iTILLING: personalized mutation screening.

One powerful approach to studying gene function is to analyze the phenotype of an organism carrying a mutant allele of a gene of interest. In order to use this experimental approach, one must have the ability to easily isolate individual organisms carrying desired mutations. A widely used method for accomplishing this task in plants and other organisms is a procedure called TILLING. A traditional TILLING project has at its foundation an ordered mutant population produced by treating seeds with a chemical mutagen. From this mutagenized seed, thousands of individual mutant lines are produced, and corresponding DNA samples are collected. For several plant species, publicly accessible screening facilities have been established that perform mutant screens on a gene-by-gene basis in response to customer requests using PCR and heteroduplex detection methods. The iTILLING method described in this chapter represents an individualized version of the TILLING process. Performing a traditional TILLING experiment requires a large investment in time and resources to establish the well-ordered mutant population. By contrast, iTILLING is a low-investment alternative that provides the individual research lab with a practical solution to mutation screening. The main difference between the two approaches is that iTILLING is not based on the establishment of a durable, organized mutant population. Instead, a system for growing Arabidopsis seedlings in 96-well plates is used to produce an ephemeral mutant population for screening. Because the intention is not to develop a long-term resource, a considerable savings in time and money is realized when using iTILLING as compared to traditional TILLING. iTILLING is not intended to serve as a replacement to traditional TILLING. Rather, iTILLING provides a strategy by which custom mutagenesis screens can be performed by individual labs using unique genetic backgrounds that are of specific interest to that research group.

Deletion of a tandem gene family in Arabidopsis: increased MEKK2 abundance triggers autoimmunity when the MEKK1-MKK1/2-MPK4 signaling cascade is disrupted.

An Arabidopsis thaliana mitogen-activated protein (MAP) kinase cascade composed of MEKK1, MKK1/MKK2, and MPK4 was previously described as a negative regulator of defense response. MEKK1 encodes a MAP kinase kinase kinase and is a member of a tandemly duplicated gene family with MEKK2 and MEKK3. Using T-DNA insertion lines, we isolated a novel deletion mutant disrupting this gene family and found it to be phenotypically wild-type, in contrast with the mekk1 dwarf phenotype. Follow-up genetic analyses indicated that MEKK2 is required for the mekk1, mkk1 mkk2, and mpk4 autoimmune phenotypes. We next analyzed a T-DNA insertion in the MEKK2 promoter region and found that although it does not reduce the basal expression of MEKK2, it does prevent the upregulation of MEKK2 that is observed in mpk4 plants. This mekk2 allele can rescue the mpk4 autoimmune phenotype in a dosage-dependent manner. We also found that expression of constitutively active MPK4 restored MEKK2 abundance to wild-type levels in mekk1 mutant plants. Finally, using mass spectrometry, we showed that MEKK2 protein levels mirror MEKK2 mRNA levels. Taken together, our results indicate that activated MPK4 is responsible for regulating MEKK2 RNA abundance. In turn, the abundance of MEKK2 appears to be under cellular surveillance such that a modest increase can trigger defense response activation.

Ice-Cap: a method for growing Arabidopsis and tomato plants in 96-well plates for high-throughput genotyping.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day (1,2). This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time. The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

iTILLING: a personalized approach to the identification of induced mutations in Arabidopsis.

TILLING (for Targeting Induced Local Lesions IN Genomes) is a well-established method for identifying plants carrying point mutations in genes of interest. A traditional TILLING project requires a significant investment of time and resources to establish the mutant population and screening infrastructure. Here, we describe a modified TILLING procedure that substantially reduces the investment needed to perform mutation screening. Our motivation for developing iTILLING was to make it practical for individual laboratories to rapidly perform mutation screens using specialized genetic backgrounds. With iTILLING, M2 seeds are collected in bulk from the mutagenized population of plants, greatly reducing the labor needed to manage the mutant lines. Growth of the M2 seedlings for mutation screening, tissue collection, and DNA extraction are all performed in 96-well format. Mutations are then identified using high-resolution melt-curve analysis of gene-specific polymerase chain reaction products. Individual plants carrying mutations of interest are transferred from the 96-well growth plates to soil. One scientist can complete an iTILLING screen in less than 4 months. As a proof-of-principle test, we applied iTILLING to Arabidopsis (Arabidopsis thaliana) plants that were homozygous for the mekk1-1 (for MAPK/ERK kinase kinase 1) mutation and also carried a MEKK1 rescue construct. The goal of our screen was to identify mutations in the closely linked MEKK2 and MEKK3 loci. We obtained five mutations in MEKK2 and seven mutations in MEKK3, all located within 20 kb of the mekk1-1 T-DNA insertion. Using repeated iterations of the iTILLING process, mutations in three or more tandemly duplicated genes could be generated.

Mutational evidence that the Arabidopsis MAP kinase MPK6 is involved in anther, inflorescence, and embryo development.

Loss-of-function, dominant-negative, and change-of-function genetic approaches were used to investigate the role played by the Arabidopsis mitogen-activated protein (MAP) kinase MPK6 throughout development. Plants homozygous for T-DNA null alleles of MPK6 displayed reduced male fertility and abnormal anther development. In addition, a portion of the seed produced by mpk6 plants was found to contain embryos that had burst out of their seed coats. To address potential functional redundancy, a dominant-negative version of MPK6 was constructed by changing the TEY activation loop motif to the amino acid sequence AEF. Plants expressing MPK6AEF via the MPK6 native promoter were found to produce excessive stomata, consistent with the recently described role of MPK6 in stomatal patterning. A novel floral phenotype characterized by abnormal sepal development was also observed in MPK6AEF lines. The gene expression pattern of the MPK6 native promoter was determined using a YFP-MPK6 fusion construct, and expression was observed throughout most plant tissues, consistent with a role for MPK6 in multiple developmental processes. The YFP-MPK6 construct was found to rescue the fertility phenotype of mpk6 null alleles, indicating that the fusion protein retains its biological activity. It was also observed, however, that plants expressing YFP-MPK6 displayed reduced apical dominance and a shortening of inflorescence internodes. These results suggest that the YFP tag modifies the activity of MPK6 in a manner that affects inflorescence development but not anther development. Taken together, the present results indicate that MPK6 is involved in the regulation of multiple aspects of plant development.