The plastid clpP1 protease gene is essential for plant development

1. The plastid clpP1 protease gene is essential for plant development Hiroshi Kuroda & Pat Maliga Sarah Koo, Grant Lee, Tania Lucinian, & Jennifer Tak Chemistry161A January 26, 2005

2. Plastids • Semi-autonomous cellular organelles • Contain own genome and transcription-translation machinery • Functions include photosynthesis and biosynthesis of starch, amino acids, lipids and pigments • 1,000 to 10,000 copies of plastid genome in a cell (polyploid)  Difficult to identify essential plastid genes

3. ClpP1 Protease Gene(Caseinolytic Protease P1) • Two component enzyme • Thought to be the main source of protein degradation in the chloroplast • Non-essential in non-photosynthetic plants (i.e. maize) • Required for shoot development in tobacco plants?

4. Tobacco Plant

5. Tobacco Plant • Cotyledon: seed leaf; the first leaf that appears on a seedling cotyledon

6. Tobacco Plant • Cotyledon: seed leaf; the first leaf that appears on a seedling • Hypocotyl: the first leaf-like structure that appears on a germinating seed; found below the cotyledon cotyledon hypocotyl

7. Question: Does the deletion of the clpP1 gene cause ablation of the shoot system of tobacco plants? Are plastid genes essential for normal plant growth?Methodology: CRE-lox site –specific recombination system

8. How does CRE work? A site-specific DNA recombinase means that the CRE protein can recombine DNA when it locates specific sites in a DNA molecule These sites are known as loxP (locus of X-over P1) sequences http://www.bioteach.ubc.ca/MolecularBiology/TargetingYourDNAWithTheCreloxSystem/#Ref2

9. Plastid Transformation aada: spectinomycin resistance clpP1: caseinolytic protease P1 Recombination! -Used vector pHK85, linked genes for clpP1 and aada RESULT: clpP1fl ptDNA

10. VECTOR TRANSFORMATION

11. CRE cyclization recombination P1 phage CRE recombinase enzyme • 38.5kDa • Allows for Rc of two loxP sites • Has 34 bp region consisting of two 13-bp inverted repeats separated by an 8bp asymmetric spacer sequence • If loxP sites in same orientation, expect recombination to result in deletion of intervening DNA • If loxP sites are in the opposite orientation, inversion of the sequence flanked by two lox P sites

12. In the lab… BOMBARDMENT OF CELLS Petri dish with plant cells

13. clpP1fl ptDNA

14. The Cre gene and protein • Source: E. coli bacteriophage P1 • Encodes for site-specific DNA recombinase (tetramer; dimer of dimers) also named Cre • Cre does both cleavage and ligation steps • loxP found only in bacteriophage P1 • Cre/lox system in use for 15+ years

15. Active site uses a tyrosine residue One CRE subunit From Gopaul, et al

16. Agrobacterium, the middleman • Transplant gene from E. coli to tobacco • First transformed Agrobacteria tumefacienswith CRE • Why Agrobacteria? It inserts its DNA into the plant’s nuclear genome, which is very efficient

17. Agrobacterium vector Fig. 2b P2’ : constitutive promoter O1, O2 : PCR primers neo : kanamycin resistance ssuTP : Rubisco small subunit aacC1 : gentamycin resistance Tnos : poly-A site

18. Excising the target in plastid, part 1 Plant cell’s machinery translates CRE in cytoplasm, which has a Rubisco SSU precursor attached. This allows CRE enzyme to be transported into plastid. CRE cleaves lox-flanked clpP1 and re-ligates plastid DNA; clpP1 is degraded.

19. Coordinate expression of nuclear and chloroplast genes Taken from Merchant 1/11/05 handout, Fig. 9.16 in textbook

20. Excising the target in plastid, part 2 Crossing transplastomic CRE tobacco with maternal clpP1-fl tobacco This gets degraded precursor CRE Because SSU is attached, CRE is not in native conformation Fig. 1b The plastid’s own machinery will cleave off SSU and allow pCRE  CRE Rubisco SSU

21. clpP1-fl Cre 2 Cre 3 Tyr loxP Tyr loxP Tyr Cre 1 Cre 4 Tyr 3’ 5’ 5’ 3’

22. Cre 2 Cre 3 Tyr Tyr Tyr Cre 1 Cre 4 Tyr 3’ 5’ 5’ 3’

23. Cre 2 Cre 3 Tyr Tyr HO OH Tyr Cre 1 Cre 4 Tyr 3’ 5’ 5’ 3’

24. Cre 2 Cre 3 Tyr Tyr Tyr Cre 1 Cre 4 Tyr 3’ 5’ 5’ 3’

25. Cre 2 Cre 3 Tyr HO Tyr Tyr OH Cre 1 Cre 4 Tyr 3’ 5’ 5’ 3’

26. Cre 2 Cre 3 Tyr Tyr Tyr Cre 1 Cre 4 Tyr Plastid DNA w/o clpP1 3’ 5’ 5’ 3’

27. Crossing of clpP1fl X CRE lines Objective: To determine the CRE line that successfully excises the clpP1f1 gene from the engineered plastid completely. Hypothesis/Expectation(s): Differences in CRE activity in the seed progeny based on the different crosses of clpP1fl with varying CRE lines created.

28. Method and Analysis • Method: Tobacco plants transformed with engineered plastids are pollinated separately with different nuclear CRE lines. • Analysis: DNA Blot Analysis - To indicate efficiency of clpP1 segment excision (Fig 3) PCR- To confirm presence of CRE gene

29. Progeny of Crossing clpP1fl and nuclear Cre Lines Independent Heterozygous nuclear Cre Lines Maternal Parent Pollinated Engineered plastid in tobacco plants Seed Progeny All seedlings carried plastid with clpP1fl After Cre excises clpP1fl ½ contain nuclear Cre gene

30. Fig 3a: clpP1fl X Cre30B • Conclusions: • Varying proportions of clpP1fl and ΔclpP1 amongst each part of the plant • Root still contains intact copies of clpP1fl due to low expression of CRE • Cotyledons carry the Cre gene (complete excision of clpP1fl) • clpP1 copy number reduced to <1% • THUS, successful excision of clpP1fl segment. CRE WT clpP1fl ΔclpP1

31. Fig 3b: clpP1fl X Cre1-100 • Conclusions: • ~50% of seed progeny carry Cre gene. • clpP1 deletion of 5-50% of plastid genome of progeny in cotyledons -[CRE protein] Cre1-100 < [CRE protein] Cre30B Presence of Cre bands correspond to ΔclpP1fl bands. Note intensities. - Less efficient excision of clpP1fl segment in comparison to Cre30B.

32. Fig 3c: clpP1fl X Cre2-100 • Conclusions: • >50% of plastid genome copies lack clpP1 gene • More efficient excision of clpP1fl in comparison to Cre1-100 • In comparison of the intensities of the bands, at 2.76kb, there is still a detectable amount of plastid genomes still containing the clpP1fl. • However, the intensity of the progeny with clpP1fl excised out is significantly more intense, thus accounting for over 50% of the plastid copies with CRE-mediated excision.

33. Fig 3d: clpP1fl X Cre2-200 • Conclusions: • Inefficient excision of clpP1 segment as shown by the high intensity of the bands at 2.76kb (representing plastids containing clpP1fl) • clpP1 deletion somewhat successful by 5-50% of seed progeny, as indicated by detected presence of plastid copies lacking in clpP1 segment in lanes 8-10.

34. Comparison of All Crosses

35. clpPlfl X Cre30B Phenotype Compare to WT • Contains both GREEN and WHITE progeny • Green seedlings developed normally with regular shoot system and leaf formation White seed progeny were unable to develop a regular shoot system even after 6 months

36. Other Crosses with Cre Lines Compare to... (Same as cross with Cre1-100) Phenotype: Green Genotype: ~50% of seed progeny contain Cre Phenotype: pale green Genotype: Cre+ - >50% of seed progeny contain Cre - Localized reduction in clpP1 levels

37. Summary of Phenotype Comparisons: White Pale Green Green WT Green w/ ~50% containing Cre Most Efficient clpP1fl excision with Cre30B Lack of efficient clpP1fl excision with Cre 2-100, Cre 1-100, and Cre 2-200.

38. Figure 5 Figure 5. Immunoblot to detect accumulation of ClpP1 in seedling cotyledons. a, 35 µg or b, 200 µg total soluble protein was loaded per lane. Wild-type protein extract serves as a reference for quantification.

39. Figure 5 Figure 5. Immunoblot to detect accumulation of ClpP1 in seedling cotyledons. a, 35 µg or b, 200 µg total soluble protein was loaded per lane. Wild-type protein extract serves as a reference for quantification. Conclusion: no clpP1 present in clpP1fl x Cre30B progeny (<2%)

40. Conclusion • The lack of the clpP1 gene does affect shoot development in tobacco plants. • The nuclear-encoded catalytic subunit genes of the tobacco plant cannot replace the clpP1 gene. • Alternatively, the lack of the clpP1 gene may be linked to the lack of degradation of a regulatory protein for a clpP1 isoform.