Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration
2001; Springer Nature; Volume: 20; Issue: 22 Linguagem: Inglês
10.1093/emboj/20.22.6550
ISSN1460-2075
Autores Tópico(s)CRISPR and Genetic Engineering
ResumoArticle15 November 2001free access Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration Haico van Attikum Haico van Attikum Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands Search for more papers by this author Paul Bundock Paul Bundock Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands Search for more papers by this author Paul J. J. Hooykaas Corresponding Author Paul J. J. Hooykaas Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands Search for more papers by this author Haico van Attikum Haico van Attikum Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands Search for more papers by this author Paul Bundock Paul Bundock Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands Search for more papers by this author Paul J. J. Hooykaas Corresponding Author Paul J. J. Hooykaas Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands Search for more papers by this author Author Information Haico van Attikum1, Paul Bundock1 and Paul J. J. Hooykaas 1 1Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:6550-6558https://doi.org/10.1093/emboj/20.22.6550 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Agrobacterium tumefaciens causes crown gall disease in dicotyledonous plants by introducing a segment of DNA (T-DNA), derived from its tumour-inducing (Ti) plasmid, into plant cells at infection sites. Besides these natural hosts, Agrobacterium can deliver the T-DNA also to monocotyledonous plants, yeasts and fungi. The T-DNA integrates randomly into one of the chromosomes of the eukaryotic host by an unknown process. Here, we have used the yeast Saccharomyces cerevisiae as a T-DNA recipient to demonstrate that the non-homologous end-joining (NHEJ) proteins Yku70, Rad50, Mre11, Xrs2, Lig4 and Sir4 are required for the integration of T-DNA into the host genome. We discovered a minor pathway for T-DNA integration at the telomeric regions, which is still operational in the absence of Rad50, Mre11 or Xrs2, but not in the absence of Yku70. T-DNA integration at the telomeric regions in the rad50, mre11 and xrs2 mutants was accompanied by gross chromosomal rearrangements. Introduction Agrobacterium tumefaciens causes crown gall disease in plants by transferring an oncogenic segment of DNA, the transferred or T-DNA, to plant cells at wound sites (Chilton et al., 1977; Tinland and Hohn, 1995). The T-DNA is derived from an ∼200 kb tumour-inducing (Ti) plasmid, which is present in the bacterium. Plant phenolic compounds, produced by the wounded plant cells, induce expression of virulence genes located elsewhere on the Ti plasmid. The virulence protein VirD2 introduces nicks at the 24 bp border repeats, which flank the T-region (Wang et al., 1987; Pansegrau, 1993). This leads to the formation of a single-stranded DNA copy from the T-region, which is called the T-strand (Stachel et al., 1986). The T-strand is bound at the 5′ end by the VirD2 protein and it is this single stranded nucleoprotein complex that is delivered into plant cells (Ward and Barnes, 1988; Chaudhury et al., 1994; Tinland et al., 1994; Yusibov et al., 1994). There it is co-operatively bound by the VirE2 protein (Citovsky et al., 1989), which is delivered separately into plant cells by the bacterium (Vergunst et al., 2000), and targeted to the nucleus by the presence of a nuclear localization signal in the VirE2 and VirD2 proteins (Tinland et al., 1992; Rossi et al., 1993; Tzfira et al., 2000). T-DNA integration occurs at random positions in the genome by a process of non-homologous recombination (NHR) (Offringa et al., 1990; Mayerhofer et al., 1991; Tinland and Hohn, 1995; Gelvin, 2000). Although the processing and transfer of T-DNA to plants is reasonably well understood, (host) factors involved in T-DNA integration are just beginning to be identified (Gelvin, 2000; Mysore et al., 2000). The T-DNA itself does not encode enzymes that are involved in integration. As the Agrobacterium proteins VirD2 and VirE2 accompany the T-DNA to the plant nucleus, it is reasonable to propose that they may be involved in T-DNA integration. The VirD2 protein indeed is important for the accuracy of the integration of T-DNA (Tinland et al., 1995). The VirE2 protein probably protects the T-DNA from nucleolytic degradation and eases its translocation into the nucleus (Rossi et al., 1996; Zupan et al., 1996). Therefore, VirD2 and VirE2 are important for T-DNA transfer and nuclear targeting, but do not seem to play an essential role in the integration process per se. In accordance with this, in vitro studies showed that a T-DNA ligation-integration reaction is mediated by plant enzymes, which implies a role for host factors in T-DNA integration (Ziemienovicz et al., 2000). T-DNA transfer can also occur, at least under laboratory conditions, to yeasts and fungi (Bundock et al., 1995; Bundock and Hooykaas, 1996; De Groot et al., 1998; Gouka et al., 1999) where, in the absence of DNA homology, integration occurs by a similar process of NHR as in plants. In contrast, integration occurs by homologous recombination (HR) when the T-DNA carries homology with the yeast genome. This was not found in plants where T-DNA sharing extensive homology with the plant genome still integrates mainly by NHR (Offringa et al., 1990). These important findings indicate that the process of T-DNA integration into the host genome is predominantly determined by host factors. Recently, Salomon and Puchta (1998) showed that T-DNA could be captured during DNA double-strand break (DSB) repair in plants. This suggests that DSB repair provides a pathway for T-DNA integration. However, as the right ends of the T-DNAs that had integrated into the DSB were all truncated, it is possible that this does not represent the most common form of T-DNA integration. Studies on the repair of DNA DSBs in the yeast Saccharomyces cerevisiae revealed that there are two general recombination mechanisms: one that requires homology between the two recombining DNA molecules (HR) and one that is independent of such homology [non-homologous end-joining (NHEJ)] (reviewed by Critchlow and Jackson, 1998; Haber, 2000). Several mechanisms have been described for the repair of DSBs by HR, most of which rely on the action of genes of the RAD52 epistasis group (RAD50-RAD59, MRE11 and XRS2) (reviewed by Sung et al., 2000). Studies on the repair of DSBs under conditions where HR was impossible revealed that at least 10 genes are required for repair by NHEJ (YKU70, YKU80, LIG4, LIF1, SIR2, SIR3, SIR4, RAD50, MRE11 and XRS2) (reviewed by Tsukamoto and Ikeda, 1998; Lewis and Resnick, 2000). Most of these NHEJ genes have additional functions in telomere length maintenance (RAD50, MRE11, XRS, YKU70, YKU80 and SIR2-SIR4; Porter et al., 1996; Boulton and Jackson, 1998; Chamankkah and Xiao, 1999; Gallego and White, 2001) and/or transcriptional silencing at the telomeres (YKU70, YKU80 and SIR2-SIR4; Aparicio et al., 1991; Boulton and Jackson, 1998). We have now studied the role of host proteins in the integration of T-DNA by NHR. As the results obtained so far on this topic with plants are controversial (Gelvin, 2000), we have now employed the yeast S.cerevisiae as a model organism to investigate which of the genes encoding for recombination enzymes are necessary for T-DNA integration. To this end T-DNA integration in wild-type was compared with that in isogenic strains carrying disruptions of these genes. The results show for the first time that the NHEJ proteins Yku70, Rad50, Mre11, Xrs2, Lig4 and Sir4 are required for the integration of T-DNA into the host genome. We discovered a minor pathway for T-DNA integration at the telomeric regions, which is still operational in the absence of Rad50, Mre11 and Xrs2, but not in the absence of Yku70. T-DNA integration at the telomeric regions in the rad50, mre11 and xrs2 mutants was accompanied by gross chromosomal rearrangements. Results A versatile T-DNA to study integration by NHR in yeast T-DNA, which lacks homology with the yeast genome, has been described to integrate by NHR (Bundock and Hooykaas, 1996). The T-DNA that was used in this study carried the URA3 gene and to prevent homology between the T-DNA and the yeast genome the URA3 gene was removed from the genome of the T-DNA recipient. In order to be able to study the integration of T-DNA by NHR in various yeast strains, independent of the genetic background, a novel T-DNA vector (pSDM8000) was constructed with the KanMX selectable marker between the T-DNA border repeats (Figure 1; Wach et al., 1994). This marker, which allows selection of transgenic yeasts resistant to G418, consists of heterologous DNA and thus the T-DNA of pSDM8000 lacks any homology with the yeast genome. Integration of the T-DNA from pSDM8000 into the yeast genome can therefore only occur by NHR. The T-DNA from pSDM8000 integrated in the wild-type yeast strains YPH250 and JKM115 at frequencies of 1.6 × 10−7 and 1.2 × 10−5, respectively (Table I). For 10 T-DNA insertions the integration site was established after retrieval of the linked genomic sequences by the Vectorette PCR (data not shown). This confirmed that integration of the T-DNA from pSDM8000 had occurred by NHR as was described previously (Bundock and Hooykaas, 1996). Figure 1.Schematic representation of the T-DNA from pSDM8000. The T-DNA from pSDM8000 was used in co-cultivation experiments to study T-DNA integration by NHR in recombination defective S.cerevisiae strains. The T-DNA contains the KanMX cassette, which consists of the kan resistance gene of the Escherichia coli transposon Tn903 under control of transcriptional and translational sequences of the filamentous fungus Ashbya gossypii TEF gene. This module allows selection of S.cerevisiae transformants resistant against the antibiotic G418 (Wach et al., 1994). Download figure Download PowerPoint Table 1. Frequencies of T-DNA integration by NHR in recombination defective yeast strains Strain Genotype Frequency of G418-resistant colonies ± SEM (×10−8)a Relative frequency of G418-resistant colonies (%)b YPH250 WT 16 ± 9.6 100 YPH250rad51 rad51Δ 14 ± 7.8 88 YPH250rad52 rad52Δ 38 ± 8.4c 238 YPH250yku70 yku70Δ <0.0075c <0.05 YPH250rad50 rad50Δ 0.80 ± 0.40c 5.0 YPH250lig4 lig4Δ 0.37 ± 0.36c 2.3 JKM115 WT 1150 ± 0.50 100 JKM129 xrs2Δ 27 ± 6.0c 2.3 JKM138 mre11Δ 29 ± 3.0c 2.5 JKM120sir4 sir4Δ 15 ± 1.9c 1.3 a All yeast strains were co-cultivated with LBA1119(pSDM8000). Averages of two or more independent experiments are shown. Frequencies are depicted as the number of G418-resistant colonies divided by the output number of yeast cells (cells/ml). SEM= standard error of the mean. b The relative frequency of T-DNA integration by NHR is (frequency in the mutant/frequency in the wild-type (WT)) × 100%. c The means of the frequency of G418-resistant colonies seen in the wild-type (WT) and the mutant were tested significantly different in a Student's t-test (p 2-fold higher in the rad52 mutant. This latter observation may be explained by the idea that Rad52 and Yku70 are competing agents, channelling recombination into either HR or NHR, respectively (Haber, 1999). Yku70-mediated integration of T-DNA by NHR may be more efficient as the competing pathway of HR is not operative in the absence of Rad52. T-DNA integrates preferentially at (sub)telomeric regions in rad50, mre11 and xrs2 mutants. The telomeres comprise ∼2% of the yeast genome (Zakian, 1996). Therefore, 2% of the T-DNA insertions would be expected at the telomeres. In fact in the wild-type we have found one telomeric T-DNA insertion in 54 analysed wild-type lines (Table II). In contrast, in the rad50, xrs2 and mre11 mutants eight out of 11 T-DNAs had integrated in this area. The Rad50, Mre11 and Xrs2 proteins play a minor role in telomeric silencing (Boulton and Jackson, 1998). Thus, an explanation might be that reduced silencing at the telomeric region makes this part of the chromosome more accessible for T-DNA, thereby facilitating T-DNA integration at (sub)telomeric regions. The Sir4 protein plays an important role in transcriptional silencing at the telomeres (Aparicio et al., 1991). However, we did not find a bias for T-DNA integration at these regions in the sir4 mutant. Therefore, we conclude that the absence of telomeric silencing in the rad50, mre11 and xrs2 mutants is not responsible for the integration of T-DNA at the telomeres. We speculate that an alternative T-DNA integration pathway, which leads to specific integration at the telomeres or ribosomal DNA repeat, can replace the normal integration pathway in the absence of an active Rad50–Mre11–Xrs2 complex. One possibility is that this pathway may operate in vivo to repair DNA aberrations occurring during DNA replication of repeated DNA. Alternatively, it may be used for the restoration of the telomeric structure. The Rad50–Mre11–Xrs2 complex and the Yku70 protein are involved in telomere length maintenance and rad50, mre11, xrs2 and yku70 mutants show shortened telomeres (Porter et al., 1996; Boulton and Jackson, 1998). The telomeric structure has to be restored in order to survive and this process may lead to the incorporation of T-DNA at the telomeres. As we did not obtain such T-DNA insertions in the yku70 mutant, it may be that YKU70 plays an important role in this pathway. It has been found that the lack of Sir genes induces the loss of silencing of cryptic mating-type genes. This leads to changes in expression of mating-type specific g
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