T-DNA as an Insertional Mutagen in Arabidopsis

Abstract

Forward genetics begins with a mutant phenotype and asks the question “What is the genotype?” that is, what is the sequence of the mutant gene causing the altered phenotype? Reverse genetics begins with a mutant gene sequence and asks the question “What is the resulting change in phenotype?” These two approaches are fundamentally different, and whereas forward genetics has been in operation for more than a century, the recent avalanche of complete genome sequences has only now created the opportunity for pursuing reverse genetics in an exhaustive and complete manner. Gene knockouts, or null mutations, are important because they provide a direct route to determining the function of a gene product in situ. Most other approaches to gene function are correlative and do not necessarily prove a causal relationship between gene sequence and function. For example, DNA chips provide an exciting means to discover conditions under which gene expression is regulated on a genomewide scale (DeRisi et al., 1997; Wodicka et al., 1997; Singh-Gasson et al., 1999). However, because factors other than mRNA level alone determine the activity of a gene product in situ, expression studies, even when done on a genomewide scale, cannot prove a causal relationship. By contrast, the availability of a null mutation for the gene of interest allows one to directly monitor the effect this deficiency has on the organism’s ability to function. There are many ways to implement targeted mutagenesis so as to compromise specific genes. In mice, knockout mutations are now routinely obtained by promoting the homologous recombination of null gene constructs with the genomic wild-type sequence in embryonic stem cells. Provided that the given mutation is not embryonic lethal, “knockout mice” can then be developed in utero by injecting such stem cells into blastocysts (Koller et al., 1989). Yeast and Escherichia coli are other organisms in which homologous recombination is the preferred means for reverse genetics. Although there has been a report of homologous recombination with intact Arabidopsis plants (Kempin et al., 1997), the frequency of this event may be so low as to preclude its use for generating knockout mutations in each of the ∼25,000 genes that comprise the 120-Mb genome. Insertional mutagenesis is an alternative means of disrupting gene function and is based on the insertion of foreign DNA into the gene of interest. In Arabidopsis, this involves the use of either transposable elements (see Parinov et al., 1999, in this issue) or T-DNA. The foreign DNA not only disrupts the expression of the gene into which it is inserted but also acts as a marker for subsequent identification of the mutation. Because Arabidopsis introns are small, and because there is very little intergenic material, the insertion of a piece of T-DNA on the order of 5 to 25 kb in length generally produces a dramatic disruption of gene function. If a large enough population of T-DNA–transformed lines is available, one has a very good chance of finding a plant carrying a T-DNA insert within any gene of interest. Mutations that are homozygous lethal can be maintained in the population in the form of heterozygous plants. Polymerase chain reaction (PCR) methods have been developed that allow one to easily isolate individual plants that carry a particular T-DNA mutation of interest (McKinney et al., 1995; Krysan et al., 1996). An advantage of using T-DNAs as the insertional mutagen, as opposed to transposons (Martienssen, 1998; Wisman et al., 1998), is that T-DNA insertions will not transpose subsequent to integration within the genome and are therefore chemically and physically stable through multiple generations. The mobility of transposons is not necessarily a bad thing, however. In situations in which multiple members of a gene family are arranged in tandem along a chromosome, the ability of transposons to “hop” to nearby locations provides a convenient method for creating mutations within all of the members of the gene family within a single plant. Several improvements in Agrobacterium-mediated transformation techniques have made T-DNA a viable method for approaching genomewide mutagenesis. The original root-explant method (Valvekens et al., 1988) allowed one to isolate many hundreds and possibly thousands of intact transformed plants, albeit via a laborious tissue culture process. Tens of thousands of transformed plants were beyond reach, however, until Feldmann and Marks (1987) elaborated a method for producing independent T-DNA transgenic lines via seed transformation. The development of transformation methods based on dipping whole plants into Agrobacterium suspensions has more recently allowed, in theory, for the production of the hundreds of thousands of insertional mutations necessary for saturation of the genome (Bechtold and Pelletier, 1998; Clough and Bent, 1998). Saturation of the Arabidopsis genome with T-DNA insertions is an experimental goal that requires