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PubMed2001

Preferential binding of Staphylococcus aureus to skin sites of Th2-mediated inflammation in a murine model.

Staphylococcus aureus is found on over 90% of atopic dermatitis skin lesions and is thought to contribute to skin inflammation via the production of potent exotoxins. In contrast, less than 5% of normal subjects harbor S. aureus. This suggests that an atopic immune response itself may play a role in preferential binding of S. aureus to the skin. To examine this issue more directly, we analyzed the S. aureus binding characteristics of skin in mice undergoing different T helper type 1 cell versus T helper type 2 cell inflammatory responses using a novel in vitro bacterial binding assay. BALB/C female mice were first sensitized to ovalbumin with alum or ovalbumin with complete Freund's adjuvant to induce T helper type 2 or T helper type 1 responses, respectively. Mice were then challenged intradermally with either saline (control) or ovalbumin. Forty-eight hours later, skin specimens were obtained from the challenge sites, and the number of S. aureus binding to each skin section was quantitated. Bacterial binding was found to be significantly greater at skin sites of BALB/C mice that had been ovalbumin/alum sensitized compared with ovalbumin/complete Freund's adjuvant sensitized (p < or = 0.01). When compared to the ovalbumin sensitized/challenged skin of wild type BALB/C mice or interferon-gamma gene knockout mice, interleukin-4, but not interferon-gamma, gene knockout mice had significantly less S. aureus binding at their ovalbumin sensitized/challenged skin sites. Mutant S. aureus strains that lacked either fibronectin- or fibrinogen-binding protein expression showed significantly reduced S. aureus binding compared with the parent wild type strain (p < 0.005). Moreover, preincubation of the wild type bacteria with fibronectin or fibrinogen, but not collagen, resulted in significantly less skin binding of S. aureus (p < 0.01). Incubation of skin with interleukin-4, and less so with interferon-gamma, led to more binding of wild type S. aureus but not of an S. aureus mutant deficient in fibronectin binding protein expression. After interleukin-4 incubation, but not interferon-gamma, epidermal immunoreactivity for fibronectin was observed in murine skin explants. These results show that a T helper type 2 inflammatory environment can promote skin binding by S. aureus and that this binding is mediated by fibronectin and fibrinogen.

Adhesins, Bacterial↗
PubMed2002

Role of IFN-gamma and tumor necrosis factor-alpha in herpes simplex virus type 1 infection.

One of the characteristics of herpes simplex virus type 1 (HSV-1) is that recurrent diseases often develop from latent infection established after acute infection. Cytokines have been proposed to play an important role in each stage of HSV-1 infection, but the exact role of cytokines remains unclear. In the present study, we investigated the role of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in acute infection and reactivation using IFN-gamma gene knockout (IFN-gamma(-/-)) mice and TNF-alpha gene knockout (TNF-alpha(-/-)) mice. We first examined the survival rate after corneal infection with HSV-1. The survival rates of wild-type C57BL/6 (B6) mice, IFN-gamma(-/-) mice, and TNF-alpha(-/-) mice were 97% (73 of 75), 57% (24 of 42), and 83% (60 of 72), respectively. These results suggest that TNF-alpha and IFN-gamma play a protective role in acute infection with HSV-1. We also examined the rate of reactivation induced by ultraviolet (UV) light in latently infected mice over 60 days postinoculation. The reactivation was confirmed by detecting viral DNA extracted from eyeballs by the polymerase chain reaction (PCR) method at day 2 after the UV light stimulation. The rates of reactivation in IFN-gamma(-/-) mice and TNF-alpha(-/-) mice were significantly higher than that in B6 mice; 16% (4 of 25) showed reactivation in B6 mice, 47% (9 of 19) in IFN-gamma(-/-) mice, and 48% (10 of 21) in TNF-alpha(-/-) mice. These results suggest that IFN-gamma and TNF-alpha play an important role in acute infection and reactivation from latency.

Acute Disease↗
PubMed2006

[Construction of Streptococcus mutans luxS gene allelic exchange plasmid].

OBJECTIVE: It is reported that Streptococcus mutans luxS gene may have an important role in the interspecies quorum sensing system. To construct the S. mutans luxS gene knockout mutant, this research aim to construct the luxS gene allelic exchange plasmid. METHODS: The upstream and downstream flank DNA fragments of S. mutans luxS gene (Xup, Xdn)and the E. coli kanamycin resistance gene (Kana) were enriched by pfu DNA polymerase with "nest PCR" methods. These fragments were ligated into pBluescript SK (+) Phagemids vector with double endonuclease reaction sequentially. RESULTS: With endonuclease reaction and DNA sequencing, it was proved that the objective plasmid, Xukd-pbsk, was constructed correctively and the kanamycin resistance gene could be expressed in vitro. CONCLUSION: The S. mutans luxS gene allelic exchange plasmid is constructed correctively in this research and can be used in the future research of S. mutans luxS gene knockout mutant.

Bacterial Proteins↗
PubMed2006

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.

Anti-Bacterial Agents↗
PubMed2001

Alternative models for carcinogenicity testing: weight of evidence evaluations across models.

Twenty-one chemicals were evaluated by standardized protocols in 6 mouse models that have been sugggested as alternatives to the 2-year mouse bioassay. Included were genotoxic and nongenotoxic chemicals, carcinogens and noncarcinogens, immunosuppressive and estrogenic agents, peroxisome proliferators, and chemicals producing cancer in rodents by other mechanisms. Mice were sacrificed at the end of 6 to 12 months, depending on the model. Standardized histopathology, biostatistical analyses, and criteria for overall evaluation of the results were employed. The TgAC transgenic (dermal and oral administration), the Tg-rasH2 transgenic, the heterozygous p53 gene knockout, the homozygous XPA and homozygous XPA-heterozygous p53 gene knockout, and the neonatal mouse models were evaluated. The chemicals were also evaluated in the in vitro SHE assay. Comparison of the results between the various in vivo models suggest that they might have usefulness as screening bioassays for hazard identification for potential human carcinogens. They have the benefits of being quicker, less expensive, and involve fewer animals than the traditional 2-year mouse bioassay. They do not appear to be overly sensitive. However, they do not definitively distinguish between genotoxic and nongenotoxic carcinogens, and they do not have 100% specificity for identifying human carcinogens. Like the 2-year bioassay, the results from these models need to be evaluated in conjunction with other information on a chemical in an overall weight-of-evidence, integrated analytical approach to assess risk for human exposures.

Animal Testing Alternatives↗
PubMed2001

Basic science research on the urinary bladder and interstitial cystitis: new genetic approaches.

This article summarizes recent genetic research that promises to advance understanding of the functioning of the urinary bladder and further our knowledge about interstitial cystitis. Results reported at the Tenth International Research Symposium on Interstitial Cystitis and Bladder Research and in the current literature are presented. Three specific areas of genetic research are summarized: gene expression via DNA arrays, development of new animal models through transgenic or gene knockout approaches, and gene therapy. Advances in genetic research (specifically in gene therapy; development of new, genetically engineered mouse models; and study of gene expression using DNA array assays) will contribute to further understanding the functioning of the urinary bladder in health and disease.

Animals↗
PubMed1997

Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression.

The signal transducer and activator of transcription, STAT5b, has been implicated in signal transduction pathways for a number of cytokines and growth factors, including growth hormone (GH). Pulsatile but not continuous GH exposure activates liver STAT5b by tyrosine phosphorylation, leading to dimerization, nuclear translocation, and transcriptional activation of the STAT, which is proposed to play a key role in regulating the sexual dimorphism of liver gene expression induced by pulsatile plasma GH. We have evaluated the importance of STAT5b for the physiological effects of GH pulses using a mouse gene knockout model. STAT5b gene disruption led to a major loss of multiple, sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male-characteristic body growth rates and male-specific liver gene expression were decreased to wild-type female levels in STAT5b-/- males, while female-predominant liver gene products were increased to a level intermediate between wild-type male and female levels. Although these responses are similar to those observed in GH-deficient Little mice, STAT5b-/- mice are not GH-deficient, suggesting that they may be GH pulse-resistant. Indeed, the dwarfism, elevated plasma GH, low plasma insulin-like growth factor I, and development of obesity seen in STAT5b-/- mice are all characteristics of Laron-type dwarfism, a human GH-resistance disease generally associated with a defective GH receptor. The requirement of STAT5b to maintain sexual dimorphism of body growth rates and liver gene expression suggests that STAT5b may be the major, if not the sole, STAT protein that mediates the sexually dimorphic effects of GH pulses in liver and perhaps other target tissues. STAT5b thus has unique physiological functions for which, surprisingly, the highly homologous STAT5a is unable to substitute.

Animals↗
PubMed2005

Infections in immunocompetent and immune-deficient mice with promastigotes of a North American isolate of Leishmania infantum.

Leishmania infantum, an etiologic agent of zoonotic visceral leishmaniasis, is widespread among foxhounds in the United States. Experimental infections with a North American isolate of L. infantum were evaluated using two inoculation routes in immunocompetent and immunosuppressed mouse strains. Groups of 2-5 interferon gamma gene knockout (IFN-gamma-KO) (BALB/c-Ifng), inducible nitric oxide synthase (NOS) gene knockout (iNOS-KO) (C57BL/6), B-cell-deficient (microMT) (C57BL/6), and BALB/c mice were intravenously (i.v.) or subcutaneously (s.c.) inoculated with various doses of promastigotes of the LIVT-1 strain of L. infantum. None of the mice developed clinical signs of leishmaniasis during the 8-9 weeks of the study. Promastigotes were cultured from spleens of all i.v.-infected mice by 3 days post culture. Spleens from s.c.-infected mice inoculated with greater than 1 x 10(6) parasites became culture positive 3-24 days post culture, but promastigotes were not cultured from mice infected with 1 x 10(5) or 5 x 10(5) LIVT-1 promastigotes. Histological lesions were prominent in the livers of i.v.-infected mice but were mild to nonexistent in s.c. infection. Serological responses were low and transient determined by indirect fluorescent antibody testing in all groups. These results indicate that the i.v. route of infection is superior to the s.c. route in a mouse model of North American leishmaniasis and that mice lacking INF-gamma, iNOS or mice that are B-cell-deficient are not more susceptible to acute infection.

Animals↗
PubMed2005

Seeing is believing! The optical properties of the eye lens are dependent upon a functional intermediate filament cytoskeleton.

Beaded filaments are the major cytoskeletal element of the eye lens and they are essential to the optical properties of the eye lens. They were discovered in 1972 by Harry Maisel and Margaret Perry and have since been found to comprise two novel intermediate filament proteins, CP49 and filensin. These proteins possess unique structure features and unusual assembly characteristics, which distinguish them from canonical IF proteins. Whilst CP49 is completely tailless, filensin has a rather short rod domain and extremely large C-terminal tail domain. In vitro, CP49 and filensin do not form IFs on their own. In vitro studies suggest that CP49 and filensin have a distinct coassembly mechanism. Whilst CP49 self-assembles into thick bundles of filaments, filensin only forms short fibrils, but when combined together they form filaments. The generation of gene knockouts by the targeted deletion of Bfsp1 and Bfsp2 that encode filensin and CP49, respectively, have been made to explore the function of beaded filaments in the lens. Our results suggest that the lens-specific beaded filaments are the key cytoskeletal element in organising and maintaining lens fibre cell architecture and are a key factor in determining the optical properties of the lens. We have also found that some common mouse strains contain a natural mutation in Bfsp2 that will effectively generate a CP49 knockout. This finding has important implications for lens research involving other gene knockouts maintained on a 129 background. It has also been observed that mutations in Bfsp2 are the genetic basis of inherited human cataract. Collectively, these data demonstrate that beaded filaments are fundamental to lens function.

Amino Acid Sequence↗
PubMed2026

Phosphoproteomic Analysis of Cortical Tissue from Mice Lacking Both CaMKII&#x3b1; and CaMKII&#x3b2; Identifies Novel In Vivo Substrates.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a critical role in calcium signaling. Several studies have shown that mice with single Camk2a or Camk2b gene knockouts are viable, yet exhibit distinct phenotypes, whereas the double knockout of both genes is lethal. These findings indicate that each gene can have distinct roles and that they also partially compensate for each other in yet unknown essential brain functions. In order to provide insight into potential novel CaMKII functions, we performed parallel phosphoproteomic analyses on nonstimulated cortex tissues from inducible Camk2a and Camk2b double knockout (Camk2af/f;Camk2bf/f;CAG-CreESR) mice and from wild type mice. A total of 5622 phosphorylated peptides derived from 2080 proteins were identified. Phosphorylation at serine/threonine residues in 130 proteins was downregulated in the double knockout mice, including residues in 113 proteins that have not previously been identified as potential CaMKII substrates. Comparison of amino acid sequences surrounding the downregulated phosphorylation residues provided new insights into the CaMKII-substrate consensus sequences in vivo. This data set provides an important resource for future studies examining novel roles for CaMKII in the brain.

Animals↗
PubMed2006

A gene trap knockout of the abundant sperm tail protein, outer dense fiber 2, results in preimplantation lethality.

Outer dense fiber 2 (Odf2) is highly expressed in the testis where it encodes a major component of the outer dense fibers of the sperm flagellum. Furthermore, ODF2 protein has recently been identified as a widespread centrosomal protein. While the expression of Odf2 highlighted a potential role for this gene in male germ cell development and centrosome function, the in vivo function of Odf2 was not known. We have generated Odf2 knockout mice using an Odf2 gene trapped embryonic stem cell (ESC) line. Insertion of a gene trap vector into exon 9 resulted in a gene that encodes a severely truncated protein lacking a large portion of its predicted coil forming domains as well as both leucine zipper motifs that are required for protein-protein interactions with ODF1, another major component of the outer dense fibers. Although wild-type and heterozygous mice were recovered, no mice homozygous for the Odf2 gene trap insertion were recovered in an extended breeding program. Furthermore, no homozygous embryos were found at the blastocyst stage of embryonic development, implying a critical pre-implantation role for Odf2. We show that Odf2 is expressed widely in adults and is also expressed in the blastocyst stage of preimplantation development. These findings are in contrast with early studies reporting Odf2 expression as testis specific and suggest that embryonic Odf2 expression plays a critical role during preimplantation development in mice.

Animals↗
PubMed2002

Characteristics of carotid body chemosensitivity in NADPH oxidase-deficient mice.

Various heme-containing proteins have been proposed as primary molecular O(2) sensors for hypoxia-sensitive type I cells in the mammalian carotid body. One set of data in particular supports the involvement of a cytochrome b NADPH oxidase that is commonly found in neutrophils. Subunits of this enzyme have been immunocytochemically localized in type I cells, and diphenyleneiodonium, an inhibitor of the oxidase, increases carotid body chemoreceptor activity. The present study evaluated immunocytochemical and functional properties of carotid bodies from normal mice and from mice with a disrupted gp91 phagocytic oxidase (gp91(phox)) DNA sequence gene knockout (KO), a gene that codes for a subunit of the neutrophilic form of NADPH oxidase. Immunostaining for tyrosine hydroxylase, a signature marker antigen for type I cells, was found in groups or lobules of cells displaying morphological features typical of the O(2)-sensitive cells in other species, and the incidence of tyrosine hydroxylase-immunopositive cells was similar in carotid bodies from both strains of mice. Studies of whole cell K(+) currents also revealed identical current-voltage relationships and current depression by hypoxia in type I cells dissociated from normal vs. KO animals. Likewise, hypoxia-evoked increases in intracellular Ca(2+) concentration were not significantly different for normal and KO type I cells. The whole organ response to hypoxia was evaluated in recordings of carotid sinus nerve activity in vitro. In these experiments, responses elicited by hypoxia and by the classic chemoreceptor stimulant nicotine were also indistinguishable in normal vs. KO preparations. Our data demonstrate that carotid body function remains intact after sequence disruption of the gp91(phox) gene. These findings are not in accord with the hypothesis that the phagocytic form of NADPH oxidase acts as a primary O(2) sensor in arterial chemoreception.

Animals↗
PubMed2005

Evolutionary programming as a platform for in silico metabolic engineering.

BACKGROUND: Through genetic engineering it is possible to introduce targeted genetic changes and hereby engineer the metabolism of microbial cells with the objective to obtain desirable phenotypes. However, owing to the complexity of metabolic networks, both in terms of structure and regulation, it is often difficult to predict the effects of genetic modifications on the resulting phenotype. Recently genome-scale metabolic models have been compiled for several different microorganisms where structural and stoichiometric complexity is inherently accounted for. New algorithms are being developed by using genome-scale metabolic models that enable identification of gene knockout strategies for obtaining improved phenotypes. However, the problem of finding optimal gene deletion strategy is combinatorial and consequently the computational time increases exponentially with the size of the problem, and it is therefore interesting to develop new faster algorithms. RESULTS: In this study we report an evolutionary programming based method to rapidly identify gene deletion strategies for optimization of a desired phenotypic objective function. We illustrate the proposed method for two important design parameters in industrial fermentations, one linear and other non-linear, by using a genome-scale model of the yeast Saccharomyces cerevisiae. Potential metabolic engineering targets for improved production of succinic acid, glycerol and vanillin are identified and underlying flux changes for the predicted mutants are discussed. CONCLUSION: We show that evolutionary programming enables solving large gene knockout problems in relatively short computational time. The proposed algorithm also allows the optimization of non-linear objective functions or incorporation of non-linear constraints and additionally provides a family of close to optimal solutions. The identified metabolic engineering strategies suggest that non-intuitive genetic modifications span several different pathways and may be necessary for solving challenging metabolic engineering problems.

Algorithms↗
PubMedDate not supplied

Role of the GABA(A)beta2, GABA(A)alpha6, GABA(A)alpha1 and GABA(A)gamma2 receptor subunit genes cluster in drug responses and the development of alcohol dependence.

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the central nervous system and it acts at the GABA(A) and GABA(B) receptors. A possible role for the GABA(A) receptors in alcohol action has been derived from in vitro cell models, animal studies and human research. GABA(A) subunit mRNA expression in cell models has suggested that the long form of the gamma2 subunit is essential for ethanol enhanced potentiation of GABA(A) receptors, by phosphorylation of a serine contained within the extra eight amino acids. Several animal studies have demonstrated that alterations in drug and alcohol responses may be caused by amino-acid differences at the GABA(A)alpha6 and GABA(A)gamma2 subunits. An Arg(100)/Glu(100) change at the GABA(A)alpha6 subunit conferring altered binding efficacy of the benzodiazepine inverse agonist Ro 15-4513, was found between the AT (alcohol tolerance) and ANT (alcohol non-tolerance) rats. Several loci related to alcohol withdrawal on mouse chromosome 11 which corresponds to the region containing four GABA(A) subunit (beta2, alpha6, alpha1 and gamma2) genes on human chromosome 5q33-34, were also identified. Gene knockout studies of the role of GABA(A)alpha6 and GABA(A)gamma2 subunit genes in mice have demonstrated an essential role in the modulation of other GABA(A) subunit expression and the efficacy of benzodiazepine binding. Absence of the GABA(A)gamma2 subunit gene has more severe effects with many of the mice dying shortly after birth. Disappointingly few studies have examined the effects of response to alcohol in these gene knockout mice. Human genetic association studies have suggested that the GABA(A)beta2, alpha6, alpha1 and gamma2 subunit genes have a role in the development of alcohol dependence, although their contributions may vary between ethnic group and phenotype. In summary, in vitro cell, animal and human genetic association studies have suggested that the GABA(A)beta2, alpha6, alpha1 and gamma2 subunit genes have an important role in alcohol related phenotypes (300 words).

Alcoholism↗
PubMed2000

Growth hormone pulse-activated STAT5 signalling: a unique regulatory mechanism governing sexual dimorphism of liver gene expression.

Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription that are regulated by the temporal pattern of pituitary GH release; this release is intermittent in male rats and nearly continuous in females. Comparisons of liver nuclear protein tyrosine phosphorylation in male and female rats have led to the discovery that the liver transcription factor STAT5b is tyrosine phosphorylated in male but not female rats in response to GH pulses. Intermittent plasma GH pulses trigger a rapid and repeated tyrosine phosphorylation and nuclear translocation of liver STAT5b in intact male rats, while the more continuous pattern of GH exposure down-regulates the STAT5b signalling pathway in female rat liver. The central importance of STAT5b for the physiological effects of GH pulses has been verified using a mouse gene knockout model. STAT5b gene disruption leads to a major loss of multiple sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male-characteristic body growth rates and male-specific liver gene expression are decreased to wild-type female levels in STAT5b-/- males, while female-predominant liver gene products are increased in males to near female levels. STAT5b is thus a liver-expressed, latent cytoplasmic transcription factor that undergoes repeated tyrosine phosphorylation and nuclear translocation in response to intermittent plasma GH stimulation, and is a key intracellular mediator of the stimulatory effects of GH pulses on male-specific liver gene transcription. Other studies indicate, however, that STAT5a and STAT5b are both required for constitutive expression in female, but not male liver, of certain GH-regulated CYP enzymes. GH activation of both STAT5 proteins, which in turn form distinct homodimeric and heterodimeric DNA-binding complexes, is thus an important determinant of the sex-dependent and gene-specific effects that GH has on the liver.

Animals↗
PubMed2003

Sequence analysis and functional characterization of the dialkylglycine decarboxylase gene DGD1 from Mycosphaerella graminicola.

Dialkylglycine decarboxylase is a pyridoxal phosphate-dependent enzyme in the aminotransferases class III group of enzymes. The enzyme is unique in terms of catalyzing both decarboxylation and transamination. Although the enzymatic activity is present in some bacteria and fungi, the biological role is unclear. We identified and disrupted the dialkylglycine decarboxylase-encoding gene DGD1 in the wheat blotch fungus Mycosphaerella graminicola by transposon-arrayed gene knockout. The DGD1 gene is highly similar to dialkylglycine decarboxylase from the soil bacterium Burkholderia cepacia. Phylogenetic analysis of various class III aminotransferases showed that dialkylglycine decarboxylases from bacteria and fungi are found in a distinct cluster. Functional analysis revealed that dgd1 disruption mutants display wild-type morphology and pathogenicity to wheat. The dgd1 mutants cannot utilize 2-methylalanine as a sole nitrogen source, as assessed by large-scale nutritional utilization analysis. This is the first description of a mutant phenotype of the fungal dialkylglycine decarboxylase gene.

Amino Acid Sequence↗
PubMed2026

CRISPR/Cas9-mediated knockout of PsLykX gene of pea (Pisum sativum L.) leads to loss of symbiotic nodules.

Pea (Pisum sativum L.) symbiosis with nodule bacteria supplying plants with additional nitrogen is a very specific plant-microbial interaction. Mutual recognition of the partners occurs through perception of bacterial signal molecules (Nod factors) by plant receptors, enabling bacterial entry via root hairs and formation of nitrogen-fixing nodules. The pea gene Sym2, described but not yet cloned, exists in different allelic forms defining the symbiotic specificity, and is therefore thought to encode a Nod factor receptor. The PsLykX gene is a strong candidate for the Sym2, since its alleles coincide with high or low symbiotic specificity; however, to date, no genetic evidence has been obtained for a role of PsLykX in symbiosis. Here, we knocked-out the PsLykX in European pea cultivar Cam&#xe9;or using Agrobacterium-mediated hairy root transformation and CRISPR-Cas9 editing. The roots with editing events confirmed by sequencing lost the ability to form nodules, providing direct functional evidence that PsLykX is essential, at least, for the symbiosis between pea cultivar Cam&#xe9;or and Rhizobium ruizarguesonis RCAM1026.

Pisum sativum↗
PubMed2005

Regulation of volatile benzenoid biosynthesis in petunia flowers.

The petunia flower has served as a model for the study of several physiological processes including floral development, self-incompatibility, anthocyanin biosynthesis and ethylene signalling during senescence. More recently, Petunia hybrida 'Mitchell' has been used to understand the complex regulation of volatile benzenoid biosynthesis, which occurs predominantly in flower petal tissues. Benzenoid biosynthesis is temporally and circadian controlled and is tightly down-regulated by ethylene during floral senescence. Using targeted transcriptomics and gene knockouts, both biosynthetic genes and a transcription factor regulating benzenoid synthesis have been recently discovered and characterized. It appears that benzenoid production is regulated predominantly by transcriptional control of the shikimate pathway, benzenoid biosynthesis genes and S-adenosyl-methionine cycle genes.

Benzene Derivatives↗