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A light-dependent selection marker system in plants Ingyu Hwang and Jae Sun Moon Seoul National University Korea Research Institute of Bioscience and Biotechnology January 15, 2012 PAG meeting Burkholderia glumae: The causal agent of rice panicle blight (bacterial rice grain rot) and bacterial wilt Burkholderia glumae • Infects roots, stems, and flowers. • Produces toxoflavin and N-octanoyl-L-homoserine lactone. • Optimum growth temperature is 37oC. • Possesses 2-4 polar flagella. C4 CH3 N Toxoflavin C6 C8 syn thet ic pur ified O 365 nm N N N H3C N O CH3 N O N N N H3C N O O H N O N N H O N H N H3C O N-octanoyl-L-homoserine lactone (C8-HSL) N O Toxoflavin is a photosensitizer. Production of hydrogen peroxide H2O2 light O2 Reduction O MeN 5 O N O Oxidation N MeN 4 8 1 N N Me Toxoflavin Oxidized type Reduction H R C O H N 4 8 N H N N Me 4,8-Dihydrotoxoflavin Reduced type Oxidation OH H Alcohols Substrate R CHO Aldehydes H2 Substrate T. Nagamatsu, Y. Hashiguchi, Y. Sakuma, and F. Yoneda, Chem. Lett., 1982, 1309. Toxoflavin is a key pathogenicity factor. Disease severity 5 5 3.5 40 μg/ml 20 μg/ml 10 μg/ml 2 5 μg/ml 0.6 0 2.5 μg/ml SDW Milyang 23 10 days after treatment Regulation of toxoflavin biosynthesis by quorum sensing Practical Application: Toxin degradation 1. 2. Developing disease resistant lines A novel selection marker for plant transformation Kim J. et. al. Mol. Microbiol. 54: 921-934 (2004) Isolation and identification of toxoflavin-degrading bacteria from natural environment Plant material LB plate (40 μg/ml toxoflavin) Soil Other sources incubation Rice seedling Identification: 16s rDNA sequencing FAME analysis Biolog analysis Pure culture of survivors Isolation of Paenibacillus polymyxa strain JH2 from rice seeds Deletion analysis of pJ9 Toxoflavin degradation activiy 1kb pJ9 pJ91 pJ90 (E) H H H B E B E B H HE (H) H H E H H tflA 1kb + + + E. coli DH5α carrying pJ9 degrades toxoflavin. 2 JH 5 DH α a 1) n ) ) 9) 1 J 4 yx 6 J J J vi (p (p a (p (p ym l l α 5 5α xof 5α 5α po H H . H H to D D P D D UV 265 nm Toxoflavin After 16 hr in LB broth (Toxoflavin 40 μg/ml) TflA is similar to ring-cleavage extradiol dioxygenases. 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Degradation of toxoflavin by TflA depends on Mn++ and DTT. 100µM toxoflavin plus 1: CK (10µM MnCl2) 2: Purified His-TflA (10µM MnCl2) 3: CK (5mM DTT) 4: Purified His-TflA (5mM DTT) 5: CK (5mM DTT/10µM MnCl2) 6: Purified His-TflA (5mM DTT/10µM MnCl2) 1 2 3 4 5 6 Comparison of TflA with other extradiol dioxygenases TflA NahC NahH ThnC BphC Specific activity (units/mg) Metal 0.04 MnCl2 25oC MnCl2 25oC FeCl2 25oC FeCl2 30oC MnCl2 25oC 1.5 7 2 0.65 Temperature pH KM Substrate (μM) 6.5 7.5 7.5 6.8 8.0 69.72 Toxoflavin 400 2,3dihydroxybiphenyl 0.25 2,3dihydroxybiphenyl 18.6 1,2dihydroxybiphenyl 7 2,3dihydroxybiphenyl Purified TflA degrades toxoflavin derivatives. Toxoflavin 3-Methyl toxoflavin 3-Phenyl toxoflavin 4,8-Dihydro toxoflavin 3-Methyl 4,8-Dihydro toxoflavin +++ +++ _ Reumycin 3-Methyl reumycin 3-Phenyl reumycin + +++ _ +++ Fervenulin + ++ 5-Deazaflavin _ Degradation activity:-0~20% degradation; +,20~50%; ++, 50~80%; +++, 80~100% Toxoflavin derivatives with phenyl group on 3 carbon were not degraded by TflA. O O CH3 H3C N H3C N O N N H N H N H N H3C N H 3-Methyltoxoflavin N N N N H O O N O O H3C N N 3 N N N N O CH3 N O N H N H3C 5-Deazaflavin O N Reumycin CH3 N H3C N N N N N CH3 O N N H N Toxoflavin O O H3C CH3 3-Phenyltoxoflavin H3C H N N N O N O N Fervenulin 3'-Methyl 4,8-Dihydrotoxoflavin O H3C N CH 3 CH3 4,8-Dihydrotoxoflavin N H H N N CH3 CH3 H3C CH3 N N N O O O N 3-Methylreumycin N O N H N 3-Phenylreumycin Extradiol dioxygenases ThnC TflA O OH N H3C H N N O N Mn2+, DTT N ? O O2 COOH OH Fe2+ 1,2-dihydroxytetralin CH3 Toxoflavin CM-2 COO- OH BphC COO- O2 O2 CHO Mn2+ COOH OH OH 3,4-dihydroxyphenylacetate OH Mn2+ OH OH 2,3-dihydroxybiphenyl O COOH OH Nature Biotechnology (2006) 20:575-580 Generation of transgenic rice plants expressing tflA XhoI XhoI SacI XbaI HindIII LB RB HygromycinR NOS 35S tflA MCS 35S genomic DNA genomic DNA Fig. 1. Genetic construction of pJ904(pCamLA::tflA). A B D I E C F G H Shoot Root Dt 1-2 T4 plants AT rich repeat region 10,021bp 3,404bp 32003573~32003575 (Chromosome 3) Muscle M-line assembly protein unc-89 (putative gypsy-type retrotransposon protein) Hypothetical protein L B water Dongjin wild-type T-DNA Dt1-2 water Dongjin wild-type Dt1-2 0 0.6 3.7 Disease degree (1~5) 0.1 10 20 30 40 50 (%) 60 0 Healthy panicle 1 Panicle 0-20% discolored 2 Panicle 21-40% discolored 3 Panicle 41-60% discolored 4 Panicle 61-80% discolored 5 Panicle 81-100% discolored 70 80 90 100 Comparison of selection markers in rice and Arabidopsis Koh et.al. Plant Biotech. J. (2010), pp.1-11 Efficiency of rice transformation Marker pCamLA (hpt) No. of calli inoculated No. of resistant calli No. of putative regenerated plants PCR positive independent plantsa Transformation frequency (%) b 300 80 20 16 80 300 134 43 38 88.37 300 84 28 25 89.28 300 192 26 21 80.76 pTflA a Independent plants : plants coming from different calli b Number of PCR-positive plants/number of putative regenerated plants Seed selection with toxoflavin (7mg/L) Dongjin wild-type (Hygromycin;30mg/L) Dongjin wild-type Dt27-4-1 Dt28-2-5 The photograph was taken 10 days after seeding. Dt7-3-5 Dt40-9-3 Spray screening of transgenic rice plants WT Dt2-7-2 Dt4-1-4 Toxoflavin (mg/L) 0 10 50 The photograph was taken after 48h after spray. Dt27-4-1 Dt40-9-1 Light-dependent sensitivity of various crops to toxoflavin Light Toxoflavin (mg/L) 0 1 2 4 6 8 10 20 Dark Toxoflavin (mg/L) 0 1 2 4 6 8 10 20 Rice Barley Corn Arabidopsis Hot pepper Soybean Tomato Tobacco Cucumber Watermelon Melon Zucchini Koh et.al. Plant Biotech. J. (2010), pp.1-11 Acknowledgments • Graduate Students Hongsup Kim, Eunhye Goo • Former Students and Postdocs Jinwoo Kim, Okhee Choi, Yun-Jung Kim • Funding: - Crop Functional Genomic Center (MEST) - The Creative Research Initiatives (NRF) • Collaborators - Tomohisa Nagamatsu (Okayama Univ.) - Jae Sun Moon, Serry Koh (KRIBB) - Nam-Soo Jwa (Sejong Univ.)