4/3/2014 An Introduction to Reverse Genetic Tools for Investigating Gene Function

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An Introduction to Reverse GeneticToolsfor Investigating Gene FunctionTierney, M.B. and Lamour, K.H. 2005. An Introduction to Reverse Genetic Tools for

Investigating Gene Function. The Plant Health Instructor. DOI: 10.1094/PHI­A­2005­1025­

01.

Melinda B. Tierney1 and Kurt H. Lamour2

1University of Tennessee and Oak Ridge NationalLaboratory Genome Science and Technology GraduateProgram; Knoxville, TN 2The University of Tennessee, Department of Entomologyand Plant Pathology; Knoxville, TN

IntroductionGenetics began in the 1860s with Gregor Mendel, anAugustinian monk who performed experiments thatsuggested the existence of genes (Griffiths et al. 2000). Thediscovery of the structure of DNA by James Watson andFrancis Crick in the 1950s (Watson and Crick 1953),followed by the development of di­deoxy terminatorsequencing by Sanger in the late 1970s (Sanger, Nicklen,

and Coulson 1977) and then of PCR (polymerase chainreaction) technology by Kary Mullis in the 1980s (Mullis andFaloona 1987; Saiki et al. 1985) set off a genetic revolution.Technological advances in sequencing have greatlyaccelerated our accumulation of genetic sequence data tothe point now where whole genome sequences are publiclyavailable for a large number of organisms including plantpathogens. The toolbox for genetic research is expandingrapidly, and this overview presents a suite of tools generallyreferred to as reverse genetics that can be used toinvestigate gene function.

Forward Genetics vs. Reverse GeneticsVariants help us understand the 'normal.' Variation can bemeasured at many scales – from macro (body size,morphology) to different levels of micro variation (crude

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protein profiles to DNA sequence variation). Forwardgenetics refers to a process where studies are initiated todetermine the genetic underpinnings of observablephenotypic variation. In many cases the observable variationhas been induced using a DNA damaging agent (mutagen)but also may be naturally occurring. The investigatoreventually ends up sequencing the gene or genes thought tobe involved (Figure 1).

Figure 1

With the advent of whole genome sequencing manyresearchers are now in a very different position. They haveaccess to all of the gene sequences within a given organismand would like to know their function. So, instead of goingfrom phenotype to sequence as in forward genetics, reversegenetics works in the opposite direction – a gene sequenceis known, but its exact function is uncertain. In reversegenetics, a specific gene or gene product is disrupted ormodified and then the phenotype is measured (Figure 1).Here we will overview some of the techniques for reversegenetics with a special emphasis on the TILLING (TargetingInduced Local Lesions IN Genomes) technique which isbeing utilized on plant pathogens in the genus Phytophthora.

Specific Reverse Genetic approachesThe goal in reverse genetics is to investigate the impact ofinduced variation within a specific gene and to infer genefunction. The process of disruption or alteration can either betargeted specifically as in the case of gene silencing orhomologous recombination or can rely on non­targetedrandom disruptions (e.g., chemical mutagenesis, transposonmediated mutagenesis) followed by screening a library ofindividuals for lesions at a specific location. Following is abrief overview of some commonly used targeted and non­targeted approaches.

Gene SilencingRNA interference (RNAi) is the process by which expressionof a target gene is inhibited by antisense and sense RNAs. Itworks based on the ability of double­stranded sequences to

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recognize and degrade sequences that are complementaryto them (Lewin 2004). RNAi was first discovered inCaenorhabditis elegans when the introduction of double­stranded RNA was observed to be an efficient method forsilencing gene expression (Fire et al. 1998; Kuttenkeuler andBoutros 2004). RNAi­ based silencing is an exciting strategyfor reverse genetics (Waterhouse, Graham, and Wang 1998).RNA interference has recently become a powerful tool to

silence the expression of genes and analyze their loss­of­function phenotype, allowing analysis of gene function whenmutant alleles are not available. Having been shown to workin a similar manner in all metazoans, RNAi has proven to beapplicable to many organisms and has been used togenerate a wide variety of loss­of­function phenotypes(Kuttenkeuler and Boutros 2004). The phenomenon of post­transcriptional gene silencing observed in plants may alsobe due to a related RNAi mechanism (Waterhouse, Graham,and Wang 1998). RNAi based silencing utilizes theendonuclease Dicer to cleave single stranded RNAs,abbreviated siRNAs, from double stranded RNA; the RISCcomplex then destroys specific target mRNAs based onsequence complementarity with the siRNA (Pattanayak et al.2005).

RNAi has been used for a systematic analysis of genefunction in C. elegans by generating loss of functionphenotypes, creating a library of worms expressing dsDNAcorresponding to different genes (Lewin 2004). Genome­wide RNAi screens against these libraries of predictedgenes have allowed study of a variety of biologicalprocesses in C. elegans. A genome­wide library of double­stranded RNAs that target every gene in the Drosophilagenome has also been published that is suitable for highthroughput cell­based assays (Kuttenkeuler and Boutros2004). One difficulty in using RNAi as a reverse genetictechnique is that throughput is limited by the ability to deliversiRNAs to target loci (Henikoff, Till, and Comai 2004). It isalso labor intensive, can give ambiguous results, and can beunsuitable for isolating mutants that have lethal or sterilephenotypes (Gilchrist and Haughn 2005).

Targeted gene disruption by homologousrecombinationHomologous recombination is a reciprocal exchange of DNAsequences, as in between two chromosomes that carry thesame genetic loci (Lewin 2004). Just as homologousrecombination has been found to be mainly initiated with adouble­strand break, gene targeting by homologousrecombination is associated with the repair of double strand

breaks. The double­strand break repair and synthesis­dependent strand­annealing models are the most generallyaccepted models to explain gene targeting (Iida and Terada2004).

Homologous recombination has been widely used inembryonic stem (ES) cells in mice, and has allowedconstruction of precise mutations in nearly every gene. A

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construction of precise mutations in nearly every gene. Areverse­genetic system using homologous recombinationhas recently been developed for Drosophila. It is promising,but is a lengthy procedure and requires generation ofspecific transgenic flies (Stemple 2004). Reproducible genetargeting by homologous recombination is now also feasiblein rice. With the combination of site­specific recombinationsystems (such as Cre­lox), the future of gene targeting byhomologous recombination as a routine procedure forengineering the genome of rice and presumably other plantsis bright (Iida and Terada 2004).

Insertional mutagenesis/transposon mediatedmutagenesisTransposons are mobile genetic elements that can relocatefrom one genomic location to another (Hayes 2003). Theyare DNA sequences that can insert themselves at a newlocation in the genome without having any sequencerelationship with the target locus (Lewin 2004). Transposon­based signature­tagged mutagenesis has been successful inidentifying essential genes as well as genes involved ininfectivity of a variety of pathogens. Strategies for insertionalmutagenesis using transposons have been developed for anumber of animal and plant models (Hayes 2003). Reverse­genetics is currently being done in Drosophila and C.elegans by utilizing libraries of individuals who carrytransposable element insertions, many of which have beenmapped, and some of which will disrupt the expression ofnearby genes. In Drosophila P­elements, imprecise excisioncan be driven to generate a mutation in the nearest gene.Transposon­based methods are also being used inArabidopsis, maize and other plants (Stemple 2004). Onedrawback of insertional mutagenesis is the low frequency ofmutations, necessitating the screening of large numbers of

individuals to find mutations in any given gene (Gilchrist andHaughn 2005). Also, insertions in essential genes willusually cause lethality, and less severe mutations must begenerated in these genes in order to understand genefunction (Till et al. 2003).

The segment of the Ti plasmid of Agrobacterium tumefaciensknown as T­DNA that carries genes to transform the plantcell has also been utilized for insertional mutagenesis. T­DNA insertional mutagenesis has been used to obtain geneknockouts for greater than 70% of Arabidopsis genes(Alonso et al 2003), but no comparable resources exist forrice or maize even as high­coverage genomic sequence isbecoming increasingly available (Henikoff, Till, and Comai2004). Unlike other successful gene targeting systems(namely mouse, Physcomitrella, and Drosophila), the precisemechanism of T­DNA integration into the plant genomeremains largely unknown (Iida and Terada 2004). Like RNAsuppression techniques, insertional mutagenesis is limitedby its host range and by its limited range of allele types(McCallum et al. 2000).

Gene replacement via transformation is a commonly usedtool for many filamentous fungi (Fang, Hanau, and

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Vaillancourt 2002; Lalucque and Silar 2004; Takano et al.2000) . The transformation can be mediated in many ways,including Agrobacterium (Zhang et al. 2003) and variousother transformation vectors (Scott­Craig et al. 1998; Takanoet al. 2000).

Chemical mutagenesis/TILLINGTwo of the most widely used mutagens for chemicalmutagenesis experiments are EMS (ethylmethanesulfonate)and ENU (ethylnitrosourea). EMS is a chemical mutagen thatalkylates guanine bases. The alkylated guanine will thenpair with thymine instead of the preferred cytosine base,ultimately resulting in a G/C to A/T transition. EMS is themost commonly used mutangen in plants. In Arabidopsis,five percent of EMS­induced mutations in targeted codingregions result in premature termination of the gene product,while fifty percent result in missense mutations that alter the

amino­acid sequence of the encoded protein (Gilchrist andHaughn 2005). This high level of missense mutationsrelative to terminated gene products is very useful inanalyzing gene function. ENU also induces point mutations,and is a more potent mutagen than EMS. It is also analkylating agent, mutagenizing by transferring an ethyl groupto oxygen or nitrogen radicals in the DNA molecule, whichleads to mispairing and ultimately results in base pairsubstitutions, and sometimes base pair losses if not repaired(Guénet 2004).

Chemical mutagenesis is attractive for reverse geneticsbecause it results in induced point mutations, which create adiverse range of alleles for genetic analysis. It induces alarge number of recessive mutations per genome that arerandomly distributed (Gilchrist and Haughn 2005) . Becausechemical mutagenesis is already widely used in manyorganisms for forward genetic screens, it promises to begenerally applicable for reverse genetics (Henikoff, Till, andComai 2004). Until recently, chemical mutagenesis has notbeen widely used as a tool for reverse genetics because ofthe lack of high­throughput techniques for detecting pointmutations (Gilchrist and Haughn 2005). The TILLINGstrategy provides a high throughput strategy to detect singlebase changes within genetic targets and can be applied to awide variety of organisms.

TILLING PhytophthoraWhole genome sequences for the soybean pathogenPhytophthora sojae and the sudden oak death pathogen P.ramorum were made public near the end of 2004. Large­scale genomic sequencing projects are underway for thepotato late blight pathogen P. infestans and the vegetablepathogen P. capsici. The compilation of these genomicsequences allows the application of many sophisticatedresearch tools and promises a better understanding of thisdevastating group of pathogens. In an effort to betterunderstand gene function within Phytophthora a project todevelop a reverse genetic TILLING resource forPhytophthora has been initiated. Figure 2 provides an

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overview of the process as it is being applied to

Phytophthora. Zoospores present an ideal life stage formutagenesis as they are uni­nucleate and single mutantindividuals can readily be isolated following mutagenesis.The mutation rate for EMS or ENU is not known forPhytophthora and lethality is being used as an indicator ofdose response. Genomic DNA is extracted from the mutantindividuals and pooled 2 to 4­fold. The genomic DNA librarycan then be repeatedly screened. Specific genes areamplified from the pools of genomic DNA, and the PCRproducts are heated up and allowed to cool slowly to formheteroduplexes between wild type and mutant strands ofDNA. The heteroduplexes are treated with the single strandspecific endonuclease CEL1 which cuts 3' of single basemismatches producing novel fragments of DNA. CEL1treated PCR products are then resolved on a polyacrylamidegel and screened for the presence of novel fragments. Poolscontaining novel fragments are then analyzed to determineexactly which mutant is carrying an induced point mutation,and the PCR product from this mutant is sequenced todetermine if the induced change is predicted to be silent,missense, or a knockout mutation.

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Figure 2Since Phytophthora is diploid through most of its life cycle(including the zoospore stage) all of the induced pointmutations exist in the heterozygous state. Once a non­silentpoint mutation has been identified the mutant isolate is takenthrough the sexual stage and the sexual progeny arescreened to identify individuals homozygous for the mutationunder investigation. Homozygous mutants are then tested todetermine if there is an altered phenotype.

Limitations/roadblocks to completing reversegeneticsCompleting reverse genetics is not without its pitfalls, andnot all techniques can be applied to all organisms. To besuccessful, there are several aspects that must be checked.For organisms that do not have efficient transformationsystems available techniques such as TILLING that can be

applied without transformation may be the only practicalchoice. In these cases, the rate of mutagenesis is animportant factor that can be difficult to determine. The load ofmutations must be balanced with the recovery of mutants(Till et al. 2003) – in other words, the genome can't be soriddled with mutations that it is impossible to see a mutantphenotype. Also to be considered is the fertility of themutagenized organism, especially in the first generation, butalso in subsequent generations (Perry et al. 2003) , bothbefore and after mutagenesis. This is especially true fordiploid organisms, because if the sexual machinery is notintact and working properly, then it is impossible to obtain ahomozygous mutant. The mutagenized organisms must alsobe kept alive long enough to screen a mutant population fora specific target. For some organisms, like Arabidopsis orPhytophthora, this is not a problem as the seeds or culturesare relatively easy to store. It presents a challenge for otherorganisms, such as zebrafish or rats, because they must bestored and kept alive through the mutant screening stage.

Supplementary information: examples ofTILLING in Action*ArabidopsisA public TILLING resource was set up in 2001 for theArabidopsis community. This effort was called theArabidopsis TILLING Project (ATP) and is now called theSeattle Tilling Project (STP) and is a joint effort between theComai Laboratory at the University of Washington and theHenikoff Laboratory at the Fred Hutchinson Cancer

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Research Center in Seattle, Washington. Users are chargeda fee that covers partial costs of the services provided torequest mutations in genes of interest (Gilchrist and Haughn2005; Till et al. 2003). Training sessions, workshops, and on­going support to researchers interested in developingTILLING in other organisms has been made availablethrough the ATP and has served to make the TILLINGtechnique more wide­spread. This group has also developedand made publicly available web­based software programsfor PCR primer design and visualization of polymorphisms

(Gilchrist and Haughn 2005).

*ZebrafishTarget selected mutagenesis using TILLING is also beingdone in zebrafish. The Hubrecht Laboratory at TheNetherlands Institute for Developmental Biology hasgenerated a library of 4608 ENU­mutagenized F1 animalsand has kept a living stock. The DNA from these animals hasbeen screened for mutations in 16 genes using TILLINGfollowed by re­sequencing. This resulted in 255 mutationsbeing identified, 14 of which resulted in a premature stopcodon, 7 in a splice donor/acceptor site mutation, and 119 inan amino acid change. They were able to knock out 13different genes in only a few months time through reversegenetics (Wienholds et al. 2003) .Thus far, mutantphenotypes have been characterized for two of the zebrafishgenes modified via TILLING ­ the gene for Dicer1 (Stemple2004; Wienholds et al. 2003) and the gene for adenomatouspolyposis, a tumor suppressor, which through was found tohave a previously unknown function for signaling in cardiac­valve formation (Hurlstone et al. 2003; Stemple 2004).

AcknowledgementsThis work was supported by a National Science Foundation,Faculty Early Career (CAREER) Development award(#0347624) to Kurt Lamour.

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