This document summarizes an internship presentation on work with the CPR5 gene in Arabidopsis thaliana. The presentation discusses: 1) Using Arabidopsis as a model organism and details about the CPR5 gene. 2) A project to uncouple the different processes CPR5 is involved in using a complementation strategy. 3) Results showing that complemented lines were successfully generated through PCR amplification and restriction enzyme digestion of the CPR5 gene.

1. Internship presentation
Sunmeet Singh Bhatia

2. Arabidopsis Thaliana
• Thale cress (Mouse-ear Cress).
• Model organism
– 120-megabase
– fully sequenced genome.
– 5 chromosomes.
– ~20,000 genes.
• Life cycle: 6 weeks
(Boyes et al, 2001; TAIR)

3. CPR5 (CONSTITUTIVE EXPRESSER OF
Pathogenesis-Related GENES 5)
Working with this specific gene in the
Arabidopsis genome.
• 2.4 kb gene size
• 564 aa long protein
• Found on the 5th chromosome
(TAIR)

4. 24-day air grown
Early leaf senescence
cpr5-2
plants
WT
cpr5-2
• ROS generation
HR execution
Resistance: Bacterial
& fungal
Aberrant Leaf trichomes
CPR5 mutant abnormalities
cpr5-2WT
Slide Courtesy: Muhammad Faisal
WT cpr5-2

5. Project
Try to uncouple the different
processes of Arabidopsis in which
CPR5 is involved.

6. How do we do that?
Complementation!

7. WT
cpr5-2
COMPLEMENTATION STRATEGY
Employed strategy for motifs/sites elucidation
Constructs
Wild type phenotype
restorationRescue ?

8. Genotyping
Genotyping the complemented lines
to confirm the success of
complementation.

9. Genotyping
• Genomic DNA
• Genotyping strategy
– PCR using gene specific primers
– Digestion of amplified product

10. Results- PCR products
1: Col-0 2:cpr5-2 3-25: Complemented lines C: -ve control
Expected amplified product size = 1.468 kb
1.5kb
1.0kb
0.8kb
0.6kb
0.4kb
0.2kb
1.5kb
1.0kb
0.8kb
0.6kb
0.4kb
0.2kb

11. Restriction Digest
Restriction
enzyme
Restriction
enzyme
Restriction
enzyme
Restriction
enzyme
1 2 3
1
Constructed CPR5 gene
cpr5-2 mutant gene
Restriction
site
PRESENT
Restriction
site ABSENT

12. Results- Restriction digest
• Used BamHI restriction enzyme
• Expected BP of fragments 460 bp + 1008 bp
1: Col-0 2:cpr5-2 3-25: Complemented lines
1.5kb
1.0kb
0.8kb
0.6kb
0.4kb
0.2kb
1.5kb
1.0kb
0.8kb
0.6kb
0.4kb
0.2kb

13. Results/Conclusions
Amplification only from complemented lines
and
Migration of expected number and size of DNA
fragments on gel
confirms successful complementation

14. Trichomes in Arabidopsis
• CPR5 associated with trichome development
(Jing et al., 2008)
• It promotes the growth of trichomes
• cpr5-2 mutants show close to no trichomes
• Col-0 (wild type) has a characteristic pattern for trichomes
(Kirk et al., 2001)
• ‘~WT’; < WT; ‘Partially complemented lines’ & ‘ Not
complemented lines’

15. Results/conclusions
-40
-20
0
20
40
60
80
100
120
140
Col-0 cpr5-2 H-IV: 5-4
(Tray 2)
J-II: 1-1 A-VI: 10-5 A-V: 5-1 H-IV: 1-5 J-II: 2-6 H-IV: 4-5
(Tray 2)
H-IV: 7-4
(Tray 2)
1-branch 2-branch 3-branch 4-branch
~WT >WT Partially complemented lineNot complemented line

16. ROS levels in Arabidopsis
• ROS (Reactive oxygen species): bi-product of
various metabolic activities of plants
• CPR5 is a master regulator of cellular ROS levels
in Arabidopsis (Jing et al, 2008)
• cpr5-2 mutants show higher levels of ROS than
the wild-type plant (Jing et al, 2008)
• Based on the ROS levels, complemented lines
– Fully complemented (WT-like)
– and not-complemented lines (cpr5-2 mutant-
like)

17. • ROS levels (Hydrogen Peroxide) in Arabidopsis:
DAB staining method
• DAB (3,3'-diaminobenzidine) is oxidized by
H2O2 : Leaves a dark brown precipitation.
• High colour intensity  High precipitation 
High ROS levels
ROS levels in Arabidopsis

18. • WT: 1-6: Closely complemented line (WT-like)
• Del1: 2-4: Not complemented line (cpr5-2-like)
Results/Conclusion
WT WT: 1-6 cpr5-2 Del1: 2-4

19. Overall experience/Things I learnt
• Plant techniques
• Molecular techniques
• Plant Physiology
• Literature

20. Acknowledgements
• Dr. Paul Dijkwel
• Muhammad Faisal
• Lab members (C5.19)
• David, Rebecca, Satendra, Pam and Pam. 