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Genetic Analysis of Tox1

Genetic Analysis of Tox1. COCHLIOBOLUS HETEROSTROPHUS. CORN N T. race O ( Tox - ). +. +. X. race T ( Tox + ). +. +++. 1 : 1. Tox1. Tox1 : a single genetic locus. (Yoder and Gracen, 1975). OH. OH. OH. OH. OH. O. O. O. O. O. O. O. O. C 41. C 39. OH.

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Genetic Analysis of Tox1

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  1. Genetic Analysis of Tox1

  2. COCHLIOBOLUS HETEROSTROPHUS CORN N T race O (Tox-) + + X race T (Tox+) + +++ 1 : 1 Tox1 Tox1: a single genetic locus (Yoder and Gracen, 1975)

  3. OH OH OH OH OH O O O O O O O O C41 C39 OH OH OH OH OH O O O O O O O C35 OH OH OH OH O O O O O O Race T produces a host selective toxin, T-toxin 1971 Discovery of T-toxin in culture filtrates (Lim & Hooker; Gracen et al)

  4. race O (Tox -) race T (Tox +) race O (Tox -) race T (Tox +) O O OH OH O O O O Mitochondrial inner membrane URF13 protein 600 m.w. N-cytoplasm corn T-cytoplasm corn

  5. RFLP map of Cochliobolus heterostrophus (Tzeng, et al, 1992, Genetics 130:81; Tasma and Bronson, 1998, Current Genetics 34:227).

  6. Reciprocal translocation 6;12 12;6 6 12 Race O Chromosomes Race T Chromosomes

  7. RFLP map of Cochliobolus heterostrophus Tox1 (Tzeng, et al, 1992, Genetics 130:81; Tasma and Bronson, 1998, Current Genetics 34:227).

  8. Generation of race T mutants deficient in ability to produce T-toxin

  9. tox1- mutants (Ctm45, REMI186, 350) tox1- mutant (PKS13) tox1- mutants X X X tox1- mutants (Ctm45, REMI186, 350) Race O tox1- mutants (Ctm45, REMI186, 350) all tox- 25% Tox+ all tox- Tox1A(Ctm45, REMI186, 350) Tox1 Tox1B(PKS13) Crosses with tox- mutants Tox1: two unlinked genetic loci PKS13 is unlinked to other mutants All map at Tox1 All are linked (G. Yang; M. Rose)

  10. 12;6 T 12 O race O race T Tox1A Tox1- Tox1+ R.C4.186 R.C4.350 Tox1B C4.PKS.13 T O Tox1 6 T O 1.2 Mb ‘extra’ DNA in race T 12;6 6;12 12 O 6 T 12;6 6 + 12 (3.7 Mb) 12;6 + 6;12 (4.9 Mb) < (Tzeng et al, 1992) Tox1 is two loci: physical evidence (Kodama et al, 1999)

  11. 1.2 Mb DNA Tox1B Tox1A Insertion Reciprocal translocation 6 12 6;12 12;6 6 12 Race O Chromosomes Hypothetical Intermediate Race T Chromosomes Tentative Model for Evolution of Race T from Race O

  12. Identification of Tox1 genes

  13. Identification of Tox1 genes • Restriction Enzyme-Mediated Integration (REMI) mutagenesis: PKS1 (Yang et al., 1996) • PKS1 = polyketide synthase • PCR amplification of conserved PKS acyl transferase (AT) domains from race T; use products to knockout PKSs in race T: DEC1 and RED1(Rose et al., 2002) • DEC1 = decarboxylase • RED1 = medium chain reductase/dehydrogenase • Subtractive AFLP/cDNA fingerprinting technique: PKS2 (Li et al, 2002; Baker et al., in preparation) • PKS2 = polyketide synthase Ge Yang Mark Rose Screen for changes in T-toxin production

  14. Tox1 genes are unique to race T C. heterostrophus race O C. heterostrophus race T C. carbonum C. victoriae C. sativus C. spicifer Probe  PKS1 9.4- 23.1- DEC1 RED1 2.3- Ge Yang Mark Rose

  15. Identification of Tox1 genes by genome sequence analysis

  16. shear and clone entire genome sequence ends assemble sequences contigs link contigs meta contig/scaffold “Shotgun” Approach to Sequencing the Cochliobolus Genome 35 Mb genomic DNA Torrey Mesa Research Institute/Syngenta

  17. These scaffolds were the smallest in the C. heterostrophus genome assembly • Genes embedded in highly repeated A+T rich DNA Identification of Tox1 genes by Genome Sequence Analysis • Query of the C. heterostrophus genome sequence database with four known Tox1 genes (PKS1, PKS2, DEC1, and RED1) • These genes mapped to three scaffolds: • 4FP (carrying PKS1) • 4LU (carrying PKS2) • 3PL (carrying DEC1 and RED1)

  18. 64 %AT 74 50 78 71 20 3616 2568 1119 PKS1 4,081 13,218 C. heterostrophus Scaffold 4FP 23,099 contig 1 contig 2 contig 3 contig 4 contig 5 1 4,287 23,099 4,307 10,089 13,706 14,908 14,477 18,634 19,754 Scaffold 4FP is at Tox1A PKS1 = polyketide synthase

  19. C. heterostrophus Scaffold 4LU 30,397 contig 1 contig 2 contig 3 contig 4 contig 5 contig 6 contig 7 76 60 57 58 52 50 45 64 73 %AT 259 176 728 405 1076 8497 1 4,038 4,297 5,753 5,930 7,293 8,022 10,410 10,816 12,290 13,367 19,622 28,120 30,397 9,360 15,974 LAM1 PKS2 LAM1 = lambda crystalline = 3-hydroxyacyl CoA dehydrogenase Scaffold 4LU is at Tox1A PKS2 = polyketide synthase

  20. C. heterostrophus Scaffold 3PL 43,533 contig 1 contig 2 contig 3 contig 4 contig 5 contig 6 contig 7 %AT 76 69 56 62 58 72 74 73 72 53 60 61 63 495 2,328 20 20 8,722 7,667 1 2,483 2,978 5,487 7,816 14,591 14,612 17,169 17,190 18,225 26,948 31,272 38,940 43,533 3,850 4,618 9,874 11,006 14,655 15,461 DEC1 RED2 RED3 RED1 in gap RED2, RED3 = short chain reductase/dehydrogenase RED1 = medium chain reductase/dehydrogenase Scaffold 3PL is at Tox1B DEC1 = decarboxylase

  21. PKS1 Tox1A LAM1 PKS2 DEC1 RED1 RED2 RED3 LAM1 = 3-hydroxyacyl CoA dehydrogenase RED2, RED3 = short chain reductase/dehydrogenase Organization of Genes for T-toxin Biosynthesis at Tox1 Tox1B PKS1, PKS2 = polyketide synthase DEC1 = decarboxylase RED1 = medium chain reductase/dehydrogenase

  22. Functional analysis of new Tox1 genes

  23. C. heterostrophus Scaffold 4LU 30,397 contig 1 contig 2 contig 3 contig 4 contig 5 contig 6 contig 7 1 4,038 4,297 5,753 5,930 7,293 8,022 10,410 10,816 12,290 13,367 19,622 28,120 30,397 9,360 15,974 LAM1 PKS2 PKS2 = polyketide synthase LAM1 = 3-hydroxyacyl CoA dehydrogenase

  24. LAM1 is required for wild type T-toxin production Phenotypes of lam1 Mutants C4 (tox+) lam1 lam1 C5 (tox -)

  25. C4 lam1 lam1 C5 C4 (Tox+) lam1 mutant lam1 mutant water T cytoplasm corn Phenotypes of lam1 Mutants LAM1 is required for high virulence on T-cytoplasm corn

  26. contig 1 contig 2 contig 3 contig 4 contig 5 contig 6 contig 7 1 2,483 2,978 5,487 7,816 14,591 14,612 17,169 17,190 18,225 26,948 31,272 38,940 43,533 3,850 4,618 9,874 11,006 14,655 15,461 DEC1 RED2 RED3 6,170 1,5111 C. heterostrophus Scaffold 3PL 43,533 RED1 in gap 6,140 7,199 DEC1 = decarboxylase RED1 = medium chain reductase/dehydrogenase RED2, RED3 = short chain reductase/dehydrogenase

  27. RED1, RED2 and RED3 are not required for T-toxin production Phenotypes of red1-red2-red3 Triple Deletion Mutants C4 (Tox+) triple red C4 C5 (tox-) C5

  28. Phenotypes of red1 and dec1 Mutants C4 (Tox+) C5 (tox-) dec1 red1 RED1 is not required for T-toxin production

  29. PKS1 Tox1A PKS2 LAM1 DEC1 RED1 RED2 RED3 Tox1B PKS1, PKS2 = polyketide synthase LAM1 = 3-hydroxyacyl CoA dehydrogenase DEC1 = decarboxylase RED1 = medium chain reductase/dehydrogenase RED2, RED3 = short chain reductase/dehydrogenase Organization of Genes for T-toxin Biosynthesis at Tox1

  30. Are there additional PKSs at Tox1 ? • 25 PKSs in C. heterostrophus, including PKS1 and PKS2 • Screen for PKSs that are only in race T to find PKSs at Tox1

  31. raceT/raceO raceT/raceO PKS1 +/- PKS14 +/+ PKS2 +/- PKS15 +/+ PKS3 +/+ PKS16 +/+ PKS4 +/+ PKS17 +/+ PKS5 +/+ PKS18 +/+ PKS6 +/+ PKS19 +/+ PKS7 +/+ PKS20 +/+ PKS8 +/+ PKS21 +/+ PKS9 +/+ PKS22 +/+ PKS10 +/+ PKS23 +/+ PKS12 +/+ PKS24 +/+ PKS13 +/+ PKS25 +/+ Presence (+)/Absence (-) in race T and race O

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