Metabolite profiling of susceptible and resistant wheat (Triticum aestivum) cultivars responding to Puccinia striiformis f. sp. tritici infection.

BACKGROUND: Puccinia striiformis f. sp. tritici (Pst) is an economically devasting disease that is prominent in cereal crops such as wheat (Triticum aestivum). The fungal pathogen can cause approximately 30-70% losses in crop productivity and yields. Pst has become difficult to manage due to its ease of transmission through wind dispersal over long distances, and intercontinental dispersal has been previously reported. The ease of transmission has resulted in further destruction because of new and more virulent strains infecting crops previously resistant to a different strain. RESULTS: In this study, a liquid chromatography-mass spectrometry-based untargeted metabolomics approach, in combination with multivariate data analytical tools, was used to elucidate the mechanistic nature of the defence systems of a Pst-resistant and a susceptible wheat cultivar infected with P. striiformis. We also investigated the time-dependant metabolic reconfiguration of infected plants over a four-week period. The untargeted metabolomic analysis revealed a time-course metabolic reprogramming involving phenylpropanoids (majority flavonoids), amino acids, lipids, benzoic acids, TCA cycle intermediates and benzoxazinoids responding to Pst infection. Interestingly, the results do not show a linear course for the decrease and increase (up-/down-regulation) of said classes of metabolites, but rather the up- or down-regulation of specific metabolites in response to the pathogen infection. The resistant Koonap cultivar had an abundance of phenolic compounds such as rutin, isoorintin-7-O-glucoside and luteolin-6-C-hexoside-O-hexoside. These compounds showed a decrease over time in control Koonap plants compared to an increase in Pst-infected plants. These metabolites were down-regulated in the susceptible Gariep cultivar, which could serve as biomarkers for plant responses to biotic stress and resistance against Pst. CONCLUSIONS: Overall, an LC-MS-based metabolomics approach allowed for the metabolic profiling and analysis of the impact of plant-pathogen interactions on the overall plant metabolome and provided a real-time snapshot of the differential significant metabolic perturbations occurring in wheat plants responding to the Pst pathogen. The Pst-resistant Koonap cultivar showed a rapid accumulation of defence metabolites in response to pathogen infection compared to the susceptible Gariep cultivar. These findings provide insight into the mechanistic biochemical nature of plant-microbe interactions and the prospects of metabolic engineering for improved plant tolerance and resistance to biotic stresses.

Genetic determinant of Bacillus pumilus lipase lethality and its application as positive selection cloning vector in Escherichia coli.

Positive selection vectors carry genes that upon expression produce proteins that cause host cell deaths. Insertion of foreign DNA fragments within the ORF of the gene disrupts the lethal effect of the expressed protein. This study described the cloning of Family I.4 Bacillus pumilus lipase gene whose expressed protein is toxic and lethal to Escherichia coli JM109 (DE3) cells. The determinant of toxicity was identified through Error-prone PCR to be the nature of amino acid residue resident at position 28 of the mature lipase protein. The presence of Thr/Ser28 within the mature lipases of B. pumilus and B. licheniformis resulted in lethality to E. coli cells. However, the Thr28Ala or Thr28Gly mutations relieved the lethal phenotype of mature Family I.4 Bacillus lipases. The toxic effect of the expressed mature B. pumilus lipase protein was exploited in the development of a positive selection cloning vector. The B. pumilus lipase gene was synthesised to contain 13 unique silent restriction sites within the ORF, and placed under the regulation of T7 promoter of the pET expression system. Insertional inactivation of the gene's toxic protein was achieved by cloning DNA fragments of different sizes within the designed multiple cloning sites. The toxic effect of the lipase protein was disrupted indicating the potential of the gene for application in suicidal positive selection cloning vectors. The results revealed that protein expression and engineering studies aimed at optimal production of mature Family I.4 Bacillus lipases in E. coli should take into consideration the nature of amino acid 28 resident.

Reduction of U(VI) by the deep subsurface bacterium, Thermus scotoductus SA-01, and the involvement of the ABC transporter protein.

In this study we investigated the effect of uranium on the growth of the bacterium Thermus scotoductus strain SA-01 as well as the whole cell U(VI) reduction capabilities of the organism. Also, site-directed mutagenesis confirmed the identity of a protein capable of a possible alternative mechanism of U(VI) reduction. SA-01 can grow aerobically in up to 1.25 mM uranium and has the capability to reduce low levels of U(VI) in under 20 h. TEM analysis performed on cells exposed to uranium showed extracellular and membrane-bound accumulation of uranium. The reductase-like protein was surprisingly identified as a peptide ABC transporter, peptide-binding protein. This study showcases the concept of protein promiscuity, where this protein with a distinct function in situ can also have the unintended function of a reactant for the reduction of U(VI).

A novel chromate reductase from Thermus scotoductus SA-01 related to old yellow enzyme.

Bacteria can reduce toxic and carcinogenic Cr(VI) to insoluble and less toxic Cr(III). Thermus scotoductus SA-01, a South African gold mine isolate, has been shown to be able to reduce a variety of metals, including Cr(VI). Here we report the purification to homogeneity and characterization of a novel chromate reductase. The oxidoreductase is a homodimeric protein, with a monomer molecular mass of approximately 36 kDa, containing a noncovalently bound flavin mononucleotide cofactor. The chromate reductase is optimally active at a pH of 6.3 and at 65 degrees C and requires Ca(2+) or Mg(2+) for activity. Enzyme activity was also dependent on NADH or NADPH, with a preference for NADPH, coupling the oxidation of approximately 2 and 1.5 mol NAD(P)H to the reduction of 1 mol Cr(VI) under aerobic and anaerobic conditions, respectively. The K(m) values for Cr(VI) reduction were 3.5 and 8.4 microM for utilizing NADH and NADPH as electron donors, respectively, with corresponding V(max) values of 6.2 and 16.0 micromol min(-1) mg(-1). The catalytic efficiency (k(cat)/K(m)) of chromate reduction was 1.14 x 10(6) M(-1) s(-1), which was >50-fold more efficient than that of the quinone reductases and >180-fold more efficient than that of the nitroreductases able to reduce Cr(VI). The chromate reductase was identified to be encoded by an open reading frame of 1,050 bp, encoding a single protein of 38 kDa under the regulation of an Escherichia coli sigma(70)-like promoter. Sequence analysis shows the chromate reductase to be related to the old yellow enzyme family, in particular the xenobiotic reductases involved in the oxidative stress response.

Identification of a lipopolysaccharide responsive erk-like MAP kinase in tobacco leaf tissue.

SUMMARY Lipopolysaccharides (LPS) are indispensable cell surface components of Gram-negative bacteria and have diverse roles in plant-microbe interactions. Treatment of Nicotiana tabacum with the LPS of an endophytic strain of Burkholderia cepacia results in an enhanced defensive capacity in the tissue. In this study the rapid and transient phosphorylation of an extracellular signal-regulated (ERK)-like mitogen-activated protein (MAP) kinase in response to LPS from B. cepacia (LPS(B.cep.)) elicitation is reported. Based on in-gel kinase assays it was found that this 43-kDa LPS(B.cep.)-responsive kinase is optimally activated following 7 min elicitation with 100 microg/mL LPS. Its identity as an ERK MAPK was supported by tyrosine-phosphorylated association with induction, immunodetection with pTEpY-specific MAPK antibodies and inhibition of phosphorylation by U0126, an upstream MAPKK inhibitor. The kinase utilized myelin basic protein, but not casein or histone, as substrate. Ca(2+) did not modulate the phosphorylation, nor did wounding. To date, other MAP kinases have been shown to act either independently or upstream from reactive oxygen intermediates produced during the oxidative burst. It was found that hydrogen peroxide is either not generated in leaf tissue in response to LPS elicitation or, if generated, does not trigger the phosphorylation of the kinase. Physicochemical characterization of the ERK-like MAPK indicated a molecular mass of 43 kDa and a pI of 6.3; two-dimensional gel analysis indicated two charge isomers. This is the first demonstration of such an LPS-responsive MAP kinase phosphorylation in plants.