Functional analysis of MAP kinases in Arabidopsis thaliana: Fully rescuing the mpk3/mpk6 mutant phenotype [abstract]

Abstract only availableMitogen-activated protein kinase (MAPK) cascades are major pathways involved in the transduction of extracellular signals into intracellular responses. A MAPK cascade consists of three kinases. MAPKK kinase (MAPKKK or MEKK) is at the top of this three-tier cascade. Upon its activation by a receptor/sensor, MAPKKK phosphorylates MAPK kinase (MAPKK or MEK), which in turn phosphorylates MAPK and activates it. The activated MAPK can then phosphorylate other protein kinases or be translocated to the nucleus where it can phosphorylate transcription factors and activate gene expression. About 20 MAPKs were identified in the fully sequenced Arabidopsis genome. To study the function of MPK3 and MPK6, the two most closely related MAPKs in Arabidopsis, we isolated the corresponding T-DNA mutants. However, no abnormal phenotype was observed in the single mutants. In order to determine if MPK3 and MPK6 have overlapping functions, we crossed the two single mutants to generate double mutants. Among the 172 F2 plants that we genotyped, no double homozygous plant was identified, indicating that this genotype is lethal. An attempt was then made to rescue this phenotype by introducing an inducible: MPK6 transgene. However, this construct led to only partial rescue of the lethal double mutants. In an attempt to attain complete rescue of these phenotypes, new MPK3 and MPK6 constructs were engineered with the following features: Transgenes regulated by endogenous promoters were used to maintain normal cell/tissue specific expression of the protein. The transgene products were tagged with YFP and GFP. Genomic DNA, as opposed to complementary DNA, was used as the coding regions in order to ensure the presence of introns. Indication of a full rescue will be verified in the T2 generation. Failure to observe completely rescued lines may indicate protein tag interference and further untagged constructs will then be attempted.MU Monsanto Undergraduate Research Fellowshi

Functional analysis of MPK3 and MPK6, two mitogen-activated protine kinases in Arabidopsis thaliana

Abstract only availableMitogen-activated protein kinase (MAPK) cascades are major pathways involved in the transduction of extracellular signals into intracellular responses. A MAPK cascade consists of three kinases; MAPK, MAPK kinase (MAPKK or MEK) and MAPKK kinase (MAPKKK or MEKK). MAPKKK is at the top of this three-tier cascade. Upon its activation by a receptor/sensor, MAPKKK phosphorylates MAPKK, which in turn phosphorylates MAPK and activates it. The activated MAPK can then phosphorylate other protein kinases or be translocated to the nucleus where it can phosphorylate transcription factors and activate gene expression. About 20 MAPKs were identified in the fully sequenced Arabidopsis genome. To study the function of MPK3 and MPK6, the two most closely related MAPKs in Arabidopsis, we isolated the corresponding T-DNA mutants from mutant libraries generated at Wisconsin Arabidopsis Knockout Facility and Salk Institute Genomic Analysis Laboratory. No morphological or developmental phenotypes were observed in the MPK3-/- and MPK6-/- single mutants. In order to determine if MPK3 and MPK6 have overlapping functions, we crossed the two single mutants (MPK3-/- and MPK6-/-) to generate double mutants. Among the 172 F2 plants that we genotyped, no double homozygous (MPK3-/-/MPK6-/-) mutant plants was identified, indicating that this genotype is lethal. We further observed that plants with the MPK3+/-/MPK6-/- genotype are a little smaller and sterile. Reciprocal back cross to wild type plants demonstrated that MPK3+/-/MPK6-/- plants are female sterile. The resilience of the pollens from such plants is still under investigation. In contrast to MPK3+/-/MPK6-/- plants, MPK3-/-/MPK6+/- plants are fertile and apparently normal. Together with the normal phenotype of MPK3-/- and MPK6-/- single mutants, we conclude that MPK3 and MPK6 perform overlapping but not identical roles in the reproduction and development of Arabidopsis thaliana.EXPRESS Progra

Functional analysis of conserved amino acid residues in the C-terminus of ACC synthase

Abstract only availableEthylene is an important plant hormone that regulates growth, development, and stress response. Synthesis of Ethylene from its immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is catalyzed by ACC oxidase. ACC is produced from S-Adenosy1-L-Methionine (SAM) in a reaction catalyzed by ACC synthase (ACS). ACS is the rate limiting enzyme of ethylene biosynthesis. Selected isoforms of ACS are substrates of MPK6 and MPK3, the two Arabidopsis stress-responsive mitogen-activated protein kinases (MAPKs). Phosphorylation of ACS6 by MPK6 stabilizes the ACS protein, thus, elevating the levels of cellular ACS activity and ethylene production. Expression of ACS6DDD, a gain-of-function ACS6 mutant that mimics the phosphorylated form of ACS6, shows constitutive ethylene production and ethylene-induced phenotypes. Analysis of Arabidopsis ACS6 and its orthologs from other species in the database revealed conserved charged amino acids (AAs) in addition to the MAPK phosphorylation sites in their C-termini. We hypothesized that these conserved residues may be involved in the regulation of ACS stability. We used site-directed mutagenesis to mutate the conserved residues to Ala, Ile, or Leu in the ACS6WT or ACS6DDD background using the polymerase chain reaction (PCR). Mutation was confirmed by DNA sequencing. ACS6 mutant gene was transformed into Arabidopsis plants. The stability of ACS6 protein was tested in vivo to determine if the mutation enhances or diminishes its stability. Ethylene production was used as an output reading and the levels of ACS6 protein were determined by immunoblot analysis. Mutation of positively charged AAs makes the ACS6 protein more stable, whereas the mutation of the negatively charged AAs which are close to the phosphorylation sites destabilizes it. Interestingly, deletion of the C-terminus stabilizes the ACS6 protein, suggesting that C-terminus is required for ACS6 degradation. We observed ethylene-regulated morphologies like short hairy main roots and epinastic leaves in ethylene-overproducing seedlings.MU Monsanto Undergraduate Research Fellowshi

Functional analysis of MAP kinases in Arabidopsis thaliana: Fully rescuing the mpk3/mpk6 mutant phenotypes

Abstract only availableMitogen Activated Protein Kinase (MAPK) cascades are three-stage modules involved in signal transduction. MAPKs function at the lower tier of these cascades and they phosphorylate transcription factors and other protein kinases upon activation, ultimately leading to cellular responses. Twenty genes coding for MAPKs were identified in the fully sequenced Arabidopsis genome. MPK3 and MPK6 are the most closely related. Analysis of T-DNA insertional lines revealed no phenotype in the mpk3 and mpk6 single mutants; however, female sterility is observed in MPK3+/-/MPK6-/- plants and embryo lethality results from knocking out both genes. This indicates overlapping function of MPK3 and MPK6. To better understand the function of these two kinases, an attempt was made to rescue these phenotypes by introducing a Dexamethasone (DEX) inducible: MPK6 transgene. This construct led to only partial rescue of the lethal double mutants, and no signs of fertility were evident in MPK3+/-/MPK6-/- plants. In an attempt to attain complete rescue of these phenotypes, new MPK3 and MPK6 constructs were engineered with the following features: • Transgenes regulated by endogenous promoters were used in order to maintain normal cell/tissue specific expression of the protein, which may be essential for normal plant function. • The transgene products were tagged with Yellow Florescent Protein and Green Florescent Protein in order to ascertain their expression patterns. • Genomic DNA, as opposed to complementary DNA, was used as the coding regions in order to ensure the presence of introns, which may be significant for gene function. Currently, T1 generation transgenic plants have been isolated and transgenic lines with good expression of the transgene proteins, in vivo, will be identified by Western Blot analysis. Indication of a full rescue will be verified in the T2 generation. Failure to observe completely rescued lines may indicate protein tag interference, and untagged constructs will then be attempted.MU Monsanto Undergraduate Research Fellowshi

Monitoring CD27 expression to evaluate Mycobacterium tuberculosis activity in HIV-1 infected individuals in vivo.

The level of bacterial activity is only poorly defined during asymptomatic Mycobacterium tuberculosis (MTB) infection. The objective was to study the capacity of a new biomarker, the expression of the T cell maturation marker CD27 on MTB-specific CD4 T cells, to identify active tuberculosis (TB) disease in subjects from a MTB and HIV endemic region. The frequency and CD27 expression of circulating MTB-specific CD4 T cells was determined in 96 study participants after stimulation with purified protein derivative (PPD) using intracellular cytokine staining for IFNgamma (IFNγ). Subjects were then stratified by their TB and HIV status. Within PPD responders, a CD27(-) phenotype was associated with active TB in HIV(-) (p = 0.0003) and HIV(+) (p = 0.057) subjects, respectively. In addition, loss of CD27 expression preceded development of active TB in one HIV seroconverter. Interestingly, in contrast to HIV(-) subjects, MTB-specific CD4 T cell populations from HIV(+) TB-asymptomatic subjects were often dominated by CD27(-) cells. These data indicate that down-regulation of CD27 on MTB-specific CD4 T cell could be used as a biomarker of active TB, potentially preceding clinical TB disease. Furthermore, these data are consistent with the hypothesis that late, chronic HIV infection is frequently associated with increased mycobacterial activity in vivo. The analysis of T cell maturation and activation markers might thus be a useful tool to monitor TB disease progression

Preferential infection and depletion of Mycobacterium tuberculosis–specific CD4 T cells after HIV-1 infection

HIV-1 preferentially infects M. tuberculosis-specific CD4+ T cells due to their increased production of IL-2

Investigating the developmental defects of rescued mpk3-/ mpk6- Arabidopsis plants [abstract]

Abstract only availableMitogen Activated Protein Kinases (MAPKs) are signaling molecules involved in transducing extracellular signals into intracellular responses. Twenty MAPK genes have been identified in the fully sequenced Arabidopsis genome. To deduce the function of MPK3 and MPK6 (the two most closely related Arabidopsis MAPKs), we obtained the corresponding T-DNA single knockout mutants. Homozygous single mutants had no obvious phenotype. However, we failed to generate double mutants (mpk3-/-mpk6-/- ) and found these plants to be lethal in the early embryo stages. Normal embryogenesis starts with elongation of the zygote followed by cell division to form two cells of different sizes and different fates. However this asymmetric cell division is defective in the double mutants. We generated conditionally rescued double mutants by introducing a steroid (DEX) inducible MPK6 transgene. The resulting (MPK3-/-/MPK6-/-/DEX:MPK6+/+ ) seedlings, however, were abnormal and unable to develop. Their growth was extremely stunted, they had irregular leaf surfaces (stomata clustering) and had no real stem or roots. To understand the molecular mechanisms underlying this embryogenesis defect in the double mutant, we are using reporter genes and Affymetrix GeneChip analysis to investigate the involvement of plant hormones.McNair Scholars Progra

Stomatal Development and Patterning Are Regulated by Environmentally Responsive Mitogen-Activated Protein Kinases in Arabidopsis

Stomata are specialized epidermal structures that regulate gas (CO(2) and O(2)) and water vapor exchange between plants and their environment. In Arabidopsis thaliana, stomatal development is preceded by asymmetric cell divisions, and stomatal distribution follows the one-cell spacing rule, reflecting the coordination of cell fate specification. Stomatal development and patterning are regulated by both genetic and environmental signals. Here, we report that Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6, two environmentally responsive mitogen-activated protein kinases (MAPKs), and their upstream MAPK kinases, MKK4 and MKK5, are key regulators of stomatal development and patterning. Loss of function of MKK4/MKK5 or MPK3/MPK6 disrupts the coordinated cell fate specification of stomata versus pavement cells, resulting in the formation of clustered stomata. Conversely, activation of MKK4/MKK5-MPK3/MPK6 causes the suppression of asymmetric cell divisions and stomatal cell fate specification, resulting in a lack of stomatal differentiation. We further establish that the MKK4/MKK5-MPK3/MPK6 module is downstream of YODA, a MAPKKK. The establishment of a complete MAPK signaling cascade as a key regulator of stomatal development and patterning advances our understanding of the regulatory mechanisms of intercellular signaling events that coordinate cell fate specification during stomatal development

Monitoring CD27 expression to evaluate mycobacterium tuberculosis activity in HIV-1 infected individuals in vivo

The work was supported by grants from the European Commission (DG XII, INCO-DC, ERBICA4-CT-2002-10035 and DG X, EuropeAID SANTE/2004/078-545/130