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The plastid clpP1 protease gene is essential for plant development Hiroshi Kuroda & Pal Maliga

The plastid clpP1 protease gene is essential for plant development Hiroshi Kuroda & Pal Maliga Nature 425 , 86 – 89. Carrie Brubaker, Michelle Gonzales, Tomo Kawashima, Cynthia Lee, Kumi Sakurai, and Julie Williams Chemistry 161/261A April 22, 2004. Plastids of Higher Plants.

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The plastid clpP1 protease gene is essential for plant development Hiroshi Kuroda & Pal Maliga

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  1. The plastid clpP1 protease gene is essential for plant development Hiroshi Kuroda & Pal Maliga Nature425, 86 – 89 Carrie Brubaker, Michelle Gonzales, Tomo Kawashima, Cynthia Lee, Kumi Sakurai, and Julie Williams Chemistry 161/261A April 22, 2004

  2. Plastids of Higher Plants • semiautonomous organelles • chloroplasts, amyloplasts, leucoplasts, chromoplasts • number of plastids per cell varies with cell type and • stage of cell cycle • self-contained genome with transcriptional and • translational machinery (double-stranded circular chromosomes) • the plastid genome is polyploid • in tobacco, only the maternal parent transmits • plastids to the next generation

  3. The Protein in Question:caseinolytic protease P1 (clpP1 protease) • two-component enzyme in E. coli: chaperone and • endopeptidase functionality • in Arabidopsis, the nuclear genome provides • additional proteolytic and regulatory subunits Do essential plastid genes exist? Might the plastid clpP1 protease gene represent a plant essential gene?

  4. Experimental Methodology • CRE-lox site-specific recombination system • Tobacco plants engineered with four types of Cre • genes encoding plastid-targeted CRE recombinases • PCR analysis • DNA gel blot analysis • Immunoblot

  5. A B LTR RTR A B A B A B How is clpP1 gene in plastid knocked out? Homologous and CRE/lox site-specific recombination systems were used.

  6. How is homologous recombination system used? Homologous recombination allows lox sequence and antibiotic resistance gene to integrate into plastid gemone (WT ptDNA), resulting in clpP1fl ptDNA.

  7. Vector clpP1fl ptDNA Plastid Select transformed cells by antibiotic How is the vector introduced into plastid? Bombardment (particle gun method) was used.

  8. The Cre-loxP system: Part 2 Generation of transgenic tobacco plants carrying a nuclear Cre gene: Agrobacterium-mediated transformation • Transformation vector: site-specific promoter + genes (Cre & Rubisco ssuTP) + selectable marker • subcloned into plasmid • Vector electroporated into disarmed Agrobacterium tumefaciens • Transformed colonies selected by growth on antibiotic medium then grown up in culture • Flowering plants dipped into solution containing the transformed bacterial cells • Seeds selected for transformants • Heterozygous transgenic plants are grown and used to pollinate the maternal parent (clpP1fl)

  9. The Transformation Vector Cre sequence encodes the CRE (cyclization recombination) recombinase Constitutive promoter targets gene expression to the nucleus selectable marker confers antibiotic resistance Rubisco small subunit transit peptide (ssuTP) targets CRE protein to the plastid nos (nopaline synthase) encodes a polyadenylation (termination) sequence

  10. Cre-lox P Recombination Promoter Cre ssuTP CRE recombinase lox P lox P A B C B C (CRE recombinase) A lox P lox P + A C B

  11. How is CRE protein targeted to the plastid? Cre coding region is translationally fused at its amino terminus with a DNA segment encoding the Rubisco small subunit transit peptide ssuTPcodes an N-terminal presequence that targets the CRE precursor protein (pCRE) to the plastid After import into the plastid, the transit peptide is cleaved off and CRE excises E2 and E3 of clpP1

  12. CRE-mediated excision of clpP1 • Seed progeny derived from the cross between: Engineered clpP1 gene carrier X a nuclear Cre gene carrier clpP1fl (♀) x Cre30B (♂) clpP1fl (♀) x Cre1-100 (♂) clpP1fl (♀) x Cre2-100 (♂) clpP1fl (♀) x Cre2-200 (♂) Was transformation successful? • Test for Cre gene: PCR analysis. • Test for clpP1fl: DNA gel blot analysis • Used total cellular DNA extract from cotyledons, hypocotyls, and roots of individual, 9-day-old seedlings. • Ncol restriction enzyme digestion • Used a 32P-labelled Ncol-Xbal clpP1 probe How about the excision of clpP1 ? DNA gel blot analysis 

  13. clpP1fl x Cre30B • Uniform transformation of plastid genome clpP1fl. • The seedlings with white cotyledons carried the Cre gene. • No clpP1 copies in white cotyledons and hypocotyls, but small fraction in roots (low CRE expression in roots).

  14. clpP1fl x Cre1-100 • About 50% of the seedlings carried the Cre (but developed green shoots, Fig4). • Insufficient excision of clpP1:clpP1 deletion in 5% to 50% of plastid genome copies in seedling cotyledons

  15. clpP1fl x Cre2-100: • >50% of plastid genome copies lacked clpP1 (apale green mottling localized reduction in ClpP1 levels, Fig4d). • clpP1fl x Cre2-200: • Insufficient excision of clpP1 • (developed green shoots, Fig4e)

  16. clpP1fl x Cre clpP1fl: clpP1 genes flanked by lox sites • Maternal parent; transmits plastids to all progeny. Heterozygous Cre lines used to pollinate • Excises sequences between lox sites • Half of all progeny should have Cre

  17. White progeny lack shoot system even after 6 months clpP1fl x Nt-Cre30B 50% green progeny 50% white progeny wild type

  18. Other Cre lines didn’t work as well as Cre30B Half of all progeny contain nuclear Cre, however all progeny appear similar to wild type clpP1fl x Nt-Cre1-100 clpP1fl x Nt-Cre2-100 clpP1fl x Nt-Cre2-200 wild type Inefficient excision of clpP1 is compensated by intact clpP1

  19. Mottled Cre+ plants: localized reduction in ClpP1 Cre- plant clpP1fl x Nt-Cre2-100 wild type

  20. clpP1fl (♀) x Cre30B (♂) no ClpP1 proteins clpP1fl (♀) x Cre1-100 (♂) ClpP1 protein present ClpP1 protein present clpP1fl (♀) x Cre2-100 (♂) ClpP1 protein present • clpP1fl (♀) x Cre2-200 (♂) Cre expression only happens when clpP1fl cross with Cre30B When Cre1-100, Cre2-100 or Cre2-200 crosses with clpP1fl, Cre expression leads to inefficiency of clpP1 excision.

  21. Experimental Findings • uniform wild-type clpP1 to clpP1fl transformation of • plastid genomes, based on a previously published • procedure (Boynton et al. 1988 and Svab & Maliga 1993, ref 17) • efficient excision of the clpP1 gene in the white • seedling offspring of clpP1fl (♀) x heterozygous • Nt-Cre30B (♂) • crosses with Nt-Cre1-100, Nt-Cre2-100, and Nt-Cre2- • 200 resulted in incomplete clpP1 excision

  22. Conclusions Related nuclear-encoded Clp protease genes cannot replace functionality provided by the plastid clpP1 gene The plastid clpP1 gene and its Clp protease subunit product are essential for normal shoot system development in tobacco plants

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