In plants, defence against specific isolates of a pathogen can be triggered by the presence of a corresponding race-specific resistance gene, whereas resistance of a more broad-spectrum nature can result from recessive, presumably loss-of-regulatory-function, mutations. An example of the latter are mlo mutations in barley, which have been successful in agriculture for the control of powdery mildew fungus (Blumeria graminis f. sp. hordei; Bgh). MLO protein resides in the plasma membrane, has seven transmembrane domains, and is the prototype of a sequence-diversified family unique to plants, reminiscent of the seven-transmembrane receptors in fungi and animals. In animals, these are known as G-protein-coupled receptors and exist in three main families, lacking sequence similarity, that are thought to be an example of molecular convergence. MLO seems to function independently of heterotrimeric G proteins. We have identified a domain in MLO that mediates a Ca2+-dependent interaction with calmodulin in vitro. Loss of calmodulin binding halves the ability of MLO to negatively regulate defence against powdery mildew in vivo. We propose a sensor role for MLO in the modulation of defence reactions.

The Ca 21-binding protein calmodulin mediates cellular Ca 21 signals in response to a wide array of stimuli in higher eukaryotes. Plants express numerous CaM isoforms. Transcription of one soybean (Glycine max) CaM isoform, SCaM-4, is dramatically induced within 30 min of pathogen or NaCl stresses. To characterize the cis-acting element(s) of this gene, we isolated an approximately 2-kb promoter sequence of the gene. Deletion analysis of the promoter revealed that a 130-bp region located between nucleotide positions 2858 and 2728 is required for the stressors to induce expression of SCaM-4. A hexameric DNA sequence within this region, GAAAAA (GT-1 cis-element), was identified as a core cis-acting element for the induction of the SCaM-4 gene. The GT-1 cis-element interacts with an Arabidopsis GT-1-like transcription factor, AtGT-3b, in vitro and in a yeast selection system. Transcription of AtGT-3b is also rapidly induced within 30 min after pathogen and NaCl treatment. These results suggest that an interaction between a GT-1 cis-element and a GT-1-like transcription factor plays a role in pathogen-and salt-induced SCaM-4 gene expression in both soybean and Arabidopsis.Plant cells, like animal cells, elevate their cytosolic free-calcium levels ([Ca 21 ] cyt ) with varying amplitude, frequency, and duration in response to a variety of external stimuli (Thomas et al., 1996; Berridge, 1997;McAinsh and Hetherington, 1998 -bound CaM transduces the signals into many cellular processes through modulation of a variety of CaM-binding proteins, including enzymes such as kinases, phosphatases, and nitric-oxide synthase, as well as receptors, ion channels, G-proteins, and transcription factors (Liao et al., 1996;Snedden and Fromm, 1998;Lee et al., 1999a;Zuhlke et al., 1999).In plant cells, in contrast to mammalian cells, multiple CaM genes code for a number of CaM isoforms. This has been shown in wheat (Triticum aestivum; Yang et al., 1996), potato (Solanum tuberosum; Takezawa et al., 1995;Poovaiah et al., 1996), and soybean (Glycine max; Lee et al., 1995a), among others. Over 30 genes encoding CaM isoforms are found in the Arabidopsis genome (The Arabidopsis Genome Initiative, 2000). We have recently cloned five CaM isoforms from soybean (SCaM-1-5). Although SCaM-1-3 are more than 90% identical to mammalian CaM, SCaM-4 and SCaM-5 exhibit only a 78% homology with SCaM-1 and are therefore the most divergent isoforms reported thus far in the plant and animal kingdoms. SCaM-4 is considered to be a bona fide CaM isoform based on the following characteristics. In its primary protein structure, SCaM-4 has four conserved putative EF-hands and a central linker region, hallmark structural features of CaM (Lee et al., 1995a). In addition, most of the nonconsensus amino acids occur outside the EF-hands, and the total number of Article, publication date, and citation information can be found at www.plantphysiol.org/cgi

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Calmodulin plays pivotal roles in the transduction of various Ca 2؉-mediated signals and is one of the most highly conserved proteins in eukaryotic cells. In plants, multiple calmodulin isoforms with minor amino acid sequence differences were identified but their functional significances are unknown. To investigate the biological function of calmodulins in the regulation of calmodulin-dependent enzymes, we cloned cDNAs encoding calmodulins in soybean. Among the five cDNAs isolated from soybean, designated as SCaM-1 to -5, SCaM-4 and -5 encoded very divergent calmodulin isoforms which have 32 amino acid substitutions from the highly conserved calmodulin, SCaM-1 encoded by SCaM-1 and SCaM-3. SCaM-4 protein produced in Escherichia coli showed typical characteristics of calmodulin such as Ca 2؉ -dependent electrophoretic mobility shift and the ability to activate phosphodiesterase. However, the extent of mobility shift and antigenicity of SCaM-4 were different from those of SCaM-1. Moreover, SCaM-4 did not activate NAD kinase at all in contrast to SCaM-1. Also there were differences in the expression pattern of SCaM-1 and SCaM-4. Expression levels of SCaM-4 were approximately 5-fold lower than those of SCaM-1 in apical and elongating regions of hypocotyls. In addition, SCaM-4 transcripts were barely detectable in root whereas SCaM-1 transcripts were as abundant as in apical and elongating regions of hypocotyls. In conclusion, the different biochemical properties together with differential expression of SCaM-4 suggest that this novel calmodulin may have different functions in plant cells.Calmodulin, a highly conserved and ubiquitous protein in eukaryotes, mediates Ca 2ϩ signals to various target proteins (1). A variety of regulatory enzymes and proteins such as protein kinases, ion channels, Ca 2ϩ pumps, nitric oxide synthetase, inositol trisphosphate kinase, cyclic nucleotide phosphodiesterase, and NAD kinase are known to be regulated by Ca 2ϩ and calmodulin (2-4). While a great deal of information has been known for biological roles of calmodulin in animal cells, very little is known about the roles of calmodulin in plant cells. This is mainly due to the absence of purified calmodulindependent enzymes and/or their genes in plants. As an effort to investigate the biological role(s) of calmodulin in plants, calmodulin genes in various plant species have been cloned and characterized recently (4). Interestingly, in Arabidopsis, cDNAs encoding multiple calmodulin isoforms have been isolated although the degree of sequence divergence is minor, only 6 amino acid differences between the two most divergent isoforms among Arabidopsis calmodulins (5-7). This is very notable because, in animal cells, only a single form of calmodulin is produced by a calmodulin multigene family (8, 9). However, it has not been determined whether plant calmodulin isoforms have the same biochemical properties such as calcium-binding abilities and activation of calmodulin-dependent enzymes. Also the biological role of multiple calmodulin isoforms...