We describe the isolation of an Arabidopsis gene that is closely related to the animal ZnT genes (Zn transporter)Several heavy metals are essential during plant growth and development, but their excess can easily lead to toxic effects. Contamination of soils with heavy metals, either by natural causes or due to pollution, often has pronounced effects on the vegetation, resulting in the appearance of metallophytes, heavy-metal-tolerant plants. The precise mechanisms of uptake, transport, and accumulation of heavy metals in plants are poorly understood, but several genes likely to be involved in these processes have been described. Recently, a family of ZIP genes that are expressed in roots upon Zn deficiency was isolated from Arabidopsis (Grotz et al., 1998). The proteins encoded by the ZIP genes have eight predicted TM regions and a high degree of similarity to the ZRT genes from yeast that are involved in Zn uptake. Expression of the ZIP genes in yeast conferred Zn-uptake activities to these cells, demonstrating that they are probably functional homologs of the yeast ZRT genes (Grotz et al., 1998). The only other metaltransporting protein recently identified in plants belongs to the large family of cation-transporting P-type ATPases (Tabata et al., 1997), but these proteins are structurally very different from the metal-transporting proteins mentioned above.Recent data have provided more insight into the mechanisms of heavy-metal tolerance. Metallophytes often exhibit tolerance to several different heavy metals, but all of these metals need not be present at toxic levels in their habitat (Schat and ten Bookum, 1992a; Schat and Vooijs, 1997, and refs. therein;Schat and Verkleij, 1998). Although such a feature is suggestive of a general mechanism of heavy-metal tolerance, recent genetic evidence has shown that a number of different mechanisms must exist, each with its own metal specificity (Schat and Vooijs, 1997). In Arabidopsis, a plant species with a typical level of tolerance to heavy metals, it has been demonstrated that the Cd-sensitive mutants cad1 and cad2 are defective in the synthesis of the metal-binding compound phytochelatin (Howden et al., 1995). cad1 plants were only slightly more sensitive to Cu and Zn, indicating that phytochelatinmediated detoxification is not sufficient for Cu and Zn detoxification (Howden et al., 1995b). Metallothioneins appear to be of major importance for constitutive Cu tolerance in Arabidopsis (Zhou and Goldsbrough, 1994).Aside from complexation of heavy metals by heavymetal-binding proteins, there is evidence that transportmediated sequestration can contribute to heavy-metal tolerance. In the Zn-tolerant plant Silene vulgaris it was shown that Zn transport across the tonoplast was about 2.5 times higher than in Zn-sensitive plants of the same species (Verkleij et al

SummaryZinc-finger nucleases (ZFNs) are artificial restriction enzymes, custom designed for induction of double-strand breaks (DSBs) at a specific locus. These DSBs may result in site-specific mutagenesis or homologous recombination at the repair site, depending on the DNA repair pathway that is used. These promising techniques for genome

Several techniques are available to study chromosomes or chromosomal domains in nuclei of chemically fixed or living cells. Current methods to detect DNA sequences in vivo are limited to trans interactions between a DNA sequence and a transcription factor from natural systems. Here, we expand live cell imaging tools using a novel approach based on zinc finger-DNA recognition codes. We constructed several polydactyl zinc finger (PZF) DNA-binding domains aimed to recognize specific DNA sequences in Arabidopsis and mouse and fused these with GFP. Plants and mouse cells expressing PZF:GFP proteins were subsequently analyzed by confocal microscopy. For Arabidopsis, we designed a PZF:GFP protein aimed to specifically recognize a 9-bp sequence within centromeric 180-bp repeat and monitored centromeres in living roots. Similarly, in mouse cells a PZF:GFP protein was targeted to a 9-bp sequence in the major satellite repeat. Both PZF:GFP proteins localized in chromocenters which represent heterochromatin domains containing centromere and other tandem repeats. The number of PZF:GFP molecules per centromere in Arabidopsis, quantified with near single-molecule precision, approximated the number of expected binding sites. Our data demonstrate that live cell imaging of specific DNA sequences can be achieved with artificial zinc finger proteins in different organisms.

Summary A library of genes for zinc finger artificial transcription factors (ZF‐ATF) was generated by fusion of DNA sequences encoding three‐finger Cys2His2 ZF domains to the VP16 activation domain under the control of the promoter of the ribosomal protein gene RPS5A from Arabidopsis thaliana. After introduction of this library into an Arabidopsis homologous recombination (HR) indicator line, we selected primary transformants exhibiting multiple somatic recombination events. After PCR‐mediated rescue of ZF sequences, reconstituted ZF‐ATFs were re‐introduced in the target line. In this manner, a ZF‐ATF was identified that led to a 200–1000‐fold increase in somatic HR (replicated in an independent second target line). A mutant plant line expressing the HR‐inducing ZF‐ATF exhibited increased resistance to the DNA‐damaging agent bleomycin and was more sensitive to methyl methanesulfonate (MMS), a combination of traits not described previously. Our results demonstrate that the use of ZF‐ATF pools is highly rewarding when screening for novel dominant phenotypes in Arabidopsis.

The VirD2 protein of Agrobacterium tumefaciens is essential for processing and transport of the T-DNA. It has at least three functional domains: a relaxase domain at the N terminus, a bipartite nuclear localization signal (NLS), and a sequence called omega at the C terminus. We confirm here that deletions of the C-terminal part of VirD2 led to lack of transfer of T-DNA but, for the first time, we report that virulence is restored when these truncations are supplemented at the C terminus by a short translocation signal from the VirF protein. The lack of virulence of C-terminal deletions suggests that the C-terminal part contains all or part of the translocation signal of VirD2. Using a novel series of mutant VirD2 proteins, the C-terminal half of VirD2 was further investigated. We demonstrate that the C-terminal 40 amino acids of VirD2, which include the NLS and omega, contain all or part of the translocation domain necessary for transport of VirD2 into plant cells, while another element is present in the middle of the protein. The finding that a type IV secretion system transport signal at the C terminus of VirD2 is necessary for virulence provides evidence for the role of VirD2 as a pilot protein driving translocation of the T-strand.