Screening of Mutant Arabidopsis Thaliana and Chlamydomonas Reinhardtii for their Potential Use as Phytosensors in 2,4,6 Trinitrotoluene (TNT) Contaminated Environments

Plant biotechnology is a diverse field that is expanding from agricultural research towards environmental applications. The focus of this project was to exploit vegetative effects, such as photosynthesis and growth in genomic model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii to 2,4,6-trinitrotoluene (TNT) with a goal to develop biomonitoring systems. Plants and algae have evolved with various biochemical pathways that have the potential to be exploited for the use of sensing explosives and chemical warfare agents in soil, water and air. The first part of the project involved characterizing the effects of TNT on germination and early seedling development of wild-type Arabidopsis thaliana. It was determined that 10 mM TNT was the tolerance level for Arabidopsis and was used to screen fast neutron irradiated mutant Arabidopsis to evaluate the phenotypic stress responses in the seedlings. TNT responsive mutant lines (lines 1, 2, 3, and 4) were selected on a basis of a leaf color change from dark green to pale green. The second part of the project was to determine the growth response of wild-type and mutant Chlamydomonas reinhardtii to TNT. Growth response studies of wild-type Chlamydomonas revealed that 3 mg/ml of TNT was the maximum TNT concentration that allowed growth. Insertional mutant lines were screened on 3 mg/ml TNT where one mutant (CL48) was selected on the basis of a color change from green to white. Growth response of CL48 in TNT indicated that this mutant line was hypersensitive to TNT compared with transformation recipient line and wild-type Chlamydomonas. The third part of the project involved using microarray technology to determine the differential gene expression of Chlamydomonas in response to TNT. Approximately 158 responsive genes were differentially expressed. Genes involved in photosynthesis and energy metabolism were up-regulated in the presence of TNT. TNT may cause oxidative stress since many oxidative stress related genes were up-regulated. Among the down-regulated genes, the expression of cell wall-related genes was repressed. Several unidentified genes were also induced or repressed. The overall study promotes future work involving the identification of the genes that are involved in TNT response

Use of the tetrazolium salt MTT to measure cell viability effects of the bacterial antagonist Lysobacter enzymogenes on the filamentous fungus Cryphonectria parasitica

Despite substantial interest investigating bacterial mechanisms of fungal growth inhibition, there are few methods available that quantify fungal cell death during direct interactions with bacteria. Here we describe an in vitro cell suspension assay using the tetrazolium salt MTT as a viability stain to assess direct effects of the bacterial antagonist Lysobacter enzymogenes on hyphal cells of the filamentous fungus Cryphonectria parasitica. The effects of bacterial cell density, fungal age and the physiological state of fungal mycelia on fungal cell viability were evaluated. As expected, increased bacterial cell density correlated with reduced fungal cell viability over time. Bacterial effects on fungal cell viability were influenced by both age and physiological state of the fungal mycelium. Cells obtained from 1-week-old mycelia lost viability faster compared with those from 2-week-old mycelia. Likewise, hyphal cells obtained from the lower layer of the mycelial pellicle lost viability more quickly compared with cells from the upper layer of the mycelial pellicle. Fungal cell viability was compared between interactions with L. enzymogenes wildtype strain C3 and a mutant strain, DCA, which was previously demonstrated to lack in vitro antifungal activity. Addition of antibiotics eliminated contributions to MTT-formazan production by bacterial cells, but not by fungal cells, demonstrating that mutant strain DCA had lost complete capacity to reduce fungal cell viability. These results indicate this cell suspension assay can be used to quantify bacterial effects on fungal cells, thus providing a reliable method to differentiate strains during bacterial/fungal interactions

A nematode effector protein similar to annexins in host plants

Nematode parasitism genes encode secreted effector proteins that play a role in host infection. A homologue of the expressed Hg4F01 gene of the root-parasitic soybean cyst nematode, Heterodera glycines, encoding an annexin-like effector, was isolated in the related Heterodera schachtii to facilitate use of Arabidopsis thaliana as a model host. Hs4F01 and its protein product were exclusively expressed within the dorsal oesophageal gland secretory cell in the parasitic stages of H. schachtii. Hs4F01 had a 41% predicted amino acid sequence identity to the nex-1 annexin of C. elegans and 33% identity to annexin-1 (annAt1) of Arabidopsis, it contained four conserved domains typical of the annexin family of calcium and phospholipid binding proteins, and it had a predicted signal peptide for secretion that was present in nematode annexins of only Heterodera spp. Constitutive expression of Hs4F01 in wild-type Arabidopsis promoted hyper-susceptibility to H. schachtii infection. Complementation of an AnnAt1 mutant by constitutive expression of Hs4F01reverted mutant sensitivity to 75mM NaCl, suggesting a similar function of the Hs4F01 annexin-like effector in the stress response by plant cells. Yeast two-hybrid assays confirmed a specific interaction between Hs4F01 and an Arabidopsis oxidoreductase member of the 2OG-Fe(II) oxygenase family, a type of plant enzyme demonstrated to promote susceptibility to oomycete pathogens. RNA interference assays that expressed double-stranded RNA complementary to Hs4F01 in transgenic Arabidopsis specifically decreased parasitic nematode Hs4F01 transcript levels and significantly reduced nematode infection levels. The combined data suggest that nematode secretion of an Hs4F01 annexin-like effector into host root cells may mimic plant annexin function during the parasitic interaction.This article is published as Patel, Nrupali, Noureddine Hamamouch, Chunying Li, Tarek Hewezi, Richard S. Hussey, Thomas J. Baum, Melissa G. Mitchum, and Eric L. Davis. "A nematode effector protein similar to annexins in host plants." Journal of Experimental Botany 61, no. 1 (2009): 235-248, doi: 10.1093/jxb/erp293. Posted with permission.</p

Comparative genomics and metabolic profiling of the genus Lysobacter

Background: Lysobacter species are Gram-negative bacteria widely distributed in soil, plant and freshwater habitats. Lysobacter owes its name to the lytic effects on other microorganisms. To better understand their ecology and interactions with other (micro)organisms, five Lysobacter strains representing the four species L. enzymogenes, L. capsici, L. gummosus and L. antibioticus were subjected to genomics and metabolomics analyses. Results: Comparative genomics revealed a diverse genome content among the Lysobacter species with a core genome of 2,891 and a pangenome of 10,028 coding sequences. Genes encoding type I, II, III, IV, V secretion systems and type IV pili were highly conserved in all five genomes, whereas type VI secretion systems were only found in L. enzymogenes and L. gummosus. Genes encoding components of the flagellar apparatus were absent in the two sequenced L. antibioticus strains. The genomes contained a large number of genes encoding extracellular enzymes including chitinases, glucanases and peptidases. Various nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) gene clusters encoding putative bioactive metabolites were identified but only few of these clusters were shared between the different species. Metabolic profiling by imaging mass spectrometry complemented, in part, the in silico genome analyses and allowed visualisation of the spatial distribution patterns of several secondary metabolites produced by or induced in Lysobacter species during interactions with the soil-borne fungus Rhizoctonia solani. Conclusions: Our work shows that mining the genomes of Lysobacter species in combination with metabolic profiling provides novel insights into the genomic and metabolic potential of this widely distributed but understudied and versatile bacterial genus