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Now showing items 1 - 4 of 4

  • Flaxseed Oil for Control of Peanut Rust and Its Disease Control Mechanism

    Chen, Wen-Yang   Ko, Wen-Hsiung  

    Severity of peanut rust caused by Puccinia arachidis was reduced by 15 edible oils tested. Flaxseed oil was the best suppressing the disease completely. Peanut oil, wheat germ oil, brown rice oil, aloe oil, olive oil and corn germ oil also caused more than 75% reduction in disease incidence. Flaxseed oil reduced the rust to a negligible level in the greenhouse and was nearly as effective as the fungicide chlorothalonil in peanut field trials. The control of peanut rust by flaxseed oil did not result from activation of the host defence mechanisms. Flaxseed oil did not affect urediniospore germination, but reduced the germ tube length and completely suppressed appressorium formation which is essential for the pathogen to form an infection peg to pass through the stomatal aperture and infect the host tissue. Although the pathogen had penetrated, flaxseed oil still exerted some inhibitory effect against the growth of the pathogen. The advantages of using flaxseed oil to control peanut rust are that it is relatively inexpensive, easy to prepare, and friendly to the environment and human health.
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  • The Two PPX-GppA Homologues from Mycobacterium tuberculosis Have Distinct Biochemical Activities

    Choi, Mei Y.   Wang, Ying   Wong, Leo L. Y.   Lu, Bing-tai   Chen, Wen-yang   Huang, Jian-Dong   Tanner, Julian A.   Watt, Rory M.  

    Inorganic polyphosphate (poly-P), guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) are ubiquitous in bacteria. These molecules play a variety of important physiological roles associated with stress resistance, persistence, and virulence. In the bacterial pathogen Mycobacterium tuberculosis, the identities of the proteins responsible for the metabolism of polyphosphate and (p) ppGpp remain to be fully established. M. tuberculosis encodes two PPX-GppA homologues, Rv0496 (MTB-PPX1) and Rv1026, which share significant sequence similarity with bacterial exopolyphosphatase (PPX) and guanosine pentaphosphate 5'-phosphohydrolase (GPP) proteins. Here we delineate the respective biochemical activities of the Rv0496 and Rv1026 proteins and benchmark these against the activities of the PPX and GPP proteins from Escherichia coli. We demonstrate that Rv0496 functions as an exopolyphosphatase, showing a distinct preference for relatively short-chain poly-P substrates. In contrast, Rv1026 has no detectable exopolyphosphatase activities. Analogous to the E. coli PPX and GPP enzymes, the exopolyphosphatase activities of Rv0496 are inhibited by pppGpp and, to a lesser extent, by ppGpp alarmones, which are produced during the bacterial stringent response. However, neither Rv0496 nor Rv1026 have the ability to hydrolyze pppGpp to ppGpp; a reaction catalyzed by E. coli PPX and GPP. Both the Rv0496 and Rv1026 proteins have modest ATPase and to a lesser extent ADPase activities. pppGpp alarmones inhibit the ATPase activities of Rv1026 and, to a lesser extent, the ATPase activities of Rv0496. We conclude that PPX-GppA family proteins may not possess all the catalytic activities implied by their name and may play distinct biochemical roles involved in polyphosphate and (p) ppGpp metabolic pathways.
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  • Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    Yang, Wen   Chen, Wen-yang   Wang, Hui   Ho, John W. S.   Huang, Jian-Dong   Woo, Patrick C. Y.   Lau, Susanna K. P.   Yuen, Kwok-Yung   Zhang, Qionglin   Zhou, Weihong   Bartlam, Mark   Watt, Rory M.   Rao, Zihe  

    Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced beta-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5'-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg(2+) or Mn(2+) ions. 5'-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 A, revealing a 'doughnut-shaped' toroidal trimeric arrangement with a central tapered channel, analogous to that of lambda-exonuclease (Exo) from bacteriophage-lambda. Active sites containing two bound Mg(2+) ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.
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  • Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae RID B-2177-2009

    Chen, Wen-yang   Ho, John W. S.   Huang, Jian-dong   Watt, Rory M.  

    Background: SXT is an integrating conjugative element (ICE) originally isolated from Vibrio cholerae, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100 kb circular double stranded DNA (dsDNA) genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo) and single strand annealing protein (S065, SXT-Bet) encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively. Results: SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA) molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn(2+) or Mg(2+) ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5' to 3' polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb). When co-expressed in E. coli cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo. Conclusions: The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected V. cholerae cells, through facilitating homologous DNA recombination events. The results presented here significantly extend our general understanding of the properties and activities of alkaline exonuclease and single strand annealing proteins of viral/bacteriophage origin, and will assist the rational development of bacterial recombineering systems.
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