SummaryTo investigate promoter strength and the tissue-specific patterns of expression, chimeric promoters incorporating subdomains of mannopine synthese (mas2') and octopine synthase (ocs) promoter and activator regions were affixed to a ~-glucuronidase reporter gene and the constructions introduced into tobacco plants. Addition of a trimer of the ocs upstream activating sequence (UAS) to a mas promoter/activator region resulted in highly elevated levels of GUS activity in ell tissues examined. In leaf tissue, this chimeric promoter is approximately 156-fold and 26-fold stronger than are the CaMV 35S and the "enhanced" double CaMV 35S promoters, respectively. Expression of GUS activity directed by the mas and ors promoters/ activators is limited to specific cell types. Addition of the ocs or mas UAS to the mas or ocs promoter/activator regions modulated these expression patterns. The addition of a trimer of the ocs UAS to the mas promoter/activator region resulted in e transcriptional control element that directed GUS expression in most cell types. In addition to the strong expression in trensgenic tobacco plants, this novel promoter directed higher levels of GUS expression than did the CaMV 35S promoter in transiently transformed tobacco leaf discs end suspension culture cells, as well as in cassava and cowpea explants. It is proposed that the strong promoter containing a trimer of the ocs UAS affixed to the mas promoter/activator will be useful for the very high level constitutive expression of linked genes in a wide variety of plant species.

Calmodulin, a ubiquitous calcium-binding protein, regulates many diverse cellular functions by modulating the activity of the proteins that interact with it. Here, we report isolation of a cDNA encoding a novel kinesin-like calmodulin-binding protein (KCBP) from Arabidopsis using biotinylated calmodulin as a probe. Calcium-dependent binding of the cDNA-encoded protein to calmodulin is confirmed by 35

We developed a sensitive procedure to investigate the kinetics of transcription of an Agrobacterium tumefaciens transferred (T)-DNA-encoded beta-glucuronidase gusA (uidA) gene soon after infection of plant suspension culture cells. The procedure uses a reverse transcriptase-polymerase chain reaction and enables detection of gusA transcripts within 18 to 24 hr after cocultivation of the bacteria with either tobacco or maize cells. Detection of gusA transcripts depended absolutely on the intact virulence (vir) genes virB, virD1/virD2, and virD4 within the bacterium. Mutations in virC and virE resulted in delayed and highly attenuated expression of the gusA gene. A nonpolar transposon insertion into the C-terminal coding region of virD2 resulted in only slightly decreased production of gusA mRNA, although this insertion resulted in the loss of the nuclear localization sequence and the important omega region from VirD2 protein and rendered the bacterium avirulent. However, expression of gusA transcripts in tobacco infected by this virD2 mutant was more transient than in cells infected by a wild-type strain. Infection of tobacco cells with an Agrobacterium strain harboring a mutant virD2 allele from which the omega region had been deleted resulted in similar transient expression of gusA mRNA. These data indicate that the C-terminal nuclear localization signal of the VirD2 protein is not essential for nuclear uptake of T-DNA and further suggest that the omega domain of VirD2 may be required for efficient integration of T-DNA into the plant genome. The finding that the initial kinetics of gusA gene expression in maize cells are similar to those shown in infected tobacco cells but that the presence of gusA mRNA in maize is highly transient suggests that the block to maize transformation involves T-DNA integration and not T-DNA entry into the cell or nuclear targeting.

We developed a sensitive procedure to investigate the kinetics of transcription of an Agrobacterium tumefaciens transferred (T)-DNA-encoded beta-glucuronidase gusA (uidA) gene soon after infection of plant suspension culture cells. The procedure uses a reverse transcriptase-polymerase chain reaction and enables detection of gusA transcripts within 18 to 24 hr after cocultivation of the bacteria with either tobacco or maize cells. Detection of gusA transcripts depended absolutely on the intact virulence (vir) genes virB, virD1/virD2, and virD4 within the bacterium. Mutations in virC and virE resulted in delayed and highly attenuated expression of the gusA gene. A nonpolar transposon insertion into the C-terminal coding region of virD2 resulted in only slightly decreased production of gusA mRNA, although this insertion resulted in the loss of the nuclear localization sequence and the important omega region from VirD2 protein and rendered the bacterium avirulent. However, expression of gusA transcripts in tobacco infected by this virD2 mutant was more transient than in cells infected by a wild-type strain. Infection of tobacco cells with an Agrobacterium strain harboring a mutant virD2 allele from which the omega region had been deleted resulted in similar transient expression of gusA mRNA. These data indicate that the C-terminal nuclear localization signal of the VirD2 protein is not essential for nuclear uptake of T-DNA and further suggest that the omega domain of VirD2 may be required for efficient integration of T-DNA into the plant genome. The finding that the initial kinetics of gusA gene expression in maize cells are similar to those shown in infected tobacco cells but that the presence of gusA mRNA in maize is highly transient suggests that the block to maize transformation involves T-DNA integration and not T-DNA entry into the cell or nuclear targeting.

The kinesin-like calmodulin binding protein (KCBP) is a new member of the kinesin superfamily that appears to be present only in plants. The KCBP is unique in its ability to interact with calmodulin in a Ca 2 ؉ -dependent manner. To study the interaction of the KCBP with microtubules, we expressed different regions of the Arabidopsis KCBP and used the purified proteins in cosedimentation assays with microtubules. The motor domain with or without the calmodulin binding domain bound to microtubules. The binding of the motor domain containing the calmodulin binding region to microtubules was inhibited by Ca 2 ؉ -calmodulin. This Ca 2 ؉ -calmodulin regulation of motor domain interactions with microtubules was abolished in the presence of antibodies specific to the calmodulin binding region. In addition, the binding of the motor domain lacking the calmodulin binding region to microtubules was not inhibited in the presence of Ca 2 ؉ -calmodulin, suggesting an essential role for the calmodulin binding region in Ca 2 ؉ -calmodulin modulation. Results of the cosedimentation assays with the N-terminal tail suggest the presence of a second microtubule binding site on the KCBP. However, the interaction of the N-terminal tail region of the KCBP with microtubules was insensitive to ATP. These data on the interaction of the KCBP with microtubules provide new insights into the functioning of the KCBP in plants.