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.The process of reverse genetics has been widely used by plant biologists to study gene function. In Arabidopsis (Arabidopsis thaliana), three approaches that have been used to generate populations of plants for reverse genetic analysis are insertional mutagenesis (Wisman et al., 1998; Alonso et al., 2003), fast neutron mutagenesis to induce deletions (Li et al., 2001), and chemical mutagenesis to induce point mutations (McCallum et al., 2000). In order to find individual plants carrying point mutations of interest, a process called TILLING (for Targeting Induced Local Lesions IN Genomes) was developed whereby genes are screened for mutations using a PCR-based assay (McCallum et al., 2000). Although originally developed for use with Arabidopsis, the TILLING process has been subsequently applied to a wide range of plants, including barley (Hordeum vulgare; Caldwell et al., 2004), Brassica napus (Wang et al., 2008), Brassica oleracea (Himelblau et al., 2009), Brassica rapa (Stephenson et al., 2010), Lotus japonicus (Perry et al., 2009), maize (Zea mays; Till et al., 2004), Medicago truncatula (Le Signor et al., 2009), oat (Avena sativa; Chawade et al., 2010), pea (Pisum sativum; Triques et al., 2007), potato (Solanum tuberosum; Elias et al., 2009), rice (Oryza sativa; Till et al., 2007), sorghum (Sorghum bicolor; Xin et al., 2008), soybean (Glycine max; Cooper et al., 2008), tomato (Solanum lycopersicum ; Gady et al., 2009), and wheat (Triticum aestivum; Dong et al., 2009). TILLING has also been used in Drosophila (Winkler et al., 2005), zebrafish (Wienholds et al., 2003), and Caenorhabditis elegans (Gilchrist et al., 2006).The chemical mutagen most commonly used to create the mutant populations used for TILLING is ethyl methanesulfonate (EMS). When working with plants, seeds are soaked in EMS to induce mutations throughout the genome. Mutagenized seeds are then planted on soil, and the resulting plants are grown to maturity to produce M2 seeds, which are collected from the plants individually or in small pools. Next, M2 seed samples from each individual plant are germinated and grown to produce tissue from which DNA can be extracted. The resulting large collection of ordered DNA samples and the corresponding M2 seeds constitute the infrastructure of a TILLING population. PCR-based screening can then be used to find individual plants in the population carrying mutations in genes of interest (McCallum et al., 2000). Once established, this type of TILLING infrastructure can serve the needs of an entire research community through a fee-for-service screening operation (Colbert et al., 2001; Martín et al., 2009).Several different strategies have been developed for identifying the mutations present in a TILLING population, but all of them involve detecting heteroduplex PCR products. A heteroduplex is formed when a mixture of wild-type and mutant PCR products are melted and reannealed, resulting in DNA duplexes that contain a single-base mismatch. TILLING was originally described using denaturing HPLC to identify mutations based on the differential retention times of heteroduplexes and homoduplexes in the chromatography column (McCallum et al., 2000). TILLING has since been modified so that endonucleases are used to cleave PCR products containing a heteroduplex. Cleavage products are then separated via gel electrophoresis to identify banding patterns indicative of mutations (Colbert et al., 2001).More recently, high-resolution melting analysis of PCR products has been used to identify heteroduplexes when performing TILLING (Dong et al., 2009; Gady et al., 2009). High-resolution melting analysis was originally developed for use in clinical settings to identify known single-nucleotide polymorphisms and small insertions/deletions potentially linked to genetic diseases (Erali et al., 2008). With high-resolution melting, the mismatch in a heteroduplex is visualized as a melting event that occurs more rapidly or at a lower temperature than the corresponding homoduplex. Montgomery et al. (2007) demonstrated that mutation scanning with high-resolution melting is a robust technique with greater than 95% sensitivity in distinguishing heteroduplexes from homoduplexes. It has also been observed that the sensitivity with which mutations in PCR products can be identified using DNA melting analysis depends on the resolution of the instrumentation used for collecting the melt-curve data (Zhou et al., 2005; Herrmann et al., 2006).Although traditional TILLING is a high-throughput method for mutation screening, the establishment of the initial screening population and the corresponding ordered DNA samples requires a substantial up-front investment of time and money. Because of this situation, TILLING resources are available for only two genetic backgrounds in Arabidopsis: wild-type Columbia-0 and Landsberg erecta (Greene et al., 2003; Martín et al., 2009). If a scientist is interested in identifying mutations in a more specialized genetic background, the costs associated with establishing a TILLING population can be prohibitive. Therefore, we were interested in determining if a modified version of the TILLING process could be developed that would substantially reduce the investment of time and resources necessary to perform mutation screening. The individualized TILLING procedure, or iTILLING, which we describe in this paper provides one solution to this challenge.     

Production of haploids and doubled haploids in oil palm

Background

Studies on host-pathogen interactions in a range of pathosystems have revealed an array of mechanisms by which plants reduce the efficiency of pathogenesis. While R-gene mediated resistance confers highly effective defense responses against pathogen invasion, quantitative resistance is associated with intermediate levels of resistance that reduces disease progress. To test the hypothesis that specific loci affect distinct stages of fungal pathogenesis, a set of maize introgression lines was used for mapping and characterization of quantitative trait loci (QTL) conditioning resistance to Setosphaeria turcica, the causal agent of northern leaf blight (NLB). To better understand the nature of quantitative resistance, the identified QTL were further tested for three secondary hypotheses: (1) that disease QTL differ by host developmental stage; (2) that their performance changes across environments; and (3) that they condition broad-spectrum resistance.

Results

Among a set of 82 introgression lines, seven lines were confirmed as more resistant or susceptible than B73. Two NLB QTL were validated in BC₄F₂ segregating populations and advanced introgression lines. These loci, designated qNLB1.02 and qNLB1.06, were investigated in detail by comparing the introgression lines with B73 for a series of macroscopic and microscopic disease components targeting different stages of NLB development. Repeated greenhouse and field trials revealed that qNLB1.06 _Tx303 (the Tx303 allele at bin 1.06) reduces the efficiency of fungal penetration, while qNLB1.02 _B73 (the B73 allele at bin 1.02) enhances the accumulation of callose and phenolics surrounding infection sites, reduces hyphal growth into the vascular bundle and impairs the subsequent necrotrophic colonization in the leaves. The QTL were equally effective in both juvenile and adult plants; qNLB1.06 _Tx303 showed greater effectiveness in the field than in the greenhouse. In addition to NLB resistance, qNLB1.02 _B73 was associated with resistance to Stewart's wilt and common rust, while qNLB1.06 _Tx303 conferred resistance to Stewart's wilt. The non-specific resistance may be attributed to pleiotropy or linkage.

Conclusions

Our research has led to successful identification of two reliably-expressed QTL that can potentially be utilized to protect maize from S. turcica in different environments. This approach to identifying and dissecting quantitative resistance in plants will facilitate the application of quantitative resistance in crop protection.     

Evolution of Novel Signal Traits in the Absence of Female Preferences in Neoconocephalus Katydids (Orthoptera,Tettigoniidae)

Background Significance

Communication signals that function to bring together the sexes are important for maintaining reproductive isolation in many taxa. Changes in male calls are often attributed to sexual selection, in which female preferences initiate signal divergence. Natural selection can also influence signal traits if calls attract predators or parasitoids, or if calling is energetically costly. Neutral evolution is often neglected in the context of acoustic communication.

Methodology/Principal Findings

We describe a signal trait that appears to have evolved in the absence of either sexual or natural selection. In the katydid genus Neoconocephalus, calls with a derived pattern in which pulses are grouped into pairs have evolved five times independently. We have previously shown that in three of these species, females require the double pulse pattern for call recognition, and hence the recognition system of the females is also in a derived state. Here we describe the remaining two species and find that although males produce the derived call pattern, females use the ancestral recognition mechanism in which no pulse pattern is required. Females respond equally well to the single and double pulse calls, indicating that the derived trait is selectively neutral in the context of mate recognition.

Conclusions/Significance

These results suggest that 1) neutral changes in signal traits could be important in the diversification of communication systems, and 2) males rather than females may be responsible for initiating signal divergence.     

Liver-specific knockdown of JNK1 up-regulates proliferator-activated receptor gamma coactivator 1 beta and increases plasma triglyceride despite reduced glucose and insulin levels in diet-induced obese mice

The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor gamma coactivator 1 beta, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice.     

Holocene fire and occupation in Amazonia: records from two lake districts

While large-scale pre-Columbian human occupation and ecological disturbance have been demonstrated close to major Amazonian waterways, less is known of sites in terra firme settings. Palaeoecological analyses of two lake districts in central and western Amazonia reveal long histories of occupation and land use. At both locations, human activity was centred on one of the lakes, while the others were either lightly used or unused. These analyses indicate that the scale of human impacts in these terra firme settings is localized and probably strongly influenced by the presence of a permanent open-water body. Evidence is found of forest clearance and cultivation of maize and manioc. These data are directly relevant to the resilience of Amazonian conservation, as they do not support the contention that all of Amazonia is a 'built landscape' and therefore a product of past human land use.     

Serum concentrations of amino acids,fatty acids,lipoproteins, vitamins A and E,and minerals in apparently healthy,free‐ranging southern giraffe (Giraffa camelopardalis giraffe)

This pilot project began establishing a nutritional profile for free‐ranging giraffe. The results will be used as a tool to begin assessing the nutritional status of captive giraffe. In October 2004 serum samples were collected opportunistically from seven adult and 17 sub‐adult giraffe being anesthetized for different studies. Seventeen animals were from Double Drift Game Reserve and seven animals were from Kariega Private Game Reserve. The serum samples were analyzed for circulating concentrations of amino acids, fatty acids, lipoproteins, vitamins, and minerals. Information from 15 serum samples collected from anesthetized giraffe in Kruger National Park during April and August 2003 was included in the calcium and phosphorus concentration data. No significant differences were identified between genders. Significant differences between locations were identified for concentrations of certain amino acids, fatty acids, and lipoproteins. Differences between locations are likely due to different nutrient concentrations of foods and possibly the result of different animal densities forcing different food choices among locations. This pilot project may expand to include changes in circulating nutrient concentrations for free‐ranging giraffe as is influenced by other locations, seasonal food availability, and different giraffe subspecies. Zoo Biol 0:1–13, 2007. © 2006 Wiley‐Liss, Inc.     

Acid-Adapted Strains of Escherichia coli K-12 Obtained by Experimental Evolution

Enteric bacteria encounter a wide range of pHs throughout the human intestinal tract. We conducted experimental evolution of Escherichia coli K-12 to isolate clones with increased fitness during growth under acidic conditions (pH 4.5 to 4.8). Twenty-four independent populations of E. coli K-12 W3110 were evolved in LBK medium (10 g/liter tryptone, 5 g/liter yeast extract, 7.45 g/liter KCl) buffered with homopiperazine-N,N′-bis-2-(ethanosulfonic acid) and malate at pH 4.8. At generation 730, the pH was decreased to 4.6 with HCl. By 2,000 generations, all populations had achieved higher endpoint growth than the ancestor at pH 4.6 but not at pH 7.0. All evolving populations showed a progressive loss of activity of lysine decarboxylase (CadA), a major acid stress enzyme. This finding suggests a surprising association between acid adaptation and moderation of an acid stress response. At generation 2,000, eight clones were isolated from four populations, and their genomes were sequenced. Each clone showed between three and eight missense mutations, including one in a subunit of the RNA polymerase holoenzyme (rpoB, rpoC, or rpoD). Missense mutations were found in adiY, the activator of the acid-inducible arginine decarboxylase (adiA), and in gcvP (glycine decarboxylase), a possible acid stress component. For tests of fitness relative to that of the ancestor, lacZ::kan was transduced into each strain. All acid-evolved clones showed a high fitness advantage at pH 4.6. With the cytoplasmic pH depressed by benzoate (at external pH 6.5), acid-evolved clones showed decreased fitness; thus, there was no adaptation to cytoplasmic pH depression. At pH 9.0, acid-evolved clones showed no fitness advantage. Thus, our acid-evolved clones showed a fitness increase specific to low external pH.     

Statistical analysis of protein structures suggests that buried ionizable residues in proteins are hydrogen bonded or form salt bridges

It is well known that nonpolar residues are largely buried in the interior of proteins, whereas polar and ionizable residues tend to be more localized on the protein surface where they are solvent exposed. Such a distribution of residues between surface and interior is well understood from a thermodynamic point: nonpolar side chains are excluded from the contact with the solvent water, whereas polar and ionizable groups have favorable interactions with the water and thus are preferred at the protein surface. However, there is an increasing amount of information suggesting that polar and ionizable residues do occur in the protein core, including at positions that have no known functional importance. This is inconsistent with the observations that dehydration of polar and in particular ionizable groups is very energetically unfavorable. To resolve this, we performed a detailed analysis of the distribution of fractional burial of polar and ionizable residues using a large set of ?2600 nonhomologous protein structures. We show that when ionizable residues are fully buried, the vast majority of them form hydrogen bonds and/or salt bridges with other polar/ionizable groups. This observation resolves an apparent contradiction: the energetic penalty of dehydration of polar/ionizable groups is paid off by favorable energy of hydrogen bonding and/or salt bridge formation in the protein interior. Our conclusion agrees well with the previous findings based on the continuum models for electrostatic interactions in proteins. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Metal ionophore treatment restores dendritic spine density and synaptic protein levels in a mouse model of Alzheimer's disease

We have previously demonstrated that brief treatment of APP transgenic mice with metal ionophores (PBT2, Prana Biotechnology) rapidly and markedly improves learning and memory. To understand the potential mechanisms of action underlying this phenomenon we examined hippocampal dendritic spine density, and the levels of key proteins involved in learning and memory, in young (4 months) and old (14 months) female Tg2576 mice following brief (11 days) oral treatment with PBT2 (30 mg/kg/d). Transgenic mice exhibited deficits in spine density compared to littermate controls that were significantly rescued by PBT2 treatment in both the young (+17%, p<0.001) and old (+32%, p<0.001) animals. There was no effect of PBT2 on spine density in the control animals. In the transgenic animals, PBT2 treatment also resulted in significant increases in brain levels of CamKII (+57%, p = 0.005), spinophilin (+37%, p = 0.04), NMDAR1A (+126%, p = 0.02), NMDAR2A (+70%, p = 0.05), pro-BDNF (+19%, p = 0.02) and BDNF (+19%, p = 0.04). While PBT2-treatment did not significantly alter neurite-length in vivo, it did increase neurite outgrowth (+200%, p = 0.006) in cultured cells, and this was abolished by co-incubation with the transition metal chelator, diamsar. These data suggest that PBT2 may affect multiple aspects of snaptic health/efficacy. In Alzheimer''s disease therefore, PBT2 may restore the uptake of physiological metal ions trapped within extracellular β-amyloid aggregates that then induce biochemical and anatomical changes to improve cognitive function.