Reduction of stability of arabidopsis genomic and transgenic DNA-repeat sequences (microsatellites) by inactivation of AtMSH2 mismatch-repair function

Highly conserved mismatch repair (MMR) systems promote genomic stability by correcting DNA replication errors, antagonizing homeologous recombination, and responding to various DNA lesions. Arabidopsis and other plants encode a suite of MMR protein orthologs, including MSH2, the constant component of various specialized eukaryotic mismatch recognition heterodimers. To study MMR roles in plant genomic stability, we used Arabidopsis AtMSH2::TDNA mutant SALK_002708 and AtMSH2 RNA-interference (RNAi) lines. AtMSH2::TDNA and RNAi lines show normal growth, development, and fertility. To analyze AtMSH2 effects on germ line DNA fidelity, we measured insertion-deletion mutation of dinucleotide-repeat sequences (microsatellite instability) at nine loci in 16 or more progeny of two to four different wild-type or AtMSH2-deficient plants. Scoring 992 total alleles revealed 23 (2.3%) unique and 51 (5.1%) total repeat length shifts ([+2], [-2], [+4], or [-4] bp). For the six longest repeat loci, the corresponding frequencies were 22/608 and 50/608. Two of four AtMSH2-RNAi plants showed similar microsatellite instability. In wild-type progeny, only one unique repeat length allele was found in 576 alleles tested. This endogenous microsatellite instability, shown for the first time in MMR-defective plants, is similar to that seen in MMR-defective yeast and mice, indicating that plants also use MMR to promote germ line fidelity. We used a frameshifted reporter transgene, (G)(7)GUS, to measure insertion-deletion reversion as blue-staining beta-glucuronidase-positive leaf spots. Reversion rates increased only 5-fold in AtMSH2::TDNA plants, considerably less than increases in MSH2-deficient yeast or mammalian cells for similar mononucleotide repeats. Thus, MMR-dependent error correction may be less stringent in differentiated leaf cells than in plant equivalents of germ line tissue.     

The Arabidopsis DNA mismatch repair gene <Emphasis Type="Italic">PMS1</Emphasis> restricts somatic recombination between homeologous sequences

The eukaryotic DNA mismatch repair (MMR) system contributes to maintaining the fidelity of genetic information by correcting replication errors and preventing illegitimate recombination events. This study aimed to examine the function(s) of the Arabidopsis thaliana PMS1 gene (AtPMS1), one of three homologs of the bacterial MutL gene in plants. Two independent mutant alleles (Atpms1-1 and Atpms1-2) were obtained and one of these (Atpms1-1) was studied in detail. The mutant exhibited a reduction in seed set and a bias against the transmission of the mutant allele. Somatic recombination, both homologous and homeologous, was examined using a set of reporter constructs. Homologous recombination remained unchanged in the mutant while homeologous recombination was between 1.7- and 4.8-fold higher than in the wild type. This increase in homeologous recombination frequency was not correlated with the degree of sequence divergence. In RNAi lines, a range of increases in homeologous recombination were observed with two lines showing a 3.3-fold and a 3.6-fold increase. These results indicate that the AtPMS1 gene contributes to an antirecombination activity aimed at restricting recombination between diverged sequences. Liangliang Li, Eric Dion contributed equally to this work.     

Effects of suppressing the DNA mismatch repair system on homeologous recombination in tomato

In plant breeding, the ability to manipulate genetic (meiotic) recombination would be beneficial for facilitating gene transfer from wild relatives of crop plants. The DNA mismatch repair (MMR) system helps maintain genetic integrity by correcting base mismatches that arise via DNA synthesis or damage, and antagonizes recombination between homeologous (divergent) DNA sequences. Previous studies have established that the genomes of cultivated tomato (Solanum lycopersicum) and the wild relative S. lycopersicoides are substantially diverged (homeologous) such that recombination between their chromosomes is strongly reduced. Here, we report the effects on homeologous recombination of suppressing endogenous MMR genes in S. lycopersicum via RNAi-induced silencing of SlMSH2 and SlMSH7 or overexpressing dominant negatives of Arabidopsis MSH2 (AtMSH2-DN) in an alien substitution line (SL-8) of S. lycopersicoides in tomato. We show that certain inhibitions of MMR (RNAi of SlMSH7, AtMSH2-DN) are associated with modest increases in homeologous recombination, ranging from 3.8 to 29.2% (average rate of 17.8%) compared to controls. Unexpectedly, only the AtMSH2-DN proteins but not RNAi-induced silencing of MSH2 was found to increase homeologous recombination. The ratio of single to double crossovers (SCO:DCO ratio) decreased by approximately 50% in progeny of the AtMSH2-DN parents. An increase in the frequency of heterozygous SL-8 plants was also observed in the progeny of the SlMSH7-RNAi parents. Our findings may contribute to acceleration of introgression in cultivated tomato.     

Are the T-DNA Mutants Amenable to Standard Recombination Analysis?

Genetic analysis with T-DNA mutants often brings difficulties resulting from instability of the transgenic phenotype. In this work three different Arabidopsis thaliana T-DNA embryonic lethals and one T-DNA morphological mutant were analyzed in F2 progeny after 15 different crosses with marker lines for individual chromosomes. F2 analysis of 44 segregation ratios revealed segregation distortion of similar character consisting in abnormal excess of nontransgenic plants to the detriment of transgenic ones. We quantified this phenotypic drift (d) on the basis of phenotypic ratios given the respective formulas. The d values indicate the rate of F1 gametes which loose the T-DNA mutation or ability of its expression. The obtained d value were relatively high, 0.4 to 0.9 for individual crosses. It makes the standard recombination analysis with insertional mutants very problematic or even impossible.     

A novel reporter for intrachromosomal homoeologous recombination in Arabidopsis thaliana

A reporter system using engineered introns as recombination substrates in the uidA (GUS) gene has been developed and characterized in Arabidopsis thaliana. The non-coding nature of the recombination substrate has allowed us to monitor recombination events between duplicated copies of the intron that are either identical (homologous recombination) or harbour sequence polymorphisms (homoeologous recombination). The effects of substrate length and divergence on the frequency of recombination events were examined. A positive correlation between substrate length and somatic recombination frequency was found as the frequency of recombination increased 183-fold when the recombination substrate was lengthened from 153 to 589 bp. The existence of 11 polymorphisms in a 589-bp recombination substrate (1.9% sequence divergence) led to an almost 10-fold reduction in the frequency of recombination. This result demonstrates that relatively modest levels of sequence divergence can substantially reduce the frequency of recombination in plants. A molecular analysis of recombination products revealed that the recombination junctions are more frequent in the central segment of the recombination substrate.     

Expression and functional analysis of AtMUS81 in Arabidopsis meiosis reveals a role in the second pathway of crossing-over

Meiotic crossovers/chiasmata, that are required to ensure chromosome disjunction, arise via the class I interference-dependent pathway or via the class II interference-free pathway. The proportions of these two classes vary considerably between different organisms. In Arabidopsis, about 85% of chiasmata are eliminated in Atmsh4 mutants, denoting that these are class I events. In budding and fission yeasts Msh4-independent crossovers arise largely or entirely via a Mus81-dependent pathway. To investigate the origins of the 15% residual (AtMSH4-independent) chiasmata in Arabidopsis we conducted a cytological and molecular analysis of AtMUS81 meiotic expression and function. Although AtMUS81 functions in somatic DNA repair and recombination, it is more highly expressed in reproductive tissues. The protein is abundantly present in early prophase I meiocytes, where it co-localizes, in a double-strand break-dependent manner, with the recombination protein AtRAD51. Despite this, an Atmus81 mutant shows normal growth and has no obvious defects in reproductive development that would indicate meiotic impairment. A cytological analysis confirmed that meiosis was apparently normal in this mutant and its mean chiasma frequency was similar to that of wild-type plants. However, an Atmsh4 / Atmus81 double mutant revealed a significantly reduced mean chiasma frequency (0.85 per cell), compared with an Atmsh4 single mutant (1.25 per cell), from which we conclude that AtMUS81 accounts for some, but not all, of the 15% AtMSH4-independent residual crossovers. It is possible that other genes are responsible for these residual chiasmata. Alternatively the AtMUS81 pathway coexists with an alternative parallel pathway that can perform the same functions.     

Reverse Genetic Approaches for Functional Genomics of Rice

T-DNA and transposable elements e.g., Ds and Tos17, are used to generate a large number of insertional mutant lines in rice. Some carry the GUS or GFP reporter for gene trap or enhancer trap. These reporter systems are valuable for identifying tissue- or organ-preferential genes. Activation tagging lines have also been generated for screening mutants and isolating mutagenized genes. To utilize these resources more efficiently, tagged lines have been produced for reverse genetic approaches. DNA pools of the T-DNA tagged lines and Tos17 lines have been prepared for PCR screening of insertional mutants in a given gene. Tag end sequences (TES) of the inserts have also been produced. TES databases are beneficial for analyzing the function of a large number of rice genes.     

Impact of the loss of AtMSH2 on double-strand break-induced recombination between highly diverged homeologous sequences in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> germinal tissues

We experimented a novel reporter system to analyze intrachromosomal recombination between homeologous sequences in Arabidopsis germ cell lineages. The recombination substrates used are the BAR and PAT genes which diverge by about 13% at the nucleotide level and confer resistance to the herbicide glufosinate. DNA double-strand breaks (DSBs) were generated by the I-Sce1 endonuclease to induce recombination. Loss of AtMSH2 induces a 3-fold increase of the frequency of recombination events indicating that AtMSH2 is involved in the anti-recombination activity that prevents exchange between highly diverged sequences in Arabidopsis. Molecular analysis of recombined alleles indicates that in wild type plants the single strand annealing (SSA) pathway can process more efficiently homologous 3′ ends than 3′ ends generated by resection of non-homologous overhangs. The loss of AtMSH2 disturbs this process, leading to a modification of the distribution of the BAR/PAT junctions and therefore showing that the MSH2 function is also involved in determining the structure of the recombined alleles. In addition, conversion tracts were observed in some alleles. They are shorter in MSH2 deficient plants than in wild-type, suggesting that a short-patch mismatch repair, not controlled by MSH2, could exist in Arabidopsis.     

Arabidopsis glucan synthase-like 10 functions in male gametogenesis

Callose or beta-1,3-glucan performs multiple functions during male and female gametophyte development. Callose is synthesized by 12 members of the glucan synthase-like (GSL) gene family in Arabidopsis thaliana. To elucidate the biological roles of Arabidopsis GSL family members during sexual development, we initiated a reverse genetic approach with T-DNA insertional mutagenesis lines. We screened T-DNA insertion lines for all members of the GSL gene family and detected homozygous mutant seedlings for all members except GSL10. Three independent alleles in GSL10, gsl10-1, gsl10-3 and gsl10-4 showed distorted segregation (1:1:0) of T-DNA inserts rather than Mendelian segregation (1:2:1). By genetic analysis through reciprocal cross, we determined that gsl10 pollen could not be transmitted to descendent. The mutant pollen of GSL10/gsl10 plants at tetrad and microspore stages were not different from that of wild type, suggesting that GSL10 is not essential for normal microspore growth. Analysis of GSL10/gsl10 hemizygous pollen during development revealed abnormal function in asymmetric microspore division. gsl10 mutant microspores failed to enter into mitosis. Unlike the previously described functions of GSL1, GSL2 and GSL5, GSL10 involves an independent process of pollen development at the mitotic division stage.     

A bidirectional gene trap construct suitable for T-DNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.)

A construct suitable for genome-wide transfer-DNA (T-DNA) and subsequent transposon-based (Ds) gene trapping has been developed for use in rice (Oryza sativa). This T-DNA/Ds construct contains: Ds terminal sequences immediately inside T-DNA borders for subsequent Ds mobilization; promoterless green fluorescent protein (sgfpS65T) and beta-glucuronidase (uidA) reporter genes, each fused to an intron (from Arabidopsis GPA1 gene) to enable bidirectional gene trapping by T-DNA or Ds; an ampicillin resistance gene (bla) and a bacterial origin of replication (ori) to serve as the plasmid rescue system; an intron-containing hygromycin phosphotransferase gene (hph) as a selectable marker or Ds tracer; and an intron-containing barnase gene in the binary vector backbone (VB) to select against transformants carrying unwanted VB sequences. More than a threefold increase over previously reported reporter gene-based gene trapping efficiencies was observed in primary T-DNA/Ds transformant rice lines, returning an overall reporter gene expression frequency of 23%. Of the plant organs tested, 3.3-7.4% expressed either reporter at varying degrees of organ or tissue specificity. Approximately 70% of the right border (RB) flanking sequence tags (FSTs) retained 1-6 bp of the RB repeat and 30% of the left border (LB) FSTs retained 5-23 bp of the LB repeat. The remaining FSTs carried deletions of 2-84 bp inside the RB or 1-97 bp inside the LB. Transposition of Ds from the original T-DNA was evident in T-DNA/Ds callus lines super-transformed with a transposase gene (Ac) construct, as indicated by gene trap reporter activity and rescue of new FSTs in the resulting double transformant lines.     

Ku80 plays a role in non-homologous recombination but is not required for T-DNA integration in Arabidopsis

Chromosomal breaks are repaired by homologous recombination (HR) or non-homologous end joining (NHEJ) mechanisms. The Ku70/Ku80 heterodimer binds DNA ends and plays roles in NHEJ and telomere maintenance in organisms ranging from yeast to humans. We have previously identified a ku80 mutant of the model plant Arabidopsis thaliana and shown the role of Ku80 in telomere homeostasis in plant cells. We show here that this mutant is hypersensitive to the DNA-damaging agent methyl methane sulphonate and has a reduced capacity to carry out NHEJ recombination. To understand the interplay between HR and NHEJ in plants, we measured HR in the absence of Ku80. We find that the frequency of intrachromosomal HR is not affected by the absence of Ku80. Previous work has clearly implicated the Ku heterodimer in Agrobacterium-mediated T-DNA transformation of yeast. Surprisingly, ku80 mutant plants show no defect in the efficiency of T-DNA transformation of plants with Agrobacterium, showing that an alternative pathway must exist in plants.     

T-DNA insertional mutagenesis for functional genomics in rice

We have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless beta-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6-2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ- or tissue-specific or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.     

Insertional mutagenesis in Arabidopsis thaliana: isolation of a T-DNA-linked mutation that alters leaf morphology

Summary We investigated the potential of the Agrobacterium tumefaciens T-DNA as an insertional mutagen in Arabidopsis thaliana. Arabidopsis lines transformed with different T-DNA vectors were generated using a leaf disc infection procedure adapted for efficient selection on either kanamycin or hygromycin medium. A standardized screening procedure was developed for the detection of recessive mutations in T2 populations of regenerated and/or transformed lines. Recessive mutations originating from the tissue culture procedure occurred at a low frequency — between 2% and 5%. Within 110 transformed lines that contained a total of about 150 T-DNA inserts, one recessive mutation, named pfl, cosegregated with a specific T-DNA copy. This pfl mutation mainly affected the morphology of the first seedling leaves under normal growth conditions and was mapped to chromosome 1. No recombination between the pfl locus and the kanamycin resistance marker on the T-DNA was detected when screening F2 and F3 populations of a mutant crossed to the wild type. The maximal genetic distance between the pfl locus and the kanamycin resistance gene, determined as 0.4±0.4 cMorgan, strongly suggests that the pfl mutation is induced by the insertion of the T-DNA. Our finding of one T-DNA-linked recessive mutation in 110 transgenic lines indicates that T-DNA can be used for mutagenization of the Arabidopsis genome under tissue culture conditions.     

Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation.

We determined whether T-DNA molecules introduced into plant cells using Agrobacterium are suitable substrates for homologous recombination. For the detection of such recombination events different mutant versions of a NPTII construct were used. In a first set of experiments protoplasts of Nicotiana tabacum SR1 were cocultivated with two Agrobacterium tumefaciens strains. Each strain contained a different T-DNA, one carrying a 5' deleted NPTII gene and the other a NPTII gene with a 3' deletion. A restored NPTII gene was found in 1-4% of the protoplasts that had been cotransformed with both T-DNAs. Restoration of the NPTII gene could only be the consequence of homologous recombination between the two different T-DNAs in the plant cell, since the possibility of recombination in Agrobacterium was excluded in control experiments. In subsequent experiments was investigated the potential use of Agrobacterium for gene targeting in plants. A transgenic tobacco line with a T-DNA insertion carrying a defective NPTII gene with a 3' deletion was transformed via Agrobacterium with a T-DNA containing a defective NPTII repair gene. Several kanamycin resistant plant lines were obtained with an intact NPTII gene integrated in their genome. In one of these lines the defective NPTII gene at the target locus had been properly restored. Our results show that in plants recombination can occur between a chromosomal locus and a homologous T-DNA introduced via A. tumefaciens. This opens the possibility of using the Agrobacterium transformation system for site directed mutagenesis of the plant genome.     

Relationship between allelic state of T-DNA and DNA methylation of chromosomal integration region in transformed <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

T-DNA insertions are currently used as a tool to introduce, or knock out, specific genes. The expression of the inserted gene is frequently haphazard and up to now, it was proposed that transgene expression depends on the site of insertion within the genome, as well as the number of copies of the transgene. In this paper, we show that the allelic state of a T-DNA insertion can be at the origin of epigenetic silencing. A T-DNA insertional mutant was characterized to explore the function of AtBP80a′, a vacuolar sorting receptor previously associated with germination. Seeds homozygous for the T-DNA do not germinate, but this can be overcome by a cold treatment and maintained by the following generations. The non-germinating phenotype is only observed in homozygous seed produced by heterozygous plants indicating that it is correlated with the allelic state of the T-DNA in parental lines. Analysis of the region between the T-DNA insertion and the ATG codon of atbp80a′ showed that cytosine methylation is highly enhanced in chromatin containing the T-DNA. Data presented here show that an unpaired DNA region during meiosis could be at the origin of a de novo cytosine methylation mechanism.     

AtMGT9基因T-DNA插入突变体表型分析

利用功能互补,对拟南芥AtMGT9基因的Mg2＋转运功能进行了研究.同时对AtMGT9的两个T-DNA插入突变体株系（mgt9-1,mgt9-2）进行表型分析,mgt9-1只能得到杂合体植株,有花粉败育现象,mgt9-2有纯合体,但没有可见表型.基因序列分析表明,mgt9-1中T-DNA插入位点在起始密码子后,mgt9-2中T-DNA插入位点在靠近C端第2个跨膜区中间位置.功能互补和63Ni2＋示踪实验证明截短的mgt9-2蛋白能够互补细菌镁离子转运突变体MM281, 具有全序列基因一样的转运特性,而mgt9-1不具有转运功能,是一个功能缺失突变体.这为进一步研究AtMGT9蛋白的结构与功能的关系奠定了基础.     

Disruption of the Arabidopsis RAD50 gene leads to plant sterility and MMS sensitivity

The Rad50 protein is involved in the cellular response to DNA-double strand breaks (DSBs), including the detection of damage, activation of cell-cycle checkpoints, and DSB repair via recombination. It is essential for meiosis in yeast, is involved in telomere maintenance, and is essential for cellular viability in mice. Here we present the isolation, sequence and characterization of the Arabidopsis thaliana RAD50 homologue (AtRAD50) and an Arabidopsis mutant of this gene. A single copy of this gene is present in the Arabidopsis genome, located on chromosome II. Northern analysis shows a single 4.3 Kb mRNA species in all plant tissues tested, which is strongly enriched in flowers and other tissues with many dividing cells. The predicted protein presents strong conservation with the other known Rad50 homologues of the amino- and carboxy-terminal regions. Mutant plants present a sterility phenotype which co-segregates with the T-DNA insertion. Molecular analysis of the mutant plants shows that the sterility phenotype is present only in the plants homozygous for the T-DNA insertion. An in vitro mutant cell line, derived from the mutant plant, shows a clear hypersensitivity to the DNA-damaging agent methylmethane sulphonate, suggesting a role of RAD50 in double-strand break repair in plant cells. This is the first report of a plant mutated in a protein of the Rad50-Mre11-Xrs2 complex, as well as the first data suggesting the involvement of the Rad50 homologue protein in meiosis and DNA repair in plants.