Sivb 2003 Congress Symposium Proceeding: Mutation- and Transposon-Based Approaches for the Identification of Genes for Pre-Harvest Sprouting in Wheat

Summary This article reviews techniques for gene identification and cloning in allohexaploid bread wheat (Triticum aestivum L.). Gene identification and cloning in wheat are complicated by the large size and high redundancy of the genome. Both classical mutagenesis and transposon tagging are important tools for the study of grain dormancy and plant hormone signaling in wheat. While classical mutagenesis can be used to identify wheat mutants with altered hormone sensitivity, it can be difficult to clone the corresponding genes. We review the techniques available for gene identification in wheat, and propose that transposon-based activation tagging will be an important tool for wheat genetics.     

A Modified Screening System for Loss-of-Function and Dominant Negative Alleles of Essential MCMV Genes

Inactivation of gene products by dominant negative mutants is a valuable tool to assign functions to yet uncharacterized proteins, to map protein-protein interactions or to dissect physiological pathways. Detailed functional and structural knowledge about the target protein would allow the construction of inhibitory mutants by targeted mutagenesis. Yet, such data are limited for the majority of viral proteins, so that the target gene needs to be subjected to random mutagenesis to identify suitable mutants. However, for cytomegaloviruses this requires a two-step screening approach, which is time-consuming and labor-intensive. Here, we report the establishment of a high-throughput suitable screening system for the identification of inhibitory alleles of essential genes of the murine cytomegalovirus (MCMV). In this screen, the site-specific recombination of a specifically modified MCMV genome was transferred from the bacterial background to permissive host cells, thereby combining the genetic engineering and the rescue test in one step. Using a reference set of characterized pM53 mutants it was shown that the novel system is applicable to identify non-complementing as well as inhibitory mutants in a high-throughput suitable setup. The new cis-complementation assay was also applied to a basic genetic characterization of pM99, which was identified as essential for MCMV growth. We believe that the here described novel genetic screening approach can be adapted for the genetic characterization of essential genes of any large DNA viruses.     

Embryonic Lethals and T-DNA Insertional Mutagenesis in Arabidopsis

T-DNA insertional mutagenesis represents a promising approach to the molecular isolation of genes with essential functions during plant embryo development. We describe in this report the isolation and characterization of 18 mutants of Arabidopsis thaliana defective in embryo development following seed transformation with Agrobacterium tumefaciens. Random T-DNA insertion was expected to result in a high frequency of recessive embryonic lethals because many target genes are required for embryogenesis. The cointegrate Ti plasmid used in these experiments contained the nopaline synthase and neomycin phosphotransferase gene markers. Nopaline assays and resistance to kanamycin were used to estimate the number of functional inserts present in segregating families. Nine families appeared to contain a T-DNA insert either within or adjacent to the mutant gene. Eight families were clearly not tagged with a functional insert and appeared instead to contain mutations induced during the transformation process. DNA gel blot hybridization with internal and right border probes revealed a variety of rearrangements associated with T-DNA insertion. A general strategy is presented to simplify the identification of tagged embryonic mutants and facilitate the molecular isolation of genes required for plant embryogenesis.     

Characterization of two genes involved in chromate resistance in a Cr(VI)-hyper-resistant bacterium

Mechanisms underlying chromate resistance in Cr(VI)-hyper-resistant Pseudomonas corrugata strain 28, isolated from a highly Cr(VI) polluted soil, were studied by analyzing its two Cr(VI)-sensitive mutants obtained by insertion mutagenesis. The mutants, namely Crg3 and Crg96, were characterized by the identification of disrupted genes, and by the high-throughput approach called Phenotype MicroArray (PM), which permitted the assay of 1,536 phenotypes simultaneously. Crg3 and Crg96 mutants were affected in a malic enzyme family gene and in a gene encoding for a RecG helicase, respectively. The application of PM provided a wealth of new information relating to the disrupted genes and permitted to establish that chromate resistance in P. corrugata strain 28 also depends on supply on NADPH required in repairing damage induced by chromate and on DNA integrity maintenance.     

Production of novel allelic variation for genes involved in starch biosynthesis through mutagenesis

Given the important role that starch plays in food and non-food uses of many crops, particularly wheat, efforts are being made to manipulate its composition through modification of the amylose/amylopectin ratio. Approaches used to achieve this goal include the manipulation of the genes involved in the starch biosynthetic pathway using natural or induced mutations and transgenic methods. The use of mutagenesis to produce novel allelic variation represents a powerful tool to increase genetic diversity and this approach seems particularly appropriate for starch synthase genes for which limited variation exists. In this work, an EMS-mutagenised population of bread wheat cv. Cadenza has been screened by combining SDS–PAGE analysis of granule bound starch proteins with a TILLING (Targeting Induced Local Lesions IN Genomes) approach at the gene level. In particular we have focused on two groups of synthase genes, those encoding the starch synthase II (Sgp-1) and those corresponding to the waxy proteins (Wx). SDS–PAGE analysis of granule bound proteins allowed the identification of single null genotypes associated with each of the three homoeologous loci. Molecular characterization of induced mutants has been performed using genome specific primer pairs for Sgp-1 and Wx genes. Additional novel allelic variation has also been detected at the different Sgp-1 homoeoloci by using a reverse genetic approach (TILLING). In particular single nucleotide substitutions, introducing a premature stop codon and creating amino acid substitutions, have been identified.     

Cryptococcus neoformans virulence gene discovery through insertional mutagenesis

Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.     

来自西尔斯山羊草的抗小麦白粉病基因Pm57抗性丧失突变体的筛选与鉴定

小麦近缘种属来源的抗白粉病基因是培育小麦抗病品种,防治白粉病危害的最重要基因来源。Pm57是位于西尔斯山羊草2S^s#l染色体长臂上的一个外源基因,对小麦白粉病具有苗期和成株期广谱抗性。为了创制Pm57白粉病抗性丧失突变体,利用基于基因突变体的植物抗病基因克隆新兴技术分离Pm57基因,选用0.625%的甲基磺酸乙酯(EMS)对1万粒小麦-西尔斯山羊草Pm57易位系89(5)69种子进行了诱变处理,M1大田密播种植,收获了1598个M2可育株系。初步对其中300个M2株系进行苗期白粉病抗性接种鉴定,并利用2个Pm57基因特异分子标记X2L4g9P4/HaeⅢ和X284274及小麦全国区试品系DUS测试所用的42对SSR核心引物对Pm57抗性丧失突变体进行鉴定,筛选出来自27个M2株系的真实抗性丧失突变体70个,Pm57基因抗性丧失突变体频率达到9.0%。本研究所获得的白粉病抗性丧失突变体为Pm57基因的后续克隆与抗白粉病分子机理研究提供了重要的材料基础。     

Deleteagene: a fast neutron deletion mutagenesis-based gene knockout system for plants

Deleteagene(trade mark) (Delete-a-gene) is a deletion-based gene knockout system for plants. To obtain deletion mutants for a specific gene, random deletion libraries created by fast neutron mutagenesis are screened by polymerase chain reaction (PCR) using primers flanking the target gene. By adjusting the PCR extension time to preferentially amplify the deletion alleles, deletion mutants can be identified in pools of DNA samples with each sample representing more than a thousand mutant lines. In Arabidopsis, knockout plants for greater than 80% of targeted genes have been obtained from a population of 51 840 lines. A large number of deletion mutants have been identified and multiple deletion alleles are often recovered for targeted loci. In Arabidopsis, the method is very useful for targeting small genes and can be used to find deletion mutants mutating two or three tandem homologous genes. In addition, the method is demonstrated to be effective in rice as a deletion mutant for a rice gene was obtained with a similar approach. Because fast neutron mutagenesis is applicable to all plant genetic systems, Deleteagene(trade mark) has the potential to enable reverse genetics for a wide range of plant species.     

Targeted mutagenesis of a conserved anther‐expressed P450 gene confers male sterility in monocots

Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.     

Random insertional mutagenesis of Leptospira interrogans, the agent of leptospirosis, using a mariner transposon

The recent availability of the complete genome sequences of Leptospira interrogans, the agent of leptospirosis, has allowed the identification of several putative virulence factors. However, to our knowledge, attempts to carry out gene transfer in pathogenic Leptospira spp. have failed so far. In this study, we show that the Himar1 mariner transposon permits random mutagenesis in the pathogen L. interrogans. We have identified genes that have been interrupted by Himar1 insertion in 35 L. interrogans mutants. This approach of transposon mutagenesis will be useful for understanding the spirochetal physiology and the pathogenic mechanisms of Leptospira, which remain largely unknown.     

An Assessment of Heavy Ion Irradiation Mutagenesis for Reverse Genetics in Wheat (Triticum aestivum L.)

Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed.     

Highly efficient Staphylococcus carnosus mutant selection system based on suicidal bacteriocin activation

Strains from various staphylococcal species produce bacteriocin peptides, which are thought to play important roles in bacterial competition and offer interesting biotechnological avenues. Many bacteriocins are secreted as inactive prepeptides with subsequent activation by specific proteolytic cleavage. By deletion of the protease gene gdmP in Staphylococcus gallinarum Tü3928, which produces the highly active lanthionine-containing bacteriocin gallidermin (lantibiotic), a strain was created producing inactive pregallidermin. On this basis, a new suicidal mutant selection system in the food-grade bacterium Staphylococcus carnosus was developed. Whereas pregallidermin was inactive against S. carnosus, it exerted potent bactericidal activity toward GdmP-secreting S. carnosus strains. To take advantage of this effect, gdmP was cloned in plasmid vectors used for random transposon mutagenesis or targeted allelic replacement of chromosomal genes. Both mutagenesis strategies rely on rare recombination events, and it has remained difficult and laborious to identify mutants among a vast majority of bacterial clones that still contain the delivery vectors. The gdmP-expressing plasmids pGS1 and pGS2 enabled very fast, easy, and reliable identification of transposon and gene replacement mutants, respectively. Mutant selection in the presence of pregallidermin caused suicidal inactivation of all clones that had retained the plasmids and allowed growth of only plasmid-cured mutants. Efficiency of mutant identification was several magnitudes higher than standard screening for the absence of plasmid-encoded antibiotic resistance markers and reached 100% specificity. Thus, the new pregallidermin-based mutant selection system represents a substantial improvement of staphylococcal mutagenesis methodology.     

RNAi for plant functional genomics

A major challenge in the post-genome era of plant biology is to determine the functions of all the genes in the plant genome. A straightforward approach to this problem is to reduce or knock out expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study, but it is limited by gene redundancy, lethal knock-outs, nontagged mutants and the inability to target the inserted element to a specific gene. RNA interference (RNAi) of plant genes, using constructs encoding self-complementary 'hairpin' RNA, largely overcomes these problems. RNAi has been used very effectively in Caenorhabditis elegans functional genomics, and resources are currently being developed for the application of RNAi to high-throughput plant functional genomics.     

Key genes involved in heavy-metal resistance in Pseudomonas putida CD2

A cadmium-resistant bacterium Pseudomonas putida CD2 was isolated from sewage sludge samples. Strain CD2 exhibited high maximal tolerant concentrations (MTC) for a large spectrum of divalent metals. Screening a library obtained using Tn5-B21 insertion mutagenesis resulted in identification of 12 mutants with a substantial decrease in resistance to 3 mM cadmium. The DNA sequences of the contiguous region from the Tn5 insertion sites were determined by inverse PCR. Six genes involved in cadmium resistance were identified. These genes were from three gene clusters: czcCBA1, cadA2R and colRS. The homologs of the first two gene clusters were predicted to be metal efflux systems, whereas the products of colRS, ColR and ColS, were thought to be a two-component signal transduction (TCST) system. In this study, we have demonstrated that ColRS also function in regulating multi-metal resistance using genetic complementation.     

一些小麦白粉病抗源抗性基因鉴定分析

研究鉴定了我国３７份小麦白粉病抗源的抗性基因,１９份材料不具有任何抗性基因;６份材料具有来自１ＢＬ／１ＲＳ易位系的抗性基因Ｐｍ８;５份材料具有抗性基因Ｐｍ５ａ;３份分别具有对目前欧洲所有生理小种均抗的抗性基因Ｐｍ２１、Ｐｍ１６和Ｐｍ１２;４份材料具有新的抗性基因。     

The Mouse MC13 Mutant Is a Novel ENU Mutation in Collagen Type II,Alpha 1

Phenotype-driven mutagenesis experiments are a powerful approach to identifying novel alleles in a variety of contexts. The traditional disadvantage of this approach has been the subsequent task of identifying the affected locus in the mutants of interest. Recent advances in bioinformatics and sequencing have reduced the burden of cloning these ENU mutants. Here we report our experience with an ENU mutagenesis experiment and the rapid identification of a mutation in a previously known gene. A combination of mapping the mutation with a high-density SNP panel and a candidate gene approach has identified a mutation in collagen type II, alpha I (Col2a1). Col2a1 has previously been studied in the mouse and our mutant phenotype closely resembles mutations made in the Col2a1 locus.     

Comprehensive mutational analysis of a herpesvirus gene in the viral genome context reveals a region essential for virus replication

Essential viral proteins perform vital functions during morphogenesis via a complex interaction with other viral and cellular gene products. Here, we present a novel approach to comprehensive mutagenesis of essential cytomegalovirus genes and biological analysis in the 230-kbp-genome context. A random Tn7-based mutagenesis procedure at the single-gene level was combined with site-specific recombination via the FLP/FLP recognition target site system for viral genome reconstitution. We show the function of more than 100 mutants from a larger library of M50/p35, a protein involved in capsid egress from the nucleus. This protein recruits other viral proteins and cellular enzymes to the inner nuclear membrane. Our approach enabled us to rapidly discriminate between essential and nonessential regions within the coding sequence. Based on the prediction of the screen, we were able to map a site essential for viral protein-protein interaction at the amino acid level.     

Current approaches to the synthesis and reconstruction of genetic material

Advanced approaches to the synthesis and reconstruction of genetic material developed in the Institutes of Molecular Biology and Genetics during the past years are summarized. The evolution of methods for oligonucleotide synthesis and scopes for their use in gene production are discussed. The principles of localised mutagenesis methods developed in the Institute are described, such as: a) mutagenesis directed to the regulatory gene regions; b) segment-localized mutagenesis; c) mutagenesis directed by phosphotriester analogues of oligonucleotides. Examples of employing these methods for induction of regulatory mutants of phage lambda, production of fused genes, mutant interferon genes, construction of new DNA vectors, construction of hybrid H1-H3 subtype haemagglutinine gene of influenza virus etc. are presented. The approach to in vivo site-directed mutagenesis is experimentally substantiated.     

Gene trap mutagenesis-based forward genetic approach reveals that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2

OVCA1 is a tumor suppressor identified by positional cloning from chromosome 17p13.3, a hot spot for chromosomal aberration in breast and ovarian cancers. It has been shown that expression of OVCA1 is reduced in some tumors and that it regulates cell proliferation, embryonic development, and tumorigenesis. However, the biochemical function of OVCA1 has remained unknown. Recently, we isolated a novel mutant resistant to diphtheria toxin and Pseudomonas exotoxin A from the gene trap insertional mutants library of Chinese hamster ovary cells. In this mutant, the Ovca1 gene was disrupted by gene trap mutagenesis, and this disruption well correlated with the toxin-resistant phenotype. We demonstrated direct evidence that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2, the target of bacterial ADP-ribosylating toxins. A functional genetic approach utilizing the random gene trap mutants library of mammalian cells should become a useful strategy to identify the genes responsible for specific phenotypes.