Nano and traditional phosphate fertilizers: exploring their effects on microbiomes and plant growth
Out of necessity, plant nutrient requirements must be locally met, which explains the importance of soil fertility and soil microbiome community structure. Soil communities were examined in long-term maintained fertilizer and warming treatments in the Arctic tundra. The microbiomes associated with Arctic birch (Betula glandulosa) within the 5 treatment plots (control, high nitrogen (N), high phosphorus (P), high N and P, and warming) near Daring Lake, NWT were sampled. Microbiome analysis of bacterial and fungal communities with 16s rRNA and ITS analysis respectively, showed some differences in bacterial communities with an increase in the family Xanthomonadaceae present in the N & P treatment, while control and warming treatments showed similar soil microbiome structures. Fungal analysis showed an increase in the genus Thelephora in the high P treatment. Microbial communities and plant growth were analyzed in the presence of nano-phosphate fertilizer. Nano-hydroxyapatite (nHA) has been proposed as a more environmentally friendly and more efficient method of P-fertilization. nHA, characterized to be needle-like in shape and ~35 nm in width, was used to evaluate the impact on early microbiome establishment in soybean (Glycine max) in an agriculturally realistic manner. The V4 region of bacterial 16s rRNA was used to determine the microbiome structure in the soil and rhizosphere. Little difference was found in the structure of the soil and rhizosphere microbiomes between controls and nHA treatments. Growth experiments implementing nHA at agricultural levels of P2O5, added to the soil at the time of planting, did not show a significant increase in growth, biomass, or yield compared with controls, suggesting that the needle-shaped nHA do not function as an effective fertilizer when implemented in this manner. The effectiveness of nano-fertilizers is likely influenced by their physicochemical properties (i.e. shape and surface) and their interactions with the soil matrix, making it important to evaluate these factors when designing new nano-phosphate fertilizers. As such, traditional P fertilization as used in the Daring Lake plots, is more effective and has a higher impact on the associated microbial community structure.
Plant immunity and phytotoxins: dissecting interactions between Fusarium graminearum and its plant hosts
Fusarium graminearum is a devastating fungal pathogen of diverse plant species including maize, barley and wheat, for which no complete resistance has been identified. Its significant economic impact worldwide is due in part to contamination of grain with diverse toxic secondary metabolites. Recent work indicates that F. graminearum may produce distinct types of unknown metabolites in different hosts. Using a reverse genetic approach, we recently identified two novel cyclic peptides, gramillin A and B, which are produced in maize but not in wheat. In addition, gramillin and expression of its biosynthetic genes are essential for fungal virulence on maize, but not wheat. Gramillin is a potent phytotoxin with specific activity within the plant kingdom, causing necrotic lesions with hours of application. Interestingly, gramillin also induces transient immune responses in plants and suppresses immunity to bacterial pathogens and elicitors. Possible mechanisms for effects on immunity and cell survival will be discussed.
Characterization of autophosphorylation of the dual-specificity Ca2+-dependent protein kinase RcCDPK1 from developing castor oil seeds
Phosphoenolpyruvate carboxylase (PEPC) is a tightly-regulated enzyme that plays essential roles in central plant metabolism, particularly the replenishment of TCA cycle intermediates withdrawn for biosynthesis or N-assimilation. In developing castor oil seeds (COS) an enigmatic ‘bacterial-type’ PEPC (BTPC) isozyme is highly expressed as a catalytic and regulatory subunit of a novel Class-2 PEPC heteromeric complex. During COS development BTPC is subject to in vivo inhibitory phosphorylation at Ser451, a highly conserved target residue occurring within an intrinsically-disordered domain. This phosphorylation event is catalyzed by RcCDPK1, a member of the castor Ca2+-dependent protein kinase (CDPK) family. Ca2+-dependent autokinase activity of CDPKs has been reported and may influence CDPK activity and substrate accessibility. However, the functions and in vivo occurrence of CDPK autophosphorylation remain poorly understood. A major objective of my MSc thesis was to test the hypothesis that autophosphorylation influences RcCDPK1’s ability to transphosphorylate its BTPC substrate at Ser451. Ca2+-stimulated autokinase activity of heterologously expressed RcCDPK1WT was readily detected by pImagoTM phosphoprotein staining, and multiple pSer, pThr, and pTyr residues were mapped via LC-MS/MS. These included Tyr30 which: (i) occurs at the interface of RcCDPK1’s N-terminal variable and catalytic domains, and (ii) is absolutely conserved and also autophosphorylated in RcCDPK1 orthologs AtCPK4 and GmCDPK-β. The influence of Tyr30 autophosphorylation was examined by generating a phosphoablative Y30F mutant (RcCDPK1Y30F). In vitro dephosphorylated RcCDPK1WT exhibited an ~13-fold increase in its rate of Ca2+-dependent BTPC transphosphorylation at Ser451 relative to autophosphorylated RcCDPK1WT. Conversely, RcCDPK1Y30F showed no obvious difference relative to RcCDPK1 WT with respect to its autokinase or BTPC Ser451 transphosphorylation activity. Although global autophosphorylation of RcCDPK1 is highly inhibitory, Tyr30 autophosphorylation appears to have little impact on its kinase activity. The results provide insights into the link between plant carbon metabolism, Ca2+-dependent signaling, and the biological significance of CDPK autophosphorylation.
Inducible GAL4 Systems for Gene Expression Control in Adult Drosophila
The ability to express a gene of interest in a directed place, time and magnitude is enabled by gene expression control tools. The UAS/GAL4 system is by far the most widely used tool inDrosophila with a collection of thousands of GAL4 drivers available within the community. Despite the system’s vast applications the UAS/GAL4 system does not allow for temporal control of gene expression. A major focus of many fly geneticists has been to create a new system by generating new transgenes based on entirely different transcriptional regulators. However it is much more convenient to take advantage of the existing UAS and/or GAL4 lines, many of which have already been spatially and temporally characterized through adult life (Seroude et al. Aging Cell, 2002). We have generated two new inducible GAL4-based gene expression systems: the Tet-Off GAL80 transgenes and the Lac Inducible UAS/GAL4 system. The Tet-Off GAL80 transgenes were designed to express GAL80 under the control of a Tet-Off promoter to regulate GAL4 activity. Two versions of the transgene were generated each with a unique ubiquitous promoter that drives expression of tTA. We exhaustively examined the repressive ability of GAL80, using both UAS-grim and UAS-lacZ reporters, finding that the best repression was achieved in genotypes with four copies and both versions of the transgene. Induction was observed in tetracycline treated animals but was found to be dependent on sex, age, and the anatomical location of expression. In this study the repressive ability of GAL80 is affected by age which limits its use to regulate expression in adult animals. The Lac Inducible UAS/GAL4 System was designed to regulate the UAS promoter by incorporating lac operator (LacO) sequences to which LacI could bind and repress UAS transcriptional activity. Two different versions of the modified LacO-UAS were built previously: pHN1 (2 operators) and pHN4 (3 operators). Here the influence of the addition of the lac operators is studied and found to negatively affect UAS transcriptional activity through age, independent of LacI.
Bio-Rad qPCR Seminar- Training: Are you MIQE Compliant?
The MIQE guidelines have helped standardize the way research projects using qPCR are performed. qPCR is the gold standard technique for quantifying levels of RNA and DNA between samples. However, there remains a knowledge gap with respect to the application of rigorous methodology for each step of qPCR to assure reproducible results with accurate conclusions. Proper primer design and validation must be performed and the correct references genes must be selected. The final analysis of the resulting data is not trivial and there are wide variations between labs who typically use EXCEL spreadsheets created by individuals who may or may not understand how to convert the raw data to normalize results to statistical means, standard deviations and their significance with p-values. Tuesday Sept. 12 11:30-12:30 BioSci Rm. 3110 Software training- 1:00-3:00 pm BioSci Rm. 3110