Clifford F. Weil

Discovery of induced point mutations in maize genes by TILLING

BackgroundGoing from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community.ResultsWe demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious.ConclusionsOur findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.

Microhomology-Dependent End Joining and Repair of Transposon-Induced DNA Hairpins by Host Factors in Saccharomyces cerevisiae

ABSTRACT The maize, cut-and-paste transposon Ac/Ds is mobile in Saccharomyces cerevisiae, and DNA sequences of repair products provide strong genetic evidence that hairpin intermediates form in host DNA during this transposition, similar to those formed for V(D)J coding joints in vertebrates. Both DNA strands must be broken for Ac/Ds to excise, suggesting that double-strand break (DSB) repair pathways should be involved in repair of excision sites. In the absence of homologous template, as expected, Ac excisions are repaired by nonhomologous end joining (NHEJ) that can involve microhomologies close to the broken ends. However, unlike repair of endonuclease-induced DSBs, repair of Ac excisions in the presence of homologous template occurs by gene conversion only about half the time, the remainder being NHEJ events. Analysis of transposition in mutant yeast suggests roles for the Mre11/Rad50 complex, SAE2, NEJ1, and the Ku complex in repair of excision sites. Separation-of-function alleles of MRE11 suggest that its endonuclease function is more important in this repair than either its exonuclease or Rad50-binding properties. In addition, the interstrand cross-link repair gene PSO2 plays a role in end joining hairpin ends that is not seen in repair of linearized plasmids and may be involved in positioning transposase cleavage at the transposon ends.

A Rice Tc1/Mariner -Like Element Transposes in Yeast

The Tc1/mariner transposable element superfamily is widely distributed in animal and plant genomes. However, no active plant element has been previously identified. Nearly identical copies of a rice (Oryza sativa) Tc1/mariner element called Osmar5 in the genome suggested potential activity. Previous studies revealed that Osmar5 encoded a protein that bound specifically to its own ends. In this report, we show that Osmar5 is an active transposable element by demonstrating that expression of its coding sequence in yeast promotes the excision of a nonautonomous Osmar5 element located in a reporter construct. Element excision produces transposon footprints, whereas element reinsertion occurs at TA dinucleotides that were either tightly linked or unlinked to the excision site. Several site-directed mutations in the transposase abolished activity, whereas mutations in the transposase binding site prevented transposition of the nonautonomous element from the reporter construct. This report of an active plant Tc1/mariner in yeast will provide a foundation for future comparative analyses of animal and plant elements in addition to making a new wide host range transposable element available for plant gene tagging.

TILLING in Grass Species

The reverse genetics detection of single base changes in genes of interest, whether induced or endogenous, is an extremely powerful tool for functional genomics. Two major thrusts of research in the grasses are characterizing the function of all the genes in various genomes and then comparing those

Functional interactions between Sae2 and the Mre11 complex

The Mre11 complex functions in double-strand break (DSB) repair, meiotic recombination, and DNA damage checkpoint pathways. Sae2 deficiency has opposing effects on the Mre11 complex. On one hand, it appears to impair Mre11 nuclease function in DNA repair and meiotic DSB processing, and on the other, Sae2 deficiency activates Mre11-complex-dependent DNA-damage-signaling via the Tel1–Mre11 complex (TM) pathway. We demonstrate that SAE2 overexpression blocks the TM pathway, suggesting that Sae2 antagonizes Mre11-complex checkpoint functions. To understand how Sae2 regulates the Mre11 complex, we screened for sae2 alleles that behaved as the null with respect to Mre11-complex checkpoint functions, but left nuclease function intact. Phenotypic characterization of these sae2 alleles suggests that Sae2 functions as a multimer and influences the substrate specificity of the Mre11 nuclease. We show that Sae2 oligomerizes independently of DNA damage and that oligomerization is required for its regulatory influence on the Mre11 nuclease and checkpoint functions.

Genetic Determinants for Enzymatic Digestion of Lignocellulosic Biomass Are Independent of Those for Lignin Abundance in a Maize Recombinant Inbred Population

Potential genetic determinants for enhancement of sugar yields from enzymatic digestion of maize biomass are unrelated to those for lignin abundance. Biotechnological approaches to reduce or modify lignin in biomass crops are predicated on the assumption that it is the principal determinant of the recalcitrance of biomass to enzymatic digestion for biofuels production. We defined quantitative trait loci (QTL) in the Intermated B73 × Mo17 recombinant inbred maize (Zea mays) population using pyrolysis molecular-beam mass spectrometry to establish stem lignin content and an enzymatic hydrolysis assay to measure glucose and xylose yield. Among five multiyear QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yield was shared. A genome-wide association study for lignin abundance and sugar yield of the 282-member maize association panel provided candidate genes in the 11 QTL of the B73 and Mo17 parents but showed that many other alleles impacting these traits exist among this broader pool of maize genetic diversity. B73 and Mo17 genotypes exhibited large differences in gene expression in developing stem tissues independent of allelic variation. Combining these complementary genetic approaches provides a narrowed list of candidate genes. A cluster of SCARECROW-LIKE9 and SCARECROW-LIKE14 transcription factor genes provides exceptionally strong candidate genes emerging from the genome-wide association study. In addition to these and genes associated with cell wall metabolism, candidates include several other transcription factors associated with vascularization and fiber formation and components of cellular signaling pathways. These results provide new insights and strategies beyond the modification of lignin to enhance yields of biofuels from genetically modified biomass.

Breadth by depth: expanding our understanding of the repair of transposon-induced DNA double strand breaks via deep-sequencing.

The transposases of DNA transposable elements catalyze the excision of the element from the host genome, but are not involved in the repair of the resulting double-strand break. To elucidate the role of various host DNA repair and damage response proteins in the repair of the hairpin-ended double strand breaks (DSBs) generated during excision of the maize Ac element in Arabidopsis thaliana, we deep-sequenced hundreds of thousands of somatic excision products from a variety of repair- or response-defective mutants. We find that each of these repair/response defects negatively affects the preservation of the ends, resulting in an enhanced frequency of deletions, insertions, and inversions at the excision site. The spectra of the resulting repair products demonstrate, not unexpectedly, that the canonical nonhomologous end joining (NHEJ) proteins DNA ligase IV and KU70 play an important role in the repair of the lesion generated by Ac excision. Our data also indicate that auxiliary NHEJ repair proteins such as DNA ligase VI and DNA polymerase lambda are routinely involved in the repair of these lesions. Roles for the damage response kinases ATM and ATR in the repair of transposition-induced DSBs are also discussed.

Tropical Maize: Exploiting Maize Genetic Diversity to Develop a Novel Annual Crop for Lignocellulosic Biomass and Sugar Production

Maize (Zea mays L.) is truly a remarkable crop species, having been adapted from its tropical origins to a wide diversity of environments and end uses. According to the Food and Agriculture Organization of the United Nations FAOSTAT webpage, 792 million metric tons of maize were produced worldwide in 2007, making it the world’s highest yielding grain crop (http://faostat.fao.org/site/339/default.aspx). When maize varieties adapted to tropical latitudes are grown in temperate environments such as the US Corn Belt, they flower later and produce little or no grain, but have higher total biomass yields compared to modern commercial corn grain hybrids (Fig. 1). Further, tropical maize also accumulates high amounts of extractable stalk sugar (sucrose, glucose, and fructose) because of reduced grain formation. Although offering potential benefits as a feedstock for biofuels, the direct use of tropical maize germplasm in temperate environments is hampered by greater lodging, less stress tolerance, and susceptibility to disease and insect pests – traits that have been greatly improved in modern US corn grain hybrids. However, hybrids derived from crossing temperate-adapted and tropical parents successfully combine the high biomass potential of tropical maize with the genetic improvements from the past century of corn breeding for high grain yields in temperate environments. Named “tropical maize,” these tropical x temperate hybrids produce greater biomass and sugar compared to current US corn hybrids using at least 50% less nitrogen (N) fertilizer inputs (Table 1)

Validation of PyMBMS as a High-throughput Screen for Lignin Abundance in Lignocellulosic Biomass of Grasses

Pyrolysis molecular-beam mass spectrometry (PyMBMS) was tested as a high-throughput method for relative abundances of guaiacyl and syringyl lignin in lignocellulosic cell-wall materials from stems of a population of maize intermated B73 × Mo17 (IBM) recombinant inbred lines. Variations of up to twofold across the population in phenylpropanoid abundance were observed. Several histochemical and quantitative biochemical assays were used to validate the mass spectrometric data for lignin, hydroxycinnamic acids, crystalline cellulose, non-cellulosic glucans, and xylans. We demonstrate PyMBMS to be a valid high-throughput screen suitable for analysis of lignin abundance in large populations of bioenergy grasses. Pentose from xylans and hexose from cellulosic and non-cellulosic glucans also varied substantially across the population, but abundances of diagnostic fragments for these monosaccharides were not well correlated with the abundance of cell-wall polysaccharides.

High-throughput Screening of EMS Mutagenized Maize for Altered Starch Digestibility

Carbohydrate research increasingly is focused on changing the biochemical nature of starch to create more efficient substrates for biofuel production; parallel work is aimed at healthier foods for human consumption. A key factor in both of these efforts is the rate at which starch is digested by amylases. Starch digestibility is influenced heavily by genetically controlled factors including starch granular and molecular structure and composition. Maize mutant varieties with increased starch digestibility would help to make more cost-efficient biofuels. To identify such mutants among segregating families of ethyl methane sulfonate-mutagenized maize, we developed a miniaturized, high-throughput single kernel preparation and starch digestion assay that can process over 500 samples per week. In a preliminary screen of 480 families, we have identified 62 mutants with faster rates of digestion as compared to wild type and, in the same screen, an additional 53 lines with slower rates of digestion, thus tremendous potential health benefits. These mutants can be used for detailed structural analysis of starch and flour physical and chemical properties, factors that interact with starch in the cell and analysis of an apparently large number of genes that can impact rates of starch digestion.