Multiple Mutant Combinations Research Articles

A haploid genetics toolbox for Arabidopsis thaliana.

Genetic analysis in haploids provides unconventional yet powerful advantages not available in diploid organisms. In Arabidopsis thaliana, haploids can be generated through seeds by crossing a wild-type strain to a transgenic strain with altered centromeres. Here we report the development of an improved haploid inducer (HI) strain, SeedGFP-HI, that aids selection of haploid seeds prior to germination. We also show that haploids can be used as a tool to accelerate a variety of genetic analyses, specifically pyramiding multiple mutant combinations, forward mutagenesis screens, scaling down a tetraploid to lower ploidy levels and swapping of nuclear and cytoplasmic genomes. Furthermore, the A. thaliana HI can be used to produce haploids from a related species A. suecica and generate homozygous mutant plants from strong maternal gametophyte lethal alleles, which is not possible via conventional diploid genetics. Taken together, our results demonstrate the utility and power of haploid genetics in A. thaliana.

BSKs are partially redundant positive regulators of brassinosteroid signaling in Arabidopsis

Arabidopsis thaliana brassinosteroid signaling kinases (BSKs) constitute a receptor-like cytoplasmic kinase sub-family (RLCK-XII) with 12 members. Previous analysis demonstrated a positive role for BSK1 and BSK3 in the initial steps of brassinosteroid (BR) signal transduction. To investigate the function of BSKs in plant growth and BR signaling, we characterized T-DNA insertion lines for eight BSK genes (BSK1-BSK8) and multiple mutant combinations. Simultaneous elimination of three BSK genes caused alterations in growth and the BR response, and the most severe phenotypes were observed in the bsk3,4,7,8 quadruple and bsk3,4,6,7,8 pentuple mutants, which displayed reduced rosette size, leaf curling and enhanced leaf inclination. In addition, upon treatment with 24-epibrassinolide, these mutants showed reduced hypocotyl elongation, enhanced root growth and alteration in the expression of BR-responsive genes. Some mutant combinations also showed antagonistic interactions. In support of a redundant function in BR signaling, multiple BSKs interacted in vivo with the BR receptor BRI1, and served as its phosphorylation substrates in vitro. The BIN2 and BIL2 GSK3-like kinases, which are negative regulators of BR signaling, interacted in vivo with BSKs and phosphorylated them in vitro, probably at different sites to BRI1. This study demonstrates redundant biological functions for BSKs, and suggests the existence of a regulatory link between BSKs and GSK3-like kinases.

SHORT INTERNODES/STYLISH genes, regulators of auxin biosynthesis, are involved in leaf vein development in Arabidopsis thaliana

Leaves depend on highly developed venation systems to collect fixed carbon for transport and to distribute water. We hypothesized that local regulation of auxin biosynthesis plays a role in vein development. To this effect, we assessed the role of the SHORT INTERNODES/STYLISH (SHI/STY) gene family, zinc-finger transcription factors linked to regulation of auxin biosynthesis, in Arabidopsis thaliana leaf vein development. Gene functions were assessed by a combination of high-resolution spatio-temporal expression analysis of promoter-marker lines and phenotypic analysis of plants homozygous for single and multiple mutant combinations. The SHI/STY genes showed expression patterns with variations on a common theme of activity in incipient and developing cotyledon and leaf primordia, narrowing to apices and hydathode regions. Mutant analysis of single to quintuple mutant combinations revealed dose-dependent defects in vein patterning affecting multiple vein traits, most notably in cotyledons. Here we demonstrate that local regulation of auxin biosynthesis is an important aspect of leaf vein development. Our findings also support a model in which auxin synthesized at the periphery of primordia affects vein development.

Inhibitory Role of Ser-425 of the α1 2.2 Subunit in the Enhancement of Cav 2.2 Currents by Phorbol-12-myristate, 13-Acetate

Voltage-gated calcium channels (Ca(v)) 2.2 currents are potentiated by phorbol-12-myristate, 13-acetate (PMA), whereas Ca(v) 2.3 currents are increased by both PMA and acetyl-beta-methylcholine (MCh). MCh-selective sites were identified in the alpha(1) 2.3 subunit, whereas the identified PMA sites responded to both PMA and MCh (Kamatchi, G. L., Franke, R., Lynch, C., III, and Sando, J. J. (2004) J. Biol. Chem. 279, 4102-4109; Fang, H., Franke, R., Patanavanich, S., Lalvani, A., Powell, N. K., Sando, J. J., and Kamatchi, G. L. (2005) J. Biol. Chem. 280, 23559-23565). The hypothesis that PMA sites in the alpha(1) 2.2 subunit are homologous to the PMA-responsive sites in alpha(1) 2.3 subunit was tested with Ser/Thr --> Ala mutations in the alpha(1) 2.2 subunit. WT alpha(1) 2.2 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits. Inward current (I(Ba)) was recorded using Ba(2+) as the charge carrier. T422A, S1757A, S2108A, or S2132A decreased the PMA response. In contrast, S425A increased the response to PMA, and thus, it was considered an inhibitory site. Replacement of each of the identified stimulatory Ser/Thr sites with Asp increased the basal current and decreased the PMA-induced enhancement, consistent with regulation by phosphorylation at these sites. Multiple mutant combinations showed (i) greater inhibition than that caused by the single Ala mutations; (ii) that enhancement observed when Thr-422 and Ser-2108 are available may be inhibited by the presence of Ser-425; and (iii) that the combination of Thr-422, Ser-2108, and either Ser-1757 or Ser-2132 can provide a greater response to PMA when Ser-425 is replaced with Ala. The homologous sites in alpha(1) 2.2 and alpha(1) 2.3 subunits seem to be functionally different. The existence of an inhibitory phosphorylation site in the I-II linker seems to be unique to the alpha(1) 2.2 subunit.

The specification of a highly derived arthropod appendage, the Drosophila labial palps, requires the joint action of selectors and signaling pathways

The remarkable diversity of form in arthropods reflects flexible genetic programs deploying many conserved genes. In the insect model Drosophila melanogaster, diversity of form can be observed between serially homologous appendages or when a single appendage is transformed by homeotic mutations, such as the adult labial mouthparts that can present alternative antennal, prothoracic, or maxillary identities. We have examined the roles of the Hox selector genes proboscipedia (pb) and Sex combs reduced (Scr), and the antennal selectors homothorax (hth) and spineless (ss) in labial specification, by tissue-directed mitotic recombination. Whereas loss of pb function transforms labium to prothoracic leg, loss of Scr, hth, or ss functions results in little or no change in labial specification. Results of analysis of single and multiple mutant combinations support a genetic hierarchy in which the homeotic pb gene possesses a primary role. It is surprising to note that while loss of ss activity alone had no detected effect, all mutant combinations lacking both pb and ss yielded the most severe phenotype observed: stunted, apparently tripartite legs that may correspond to a default state. The roles of the four selector genes are functionally linked to a cell nonautonomous mechanism involving the coupled activities of the decapentaplegic (dpp)/TGF-beta and wingless (wg)/Wnt signaling pathways. Accordingly, several mutant combinations impaired in dpp signaling were seen to reorient labial-to-leg transformations toward antennal aristae. A crucial aspect of selector function in development and evolution may be in regulating diffusible signals, including those emitted by dpp and wg.

Suicide vectors for antibiotic marker exchange and rapid generation of multiple knockout mutants by allelic exchange in Gram-negative bacteria

Allelic exchange is frequently used in bacteria to generate knockout mutants in genes of interest, to carry out phenotypic analysis and learn about their function. Frequently, understanding of gene function in complex processes such as pathogenesis requires the generation of multiple mutant strains. In Pseudomonads and other non-Enterobacteriaceae, this is a time-consuming and laborious process based on the use of suicide vectors and allelic exchange of the appropriate mutant version of each gene, disrupted by a different antibiotic marker. This often implies the generation of a series of mutants for each gene, each disrupted by a different antibiotic marker, in order to obtain all possible double or multiple mutant combinations. In this work, we have modified this method by developing a set of 3 plasmid derivatives from the previously described suicide vector for allelic exchange, pKAS32, to make antibiotic marker exchange easier and thus accelerate the entire process. Briefly, the construction of each single gene knockout mutant is carried out by allelic exchange of the chromosomal gene with a mutant allele disrupted by the insertion of a kanamycin resistance cassette. When a double mutant strain is required, antibiotic marker exchange is performed in either one of the single mutants, using any of the three plasmid derivatives that carry the kanamycin resistance gene disrupted by either a chloramphenicol, gentamycin, or streptomycin resistance cassette. The single mutant strain, carrying now an antibiotic resistance marker other than kanamycin, can be used to introduce a second mutation using the original plasmid constructs, to generate a double mutant. The process can be repeated sequentially to generate multiple mutants. We have validated this method by generating strains carrying different combinations of mutations in genes encoding different transcriptional regulators of the Hrp type III secretion system in Pseudomonas syringae. We have also tested the genetic organisation and stability of the resulting mutant strains during growth in laboratory conditions as well as in planta.

Endogenous and Synthetic MicroRNAs Stimulate Simultaneous, Efficient, and Localized Regulation of Multiple Targets in Diverse Species

Recent studies demonstrated that pattern formation in plants involves regulation of transcription factor families by microRNAs (miRNAs). To explore the potency, autonomy, target range, and functional conservation of miRNA genes, a systematic comparison between plants ectopically expressing pre-miRNAs and plants with corresponding multiple mutant combinations of target genes was performed. We show that regulated expression of several Arabidopsis thaliana pre-miRNA genes induced a range of phenotypic alterations, the most extreme ones being a phenocopy of combined loss of their predicted target genes. This result indicates quantitative regulation by miRNA as a potential source for diversity in developmental outcomes. Remarkably, custom-made, synthetic miRNAs vectored by endogenous pre-miRNA backbones also produced phenocopies of multiple mutant combinations of genes that are not naturally regulated by miRNA. Arabidopsis-based endogenous and synthetic pre-miRNAs were also processed effectively in tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum). Synthetic miR-ARF targeting Auxin Response Factors 2, 3, and 4 induced dramatic transformations of abaxial tissues into adaxial ones in all three species, which could not cross graft joints. Likewise, organ-specific expression of miR165b that coregulates the PHABULOSA-like adaxial identity genes induced localized abaxial transformations. Thus, miRNAs provide a flexible, quantitative, and autonomous platform that can be employed for regulated expression of multiple related genes in diverse species.

TheArabidopsis-mei2-LikeGenes Play a Role in Meiosis and Vegetative Growth inArabidopsis

The Arabidopsis-mei2-Like (AML) genes comprise a five-member gene family related to the mei2 gene, which is a master regulator of meiosis in Schizosaccharomyces pombe and encodes an RNA binding protein. We have analyzed the AML genes to assess their role in plant meiosis and development. All five AML genes were expressed in both vegetative and reproductive tissues. Analysis of AML1-AML5 expression at the cellular level indicated a closely similar expression pattern. In the inflorescence, expression was concentrated in the shoot apical meristem, young buds, and reproductive organ primordia. Within the reproductive organs, strong expression was observed in meiocytes and developing gametes. Functional analysis using RNA interference (RNAi) and combinations of insertion alleles revealed a role for the AML genes in meiosis, with RNAi lines and specific multiple mutant combinations displaying sterility and a range of defects in meiotic chromosome behavior. Defects in seedling growth were also observed at low penetrance. These results indicate that the AML genes play a role in meiosis as well as in vegetative growth and reveal conservation in the genetic mechanisms controlling meiosis in yeast and plants.

RNA-binding protein Csx1 mediates global control of gene expression in response to oxidative stress.

Fission yeast Spc1 (Sty1), a stress-activated mitogen-activated protein kinase (MAPK) homologous to human p38, orchestrates global changes in gene expression in response to diverse forms of cytotoxic stress. This control is partly mediated through Atf1, a transcription factor homologous to human ATF2. How Spc1 controls Atf1, and how the cells tailor gene expression patterns to different forms of stress, are unknown. Here we describe Csx1, a novel protein crucial for survival of oxidative but not osmotic stress. Csx1 associates with and stabilizes atf1+ mRNA in response to oxidative stress. Csx1 controls expression of the majority of the genes induced by oxidative stress, including most of the genes regulated by Spc1 and Atf1. These studies reveal a novel mechanism controlling MAPK-regulated transcription factors and suggest how gene expression patterns can be customized to specific forms of stress. Csx1-like proteins in humans may perform similar tasks.

Genetic analysis of the wing vein pattern of Drosophila.

Of the many mutations known to affect the wing vein pattern we have selected the most extreme in 29 genes for study. Their phenotype can be classified in two major classes: lack-of-veins and excess-of-veins, and in several internally coherent groups. The study of multiple mutant combinations, within groups and between groups, reveals several genetic operations at work in the generation of the vein pattern. The finding that some of these mutations also affect cell proliferation in characteristic ways has prompted a generative model of wing morphogenetic and pattern formation based on cell behaviour properties defined by the corresponding wild-type genes.

Genetic analysis of muscle development in Drosophila melanogaster

The different thoracic muscles of Drosophila are affected specifically in the mutants: stripe (sr), erect wing (ewg), vertical wings (vtw), and nonjumper (nj). We have tested the extent of this specificity by means of a genetic analysis of these loci, multiple mutant combinations, and gene dosage experiments. A quantitative, rather than a qualitative, specificity is found in the mutant phenotypes. All muscles are altered by mutations in any given gene, but the severity of these alterations is muscle specific. The locus stripe seems to have a polar organization where different allelic combinations show quantitative specificity in the muscle affected. In addition to the muscle phenotypes, neural alterations are detected in these mutants. The synergism found between ewg, vtw and ewg, sr as well as the dosage effect of the distal end of the X chromosome upon the expression of ewg and sr suggests the existence of functional relationships among the loci analyzed.

Radiation-induced polygenic mutation in Arabidopsis thaliana

Three experiments were carried out designed to examine further the properties of radiation-induced mutations affecting flowering time in Arabidopsis thaliana and some of the variables which influence these properties, such as the genotype of the variety that was irradiated (first experiment) and the factors which influence the accumulation of mutations within a single inbred line (second and third experiments). The average additive effect of the induced mutations was found to be independent of parental genotype, supporting the view that this property depends on the relation between the particular character studied and fitness rather than the frequency of “plus” and “minus” alleles in the parental genome as suggested by Brock (1965). Considerable response to selection was obtained in families derived by crossing inbred lines which had been previously irradiated and selected, indicating that few of the mutations fixed in these lines were allelic or linked. This result also suggests that each line contained few mutations, each of relatively large effect. Lines which were irradiated and selected every generation showed a linear relationship between response and accumulated dose in both the early and late flowering direction during generations one to five, but a negative response in the early direction and disproportionately large response in the late direction among generations six to nine inclusive. It is suggested that mutations induced in the later generations interact with those fixed in the early generations giving an adverse effect of multiple mutant combinations on fitness. Such interactions may impose limits on the usefulness of induced mutations in plant breeding.