UCOL talk (031214)

Internship presentation

Sunmeet Singh Bhatia

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)

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)

24-day air grownEarly leaf senescence

cpr5-2 plants

WT

cpr5-2

• ROS generation

HR execution

Resistance: Bacterial & fungal

Aberrant Leaf trichomes

CPR5 mutant abnormalitiescpr5-2WT

Slide Courtesy: Muhammad Faisal

WT cpr5-2

Project

Try to uncouple the different processes of Arabidopsis in which

CPR5 is involved.

How do we do that?

Complementation!

WTcpr5-2

COMPLEMENTATION STRATEGY

Employed strategy for motifs/sites elucidation

Constructs

Wild type phenotype restorationRescue ?

Genotyping

Genotyping the complemented lines to confirm the success of

complementation.

Genotyping

• Genomic DNA

• Genotyping strategy

– PCR using gene specific primers

– Digestion of amplified product

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

1.0kb0.8kb0.6kb0.4kb0.2kb

1.5kb

1.0kb0.8kb0.6kb0.4kb0.2kb

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

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

1.5kb1.0kb0.8kb0.6kb0.4kb0.2kb

1.5kb1.0kb0.8kb0.6kb0.4kb0.2kb

Results/Conclusions

Amplification only from complemented linesand

Migration of expected number and size of DNA fragments on gel

confirms successful complementation

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’

Results/conclusions

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)

0

20

40

60

80

100

120

140

1-branch 2-branch 3-branch 4-branch

~WT >WT Partially complemented lineNot complemented line

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)

• 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

• 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

Overall experience/Things I learnt

• Plant techniques

• Molecular techniques

• Plant Physiology

• Literature

Acknowledgements

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