isolated after three rounds of screening. cDNA inserts were recovered by in vivo excision with helper phage (ExAssist, Stratagene). To confirm the binding of positive clones to CaM, we expressed positive clones as ␤-galactosidase fusion proteins in E. coli and examined the clones for CaM binding by a CaM:HRP overlay assay as described above. In brief, we transformed positive clones into XL1-Blue (Stratagene) and induced the expression of ␤-galactosidase fusion proteins with IPTG treatment. Twenty micrograms of IPTG-induced E. coli crude protein were separated on a 10% SDSpolyacrylamide gel and transferred onto an Immobilon-MP membrane (PVDF, Millipore). The membrane was rinsed in TBS-T, blocked by incubation in 7% (w/v) non-fat dry milk/TBS-T overnight, and processed as described above. For determination of Ca 2ϩ -independent binding of CaM, 5 mM EGTA was substituted for the 1 mM CaCl 2 in all overlay buffers. The cDNA sequences of the resulting positive clones were determined from both strands of cDNA by dideoxynucleotide chain termination using an automatic DNA sequencer (ABI 373A, Applied Biosystems Inc.), and BLAST searches were performed at the National Center for Biotechnology Information web site.
5Ј-Rapid Amplification of cDNA Ends-One of the positive clones contained a partial coding region that was homologous to the barley Mlo gene. To obtain the truncated 5Ј region of OsMlo cDNA, we performed 5Ј-rapid amplification of cDNA ends (RACE) with a Marathon cDNA amplification kit (CLONTECH) according to the manufacturer's instructions. The truncated 5Ј region of OsMlo cDNA was amplified from cDNA synthesized from mRNA of rice suspension cells treated with a fungal elicitor by using an OsMlo gene-specific oligonucleotide (5Ј-GCCCTAAACCCATGGTGATGA-CGC-3Ј) and an adaptor primer from CLONTECH. The amplified product was subcloned into the SmaI site of pBluescript II SK(Ϫ) (Stratagene) and verified by sequencing. To obtain a full-length clone, the 5Ј-RACE product was subcloned into the 5Ј region of the partial OsMlo cDNA clone.
Expression of Recombinant Proteins in E. coli and the CaM Binding Assay-All the clones were introduced into E. coli BL21(DE3)pLysS and expressed. Expression of the GST fusion proteins was induced by application of 1 mM IPTG for 5 h at 25°C. The cells were harvested, resuspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 2 mM PMSF, 1 mM DTT, 100 g/ml lysozyme), and incubated on ice for 20 min. The mixture was sonicated for 1 min at 50% pulse and then centrifuged at 6,000 ϫ g for 10 min to remove the cell debris. The supernatant (containing the E. coli crude protein) was used for Western blotting and a CaM:HRP gel overlay assay. Twenty micrograms of E. coli crude protein were separated on 10% SDS-polyacrylamide gels and transferred onto Immobilon-P membranes (PVDF, Millipore); expressed GST fusion proteins were detected by a polyclonal GST-specific antiserum. To examine the CaM binding ability of recombinant proteins, a duplicate blot was probed with an SCaM-1:HRP conjugate in the presence of 1 mM CaCl 2 or 5 mM EGTA. The CaM:HRP overlay assay was carried out as described above. The bound CaM was visualized using an ECL detection system (Amersham Biosciences).
CaM Mobility Shift Assay with a Synthetic Peptide-A peptide ( 443 MKALMNWRKKAMEKKKVRDA 462 ) corresponding to a stretch of 20 amino acids in the CaMBD of OsMlo was synthesized at a peptide synthesis facility (Genosys Biotechnologies, Inc.). The CaM binding ability of the synthetic peptide was detected by a relative mobility shift of CaM in the presence of the peptide (32). SCaM-1 (303 pmol) was incubated with increasing concentrations of the peptide (molar ratio: 0.0, 0.5, 0.75, 1.0, 1.5, 2.0, and 3.0) in a binding buffer (100 mM Tris-HCl (pH 7.2), plus 0.1 mM CaCl 2 or 2 mM EGTA) at room temperature for 1 h. One-half volume of 50% glycerol plus the tracer bromphenol blue was added, and the mixtures were electrophoresed in 15% nondenaturing polyacrylamide gels containing 0.375 M Tris-HCl (pH 8.8), and either 0.1 mM CaCl 2 or 2 mM EGTA. The gels were run at a constant voltage of 100 V in an electrode buffer (25 mM Tris-HCl (pH 8.3), 192 mM glycine, and either 0.1 mM CaCl 2 or 2 mM EGTA) and stained with Coomassie Brilliant Blue.
Phosphodiesterase Competition Assay-Cyclic nucleotide phosphodiesterase (PDE) assays were performed using commercially available bovine heart CaM-deficient PDE (Sigma). The initial 500-l reaction volume contained buffer (100 mM imidazole HCl (pH 7.5), 2.56 mM cAMP, 5.13 mM MgSO 4 , 1.28 mM CaCl 2 ) with concentrations of SCaM-1 (5-200 nM) in the presence (100 nM) or absence of peptide. The reaction was started with the addition of PDE (0.5 milliunit/l). The basal level of enzyme activity was determined in the absence of SCaM-1, and the stimulated activity was determined in the presence of SCaM-1 and CaCl 2 . After incubation at 30°C for 30 min, the reaction was stopped by placing the reaction tubes into a boiling water bath for 5 min and on ice for 2 min. Following a brief centrifugation, 50 l of alkaline phosphatase (10 units) was added and incubated at 37°C for 10 min. The reaction was stopped by adding 500 l of 10% trichloroacetic acid. After vortexing, the precipitates were spun down, and the supernatant (400 l) was transferred to a new tube. After adding 1 ml of phosphate reagent (19), the supernatant was incubated at 37°C for 30 min and assayed for P i at OD 660 . The dissociation constant (K d ) of SCaM-1 for the peptide was calculated from the concentration of SCaM-1 (nM) required to obtain half-maximal (50%) PDE activity either in the presence (100 nM) or absence of peptide. The following equation was used to calculate dissociation constants (32): is the total concentration of peptide added, and [CaM] and K are the concentrations of CaM required to obtain half-maximal activation of PDE in the presence or absence of peptides, respectively.
Northern Blot Analysis-Fungal spores were isolated from rice blast fungus (Magnaporthe grisea) and inoculated into rice suspension-cultured cells (5 ϫ 10 5 spores/ml) as described by Kim et al. (29). All of the chemicals used in the procedure were introduced into the rice cell suspension culture, and total RNA was isolated by the guanidinium isothiocyanate method with subsequent ultracentrifugation (31). RNA was separated on 1.2% agarose-formaldehyde gels and blotted onto a nylon membranes (GeneScreen Plus, PerkinElmer Life Sciences). The membranes were incubated with 32 P-labeled full-length OsMlo cDNA at 65°C overnight and washed under high stringency conditions according to the method of Church and Gilbert (33).
Genomic Southern Blot Analysis-Rice genomic DNA was extracted as described (34). Ten micrograms of DNA were digested with EcoRI, HindIII, or XbaI, fractionated on a 0.8% agarose gel, and transferred onto a nylon membrane (GeneScreen Plus, PerkinElmer Life Sciences). The membrane was probed with 32 P-labeled full-length OsMlo cDNA. Hybridization and washing conditions were as described above for Northern blot analysis.
Preparation of the Polyclonal Antibody-A multiple antigenic peptide (OsMloAg, 103 KGLKGKKDHRRRLLW 117 in OsMlo) was synthesized at the Peptide Synthesis Facility (Peptron, Daejun, South Korea). The polyclonal antibody against the OsMloAg peptide was raised in a New Zealand White rabbit, and the polyclonal antiserum was purified by antigen affinity chromatography on an OsMloAg peptide-Sepharose column as described (35). The column was prepared by conjugating the OsMloAg peptide to CNBr-activated Sepharose 4B according to the procedure recommended by the manufacturer (Amersham Biosciences).
Preparation of Rice Membrane Fractions-A microsomal membrane fraction was prepared from 10-day-old rice leaves according to the procedure described by Chung et al. (35). Rice leaves were ground on ice in prechilled homogenization buffer of 50 mM Tris-HCl (pH 8.0), 20% (w/w) sucrose, 2 mM EDTA, 4 mM DTT, 2 mM PMSF, and complete protease inhibitor mixture (1 tablet per 10 ml of lysis buffer) (Roche Molecular Biochemicals). The cell homogenate was filtered through two layers of Miracloth (Calbiochem) and then centrifuged at 12,000 ϫ g for 10 min to remove intact organelles and cell walls. The supernatant was divided into a cytosolic protein fraction (supernatant) and a microsomal fraction (pellet) by further centrifugation at 100,000 ϫ g for 1 h. The resulting microsomal pellets were resuspended in homogenization buffer (1 ml per 10 g of starting material) using glass homogenizers. Microsomes were applied to a continuous sucrose gradient from 15 to 45% (w/w) in centrifugation buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM DTT, 2 mM PMSF) and centrifuged at 110,000 ϫ g for 18 h. Fractions (0.8 ml) were collected from the top, frozen in liquid nitrogen, and stored at -80°C until used.

RESULTS
Cloning of Rice OsMlo-To identify molecular components of the Ca 2ϩ /CaM-mediated signaling pathways associated with plant defense responses, we screened a rice cDNA expression library constructed from a fungal elicitor-treated rice suspension cell culture using HRP-conjugated SCaM-1 as a probe (29). So far, seven highly conserved CaMs (GenBank TM accession numbers X65016 (36), AF441190, AF042839, AF441191, AF042840, L18913, and Z12827) and a divergent CaM isoform containing an unusual 12-amino acid insertion, between the second and third calcium-binding motifs (AB060552), have The amino acid sequences of 32 Mlo proteins were obtained from GenBank TM data base searches. The sequences were aligned and compared to construct a phylogenetic tree by the Clustal method using the MecAlign program (DNAstar Inc.). The GenBank TM accession numbers used in building the phylogenetic tree are as follows: The hydrophobic amino acids corresponding to a Ca 2ϩ -dependent CaM-binding motif (1-8-14 motif) and the highly conserved Trp residues are indicated with gray boxes and a black box, respectively. C, helical wheel projection of the putative CaM-binding region of OsMlo. Hydrophobic and basic amino acid residues are marked with * and ϩ, respectively. The dashed line indicates that such a helix is amphipathic, with one side being hydrophilic and the other side hydrophobic. D, a topology of OsMlo. The plasma membrane is represented by a gray bar. Open circles represent the amino acids of OsMlo, and the 20 amino acids corresponding to the putative CaMBD of OsMlo are denoted by closed circles.
been identified from rice. SCaM-1 is nearly identical to the conserved type of rice CaMs, showing only a single amino acid difference compared with OsCaM (Ser 11 3 Ala (37)) and two amino acid differences compared with the other conserved rice CaMs (GenBank TM accession numbers AF441190 and AF042839: Ser 11 3 Ala and Arg 75 3 Lys; GenBank TM accession numbers AF441191, AF042840, L18913, and Z12827: Glu 8 3 Asp and Ser 11 3 Ala). High conservation of amino acid sequences between conserved rice CaMs and SCaM-1 may suggest that they can share their target/binding proteins, leading us to expect a similar result in the protein-protein interactionbased screening in planta. Fifty positive clones were obtained from about 5 ϫ 10 5 recombinant phages. DNA sequencing of the clones and comparisons to known sequences in GenBank TM revealed that they included known plant CaMBPs such as kinesin-like proteins (37), CaM-binding heat-shock proteins (38), multidrug resistance associated proteins (39), and two different CaMBPs of unknown function (40,41). They also included several novel CaMBPs with high homology to known proteins (e.g. OsMlo, in this report) and other proteins with no significant homology to any other reported proteins in Gen-Bank TM data base (data not shown).
One of the clones had high homology to barley Mlo (27). The nucleotide sequence of the 1,302-bp cDNA clone contained a partial coding region (encoding 403 amino acids) and a full 3Ј-untranslated region. The 5Ј-truncated region of the cDNA was recovered by RACE-PCR and sequenced. The 1,662 nucle-otide full-length cDNA contained an open reading frame encoding a 62-kDa protein consisting of 554 amino acid residues that was designated OsMlo (Fig. 1). The deduced amino acid sequence of OsMlo shared 65% identity with barley Mlo and revealed seven putative transmembrane (TM) domains predicted by hydrophobicity analysis (data not shown). At present, 32 plant genes encoding homologues of Mlo protein have been identified in both dicot and monocot plant species such as Arabidopsis (15 genes), rice (3 genes), barley (2 genes), wheat (3 genes), and maize (9 genes). Interestingly, Mlo sequence-related genes were found only in the plant kingdom, suggesting that Mlo is involved in plant-specific cellular processes. In an effort to determine the evolutionary relationships among Mlo family members, a phylogenetic tree was generated using the amino acid sequences of 32 Mlo proteins (Fig. 2A). The phylogenetic analysis showed that plant Mlo families are divided into five groups, and monocot Mlo proteins, with the exception of maize Mlo proteins, are clustered in a separate group. However, the biological meaning of these groups is not yet clear.
Mapping of the CaMBD within OsMlo-Comparative analysis of the CaM-binding regions of the many reported CaMBPs has provided multiple sequence motifs required for CaM complex formation (42). Based on the structural characteristics of known CaMBDs, a putative CaMBD was located in OsMlo in the last cytosolic region between Met 443 and Ala 462 (Fig. 2, B  and D). Within this 20-amino acid stretch, hydrophobic amino acids are present at positions 1 (Leu 446 ), 8 (Ala 453 ), and 14 (Val 459 ), and several basic residues (five lysines and one arginine) are interspersed between the hydrophobic residues (Fig.  2B). These characteristics of the putative OsMlo CaMBD match a consensus Ca 2ϩ -dependent CaM-binding motif designated "1-8-14" (42). Furthermore, a Trp residue (amino acid 449) that is known to play a critical role in the CaM binding of many CaM target proteins (43)(44)(45) was also found within this region (Fig. 2B). A sequence comparison of the OsMlo CaMBD with the corresponding region of plant Mlo homologues showed that the positions of critical hydrophobic amino acids, espe-cially the first hydrophobic residue in the 1-8-14 motif and the Trp residue, are strictly conserved among all of the Mlo families (Fig. 2B). Most of the characterized CaM-binding proteins possess a basic amphiphilic ␣-helical region (13,46). A helical wheel projection of the peptide sequences of the OsMlo CaMBD (20 amino acids, Met 443 to Ala 462 ) shows a typical CaMBD feature, segregation of basic and hydrophobic residues on opposite sides of the helix (Fig. 2C (48, 49)).
To confirm the location of the putative CaMBD of OsMlo, we made a series of GST fusion constructs containing the fulllength cDNA, four serial C-terminal deletion mutants, and a construct harboring the presumptive binding site. The recombinant proteins were produced in E. coli, separated by SDS-PAGE, and transferred to PVDF membranes for Western blotting and subsequent CaM:HRP overlay assays. Expression of the GST fusion proteins was verified by probing the blot with an anti-GST antibody (data not shown). Three recombinant proteins (designated D0, D1, and C) that contained the putative CaMBD interacted with the SCaM-1:HRP conjugate, whereas GST only (G) and the GST fusion proteins lacking the predicted CaM-binding region (designated D2, D3, and D4) did not bind to CaM. CaM bound to OsMlo in the presence but not in the absence of Ca 2ϩ (Fig. 3B). These results demonstrate that CaM binds to the predicted OsMlo CaMBD in the Cterminal region between Met 443 and Ala 462 in a Ca 2ϩ -dependent manner.
Binding of a Synthetic Peptide to CaM-To analyze further whether CaM binds to the 20-amino acid stretch from Met 443 to Ala 462 of OsMlo, a peptide corresponding to this region was synthesized and used for a gel shift assay in which the larger peptide-bound form of CaM should migrate with a lower mobility than free CaM under non-denaturing conditions (32). As shown in Fig. 4A, the intensity of a higher molecular mass band representing the peptide-CaM complex increased with increasing concentration of the synthetic peptide in the presence of Ca 2ϩ , whereas the higher molecular weight complex was undetectable when EGTA was substituted for Ca 2ϩ . At a molar ratio of 1:1 (peptide:CaM), about 50% of the CaM was shifted, and all of the CaM formed a complex with the peptide at molar ratios of 2:1 and 3:1 (peptide:CaM). This result shows that the 20-mer peptide from Met 443 to Ala 462 of the predicted OsMlo CaMBD is sufficient for Ca 2ϩ -dependent CaM binding and provides evidence for a complex stoichiometry.
We further analyzed the binding of the synthetic peptide to CaM by a competition assay with PDE, a Ca 2ϩ /CaM-dependent enzyme. To determine K d values of the peptide for the activation of PDE by CaM, the CaM dose-dependent activation of PDE was monitored either in the presence (100 nM) or absence of the peptide (Fig. 4B). The activation curves shifted to the right in the presence of the peptide, indicating that competition occurred between PDE and the peptide for binding to CaM. The concentrations of SCaM-1 needed to achieve half-maximal activation of PDE activity in the absence and presence of the 20-mer peptide were 10.1 and 45.8 nM, respectively, a 4.5-fold difference. The K d value of the peptide for the activation of PDE by SCaM-1 was determined to be 18.2 nM. Critical Residues of the CaM-binding Motif-We made amino acid substitutions at several residues of the CaMBD that were expected to play a critical role in the interaction between CaM and OsMlo, and we analyzed the variants for CaM binding in a CaM overlay assay (Fig. 5). The hydrophobic residue Leu 446 at the first position of the 1-8-14 motif and Trp 449 , which is strictly conserved in all known Mlo proteins, were separately replaced by Arg (denoted by L446R and W449R, respectively). Two additional amino acids, Asn 448 and Glu 455 , were replaced by another basic amino acid, Lys (denoted by N448K and E455K, respectively). Each of the mutations were introduced into the GST::OsMlo CaMBD fusion construct (Fig. 3A, denoted by C), expressed in E. coli, and examined for CaM binding. Expression levels of the mutant proteins in E. coli were similar to that of the wild-type CaMBD of OsMlo, suggesting that these mutations did not affect protein stability (Fig. 5B). However, as shown in Fig. 5C, the single amino acid substitutions L446R and W449R completely abolished Ca 2ϩ -dependent CaM-binding of OsMlo. In contrast, the N448K and E455K substitutions did not affect complex formation with CaM. These results reflect sequence-specific complex formation and demonstrate the crucial roles of the conserved CaMBD residues Leu 446 and Trp 449 for binding.

Induction of OsMlo Gene Expression by Pathogen and Plant Defense Signaling
Molecules-To investigate the potential responsiveness of OsMlo gene expression to pathogen invasion, rice suspension cells were inoculated with fungal spores isolated from the rice blast fungus (M. grisea). The low transcript level of OsMlo increased rapidly and transiently in response to fungal spore inoculation (Fig. 6A, 1st panel). The OsMlo transcript level peaked at 1 h and then declined to basal levels by 12 h. The rapid induction of OsMlo gene expression in response to pathogen invasion suggests a direct role of OsMlo in plant defenses. We also examined the effect of several potential inducers of plant defense-related genes on OsMlo expression (Fig.  6A). The oligosaccharide elicitor glycol chitin (49) effectively induced OsMlo gene expression. Application of exogenous SA, H 2 O 2 , or an H 2 O 2 -generating system (glucose and glucose oxidase) also increased the transcript accumulation. Furthermore, two unrelated signaling molecules, jasmonic acid and ethephon, induced OsMlo gene expression, whereas Ca 2ϩ /ionomycin treatment had no effect. Thus, OsMlo expression appears to be inducible by multiple defense/stress response pathways.
To gain further insight into potential mechanisms mediating OsMlo gene induction upon challenge by a pathogen, we carried out pharmacological studies. As shown in Fig. 6B, cycloheximide, a protein synthesis inhibitor, did not block OsMlo transcript accumulation, indicating that transcriptional activation of OsMlo does not require protein synthesis. Likewise, diphenyleneiodonium, an inhibitor of neutrophil-like NADPH oxidase that is known to block the oxidative burst in plant defense (50,51), did not affect OsMlo gene induction. In contrast, 2,5dihydroxycinnamic acid methyl ester and N,NЈ-dimethylthiourea, H 2 O 2 -trapping chemicals, inhibited the elicitor-mediated induction of OsMlo expression up to 56 and 24% of the control expression level, respectively. These results suggest that H 2 O 2 generated by cell wall peroxidases (52) or germine-like oxalate oxidases (53) is more effective for OsMlo induction than the NADPH oxidase system (54). In addition, inhibition of an external Ca 2ϩ influx by the application of an extracellular Ca 2ϩ chelator, BAPTA, or a Ca 2ϩ channel blocker, lanthanium chloride, greatly reduced OsMlo transcript accumulation induced by the oligosaccharide elicitor (67 and 60% reduction, respectively). Rice suspension cells were also pretreated with inhibitors of protein kinase and protein phosphatases to address whether protein phosphorylation and dephosphorylation are involved in the induction of OsMlo expression. As shown in Fig.  6, B and C, pretreatment with K-252a and staurosporine, inhibitors of protein kinases, equally diminished the effect of glycol chitin on OsMlo induction (43%). However, the activation of OsMlo by glycol chitin showed a different sensitivity to specific protein phosphatase inhibitors. OsMlo induction was inhibited about 40% by calyculin A, a protein phosphatase 1A and 2A inhibitor, and about 20% by cyclosporin A, a protein phosphatase 2B inhibitor. In contrast, cantharidin, a protein phosphatase 2A inhibitor, had no effect.
Taken together, these pharmacological studies suggest that both an influx of Ca 2ϩ ions and an oxidative burst play significant roles in the induction of OsMlo transcription and that protein kinases and phosphatases participate in this process.
Genomic Southern Blot Analysis-To examine the OsMlo copy number in the rice genome, Southern blot analysis was carried out with rice genomic DNA digested with EcoRI, Hin-dIII, or XbaI. The hybridization was performed with the fulllength OsMlo cDNA as a probe. Under high stringency conditions, only one strong band was observed from each restriction (Fig. 7), indicating that the transcript accumulation profile detected by the OsMlo cDNA probe is indeed gene-specific. However, two weakly hybridizing bands were observed in the HindIII digest, indicating the existence of potentially related gene(s). This result suggests that OsMlo may belong to a small gene family.
OsMlo Is Located at the Plasma Membrane-A polyclonal OsMlo antibody was generated using a multiply antigenic peptide, corresponding to OsMlo amino acids Lys 103 to Trp 117 , and purified through immunoaffinity chromatography to remove any minor cross-reactivities (see "Experimental Procedures"). To test the specificity of the affinity-purified antibody, we carried out Western blot analysis with several truncated GST::OsMlo recombinant proteins expressed in E. coli (Fig. 3A, D0 -D4 and C). We observed anti-OsMlo immunoreacting bands only in the presence of recombinant proteins containing the region corresponding to the multiply antigenic peptide (D0, D1, D2, and D3). These results indicate that the anti-OsMlo antibody is specific for the peptide.
To determine the subcellular localization of OsMlo, we fractionated cytosolic and microsomal membrane proteins by centrifugation at 100,000 ϫ g, and we tested for the presence of OsMlo by immunoblotting with the affinity-purified anti-OsMlo antibody. As shown in Fig. 8A, a specific signal was detected only in the microsomal fraction; this signal corresponded to a mass of ϳ65 kDa. To define further the endomembrane location of OsMlo, microsomal membranes were fractionated on sucrose gradients and characterized by Western blot analysis. The fractionation of endomembranes was confirmed by Western blotting with anti-H ϩ -ATPase and anti-BIP antibodies. H ϩ -ATPase is commonly used as a marker for plasma membrane proteins and is abundant in fractions with a sucrose content between 34 and 45% (55). In contrast, BIP, an ER marker, is usually found in lighter (less sucrose content) fractions (56). Co-fractionation of OsMlo with H ϩ -ATPase (Fig. 8B) indicates that OsMlo is a plasma membrane protein. DISCUSSION Plant cells generate Ca 2ϩ signals with different amplitudes, frequencies, and durations in response to a variety of external stimuli and use these signals to control many cellular processes, including plant defense responses (1)(2)(3)(4)(5). CaM plays a vital role in transducing the Ca 2ϩ signals by modulating the activity of numerous target proteins (13,14). To understand the role(s) of CaM in plant defense responses, we isolated potential CaM-binding proteins by screening a cDNA expression library prepared from fungal elicitor-treated rice suspension cells using HRP-conjugated CaM as a probe. In this study, we isolated and characterized a novel CaM-binding protein encoded by OsMlo, which is highly homologous to barley Mlo, a plant defense and cell death modulator (25,27).
Most of the 50 or more CaMBDs isolated thus far are stretches of 16 -35 amino acid residues that, in the ␣-helical wheel representation, show a segregation of basic and polar residues on one side and hydrophobic amino acids on the other side (46,47,57,58). Recently the Ca 2ϩ -dependent CaM-binding motifs have been classified into two major groups, the 1-8-14 and 1-5-10 motifs, in which the numbers indicate positions of conserved hydrophobic residues (42). Based on the conserved structural features of CaMBDs, the CaM-binding motif of OsMlo was predicted to be located in the C-terminal last cytosolic region between Met 443 and Ala 462 in a 20-amino acid stretch showing the 1-8-14 motif (Fig. 2B). The location of the CaMBD of OsMlo was identified by cDNA expression-deletion mapping (Fig. 3, A and B) and was confirmed by CaM mobility shift and PDE enzyme competition assays with a synthetic peptide corresponding to the 20-amino acid stretch from Met 443 to Ala 462 (Fig. 4). We further verified that the predicted CaMbinding motif is correct by showing that the substitution of a single amino acid, Leu 446 3 Arg (corresponding to the first conserved hydrophobic residue in the 1-8-14 motif) or Trp 449 3 Arg (which has been known to play a key role in interactions between Ca 2ϩ -loaded CaM and CaMBPs (43)(44)(45)) resulted in the loss of the Ca 2ϩ -dependent CaM-binding ability of OsMlo (Fig. 5).
Thirty two Mlo sequence-related genes have been found in monocot and dicot plant species, showing a striking sequence conservation in each of the seven TM regions, in cytoplasmic loops 1-3, and in the first 30 residues of the C terminus adjacent to TM 7 (25). Interestingly, the CaMBD of OsMlo is located within the conserved stretch of the C-terminal cytoplasmic tail, proximal to TM 7. Sequences adjacent to the CaMBD on the C-terminal side are hypervariable between Mlo family mem-  (52)), or an anti-BIP antibody (1:3000, ER marker (53)). The immune complexes were visualized using the ECL system (Amersham Biosciences). bers. A sequence comparison of the putative OsMlo CaMBD with the corresponding regions in Mlo proteins of the other plant species listed in Fig. 2B shows conservation of hydrophobic residues located at positions 1, 8, and 14 and an average net charge of ϩ4 to ϩ7. Another striking feature of the putative CaMBDs of plant Mlo proteins is that the Trp residue is strictly conserved. A critical role for Trp residues in recognizing CaM has been demonstrated in several CaMBPs isolated from animals and plants (43, 59 -63). Alteration of this Trp to other amino acid residues abolishes either the binding of CaM to target proteins or the CaM-dependent activation of target proteins (43, 59 -61). We used site-specific mutagenesis to show the critical roles of the highly conserved Leu 446 and Trp 449 residues of OsMlo in the interaction with CaM (Fig. 5). Thus, it seems likely that all Mlo proteins are potential CaM-binding proteins.
Devoto et al. (25) suggested that plant Mlo proteins are reminiscent of the most abundant class of metazoan membrane resident receptors, the GPCRs. Interestingly, recent evidence suggests that CaM participates in GPCR-mediated signaling by direct binding to the receptors (64 -66). CaM interacts with the metabotropic glutamate receptor subtype 5 (mGluR5, one of the GPCR members) in a Ca 2ϩ -dependent manner in vitro (64). The opioid and D2-dopamine receptors, members of another subclass of GPCRs, also contain a CaMBD in their third intracellular loop, and CaM binding inhibits receptor-mediated signal transduction (65,66). Although the conservation of the CaMBDs in the C-terminal tails of Mlo family members provides evidence for their functional significance in vivo, further experiments must be performed to determine whether and how CaM binding regulates Mlo activity.
A great deal of genetic and cytological analysis of barley mlo mutant plants suggests that the wild-type Mlo protein has a regulatory function in cell death protection and plant defense (26 -28). However, it is not yet understood at the molecular level how Mlo modulates plant defense processes. To examine a possible link between OsMlo and plant defense responses, we monitored OsMlo gene expression following pathogen challenge. The observed responsiveness to pathogens and the upregulation of OsMlo transcription following treatment with the plant defense signaling compounds SA and H 2 O 2 support a putative involvement of the rice gene in plant defense modulation (Fig. 6A). The increase in transcript abundance following treatment with ethephon and jasmonic acid (Fig. 6A) may be indirect evidence for an additional role of OsMlo in mechanical wounding and senescence (67,68). Inhibition of cellular processes that are involved in defense signaling, such as the generation of reactive oxygen species (54), influx of extracellular Ca 2ϩ (6 -9), and protein phosphorylation (69), greatly reduced elicitor-induced OsMlo gene expression. Interestingly, inhibition of extracellular Ca 2ϩ influx efficiently reduced oligosaccharide elicitor-induced OsMlo gene expression, whereas application of Ca 2ϩ /ionomycin did not induce OsMlo gene expression. Plants respond to pathogen attack as well as elicitor treatment by activating various early responses such as ion fluxes across the plasma membrane (influx of Ca 2ϩ and H ϩ and efflux of Cl Ϫ and K ϩ ), generation of reactive oxygen species, and protein phosphorylation and dephosphorylation, sequentially followed by defense gene activation and phytoalexin accumulation (6, 70 -72). The critical role of elicitor-mediated Ca 2ϩ influx in the activation of downstream plant defense signaling has been shown by pharmacological studies in various plants. The Ca 2ϩ chelator BAPTA and the Ca 2ϩ channel inhibitor La 3ϩ efficiently inhibit elicitor-stimulated reactions, such as Ca 2ϩ influx and phytoalexin accumulation in parsley (6), soybean (73), carrot (74), and tobacco (75). But in the absence of elicitor, the effects of Ca 2ϩ influx on the activation of plant defense responses were slightly different depending on the plant cells. In soybean (73) and carrot cells (74), the treatment of Ca 2ϩ ionophore alone stimulated phytoalexin accumulation, but this did not happen in tobacco cells (75). These results suggest that in some systems, these Ca 2ϩ fluxes are apparently sufficient, whereas in others additional elements seem to be required for the induction of downstream effects. From these results we propose that extracellular Ca 2ϩ influx is a necessary element for the induction of OsMlo gene expression in response to pathogen but is not sufficient to induce OsMlo gene expression by itself in rice suspension cultured cells. Our results provide biological cues for understanding the role of OsMlo in plant defense responses. Further characterization of the mode of CaM action in Mlo function(s) should help to elucidate the role of Ca 2ϩ -CaM in plant defense and stress responses.