1. Presented By: Janesse Holmes and Maziar Hamzeh
Knockout Analysis of Arabidopsis Transcription Factors TGA2, TGA5, and TGA6 Reveals Their Redundant and Essential Roles in Systemic Acquired Resistance
Researchers: Yuelin Zhang, Mark J. Tessaro, Michael Lassner, and Xin Li
Presented By: Janesse Holmes and Maziar Hamzeh

2. Systemic Acquired Resistance (SAR)
Non-infected regions gain resistance after other regions exposed to pathogen
First, the plant is infected with a pathogen. The plant is then exposed a second time in a different area to that pathogen but no infection occurs because its defense system has been turned on throughout the entire plant. SAR = areas throughout the plant that were not inoculated with the pathogen gain resistance to the pathogen that the inoculated areas were exposed to.
Conrath, 2006

3. Salicylic Acid SAR signal molecule Major player in HR
Accumulates in diseased tissue/necrotic regions

4. INA 2,6 dichloroisonicotinic acid biologically active analog of SA
Used in addition to SA in SAR experiments

5. npr1 Phenotype npr1 plants: Reduced SA accumulation
Reduced induction of PR genes
Increased susceptibility to pathogens
mutants defective in SAR responses led to identification of NPR1
Cao et al., 1994

6. NPR1 Gene Structure

7. What do NPR1’s Ankyrin domains do?
QUESTION
What do NPR1’s Ankyrin domains do?
Answer = they interact with TGA TFs using their ankyrin domains

8. NPR1 Ankyrin Structure Involved in protein-protein interactions
NPR1 has 4 ankyrin repeats
Interact with TGA transcription factors
Nair et al., 2013

9. NPR1 Gene Structure
C-terminal (binding SA – Yuelin Zhang results publishing in progress)

10. Previously from Tutorial #9
Previous Y2H (Yeast Two Hybrid) assay used tomato homolog of NPR1 and found bZIP Transcription factor NIF1

11. What is a Yeast Two Hybrid Assay?
QUESTION
What is a Yeast Two Hybrid Assay?
How does it work?
It is a method of finding protein-protein interactions

12. TGA proteins (tutorial #9)
BLAST results of NIF1 to Arabidopsis genome found...
3 Arabidopsis homologs of NIF1:
TGA2 = AHBP-1b (strong interaction with NPR1)
TGA5 = OBF5 (weak interaction with NPR1)
TGA 6 (strong interaction with NPR1)
→ these are also bZIP TFs
→ bind to as-1 domain (tgacg) in PR-1 promoter
→ interact with NPR1 through NPR1’s ankyrin repeat domains

13. TGA proteins Group of Transcription Factors (TFs)
Recognize core sequence 5'-TGACG-3' → as-1 motiff in PR-1 promoter
NPR1 = transcriptional activation domain
TGA = DNA binding domain.
NPR1
TGA2
PR-1 Gene
as-1 motiff

14. TGA2/5/6 found to be closely related
QUESTION
Objective = Determine biochemical functions of TGA2/5/6
Relatedness can tell us if proteins could have redundant functions (if proteins share >80% identity then high chance of redundancy)
What is the objective of this paper?
What can relatedness of TGAs tell us?

15. Deleteagene Workflow Used deletion mutants to study protein functions
Screened a deletion library created using Deleteagene method
Li et al., 2001

16. Deleteagene PCR Strategy
PCR primers are designed well into the flanking regions surrounding the Target Gene.
Amplification time is shortened so that only the deletion mutants may be sequenced
Li et al., 2001
Li et al., 2002

17. How did the researchers screen for tga6-1?
QUESTION
How did the researchers screen for tga6-1?
Used Deleteagene PCR method where they designed primers spanning a very large region.

18. Identifying tga6-1 Deletion Mutant
Screened an Arabidopsis fast neutron-induced deletion mutant population for a TGA6 mutant using PCR.
tga6-1 mutant found
Primers designed to amplify a ~ 9kb region
~ 9 kb
TGA6 gene
The extension time was set to 1.5 mins in order to avoid the amplification of WT DNA.

19. tga6-1 Mutant tga6-1 mutant found on BAC clone F28J15.
Found an ~2.7kb deletion
cDNA = 30, ,651 bp on F28J15
Coding region = 31, ,264bp on F28J15
TGA6 coding region was deleted!
Figure 1

20. QUESTION
If the TGA6 coding region could fit within the 2.7kb deletion then why were primers designed to amplify a ~9kb region?
Researchers were not sure how big the deletion would be. Need primers to cover a potentially large region.

21. tga2-1tga5-1 Also used Deleteagene protocol
TGA2 and TGA5 located <2kb apart
Primers designed to span ~17kb
Found a deletion of ~9.7kb which encompassed TGA2 and TGA5
~ 17 kb
TGA2
TGA5

22. Why did they use Deleteagene method to find tga2-1 tga5- 1 mutant?
QUESTION
Why did they use Deleteagene method to find tga2-1 tga5- 1 mutant?

23. Triple Mutant tga2-1/tga5-1 x tga6-1 (pollen) F1 (self-crossed)
F2 homozygous for both loci → used for phenotype analysis
F2 heterozygous for tga6-1 but homozygous for tga2-1 tga5-1 → used for cosegregation analysis

24. Why did they use the F2 generation for analysis?
QUESTION
Why did they use the F2 generation for analysis?

25. Triple Mutant Shares npr1 Phenotype
Plants grown on MS medium with 0.2mM SA
Photos taken 14 days after germination
Figure 2

26. SA Toxicity npr1-1 highly sensitive to SA toxicity
Triple mutant showed similar response to npr1-1
Only cotyledon stage was reached and seedlings were bleached → npr1-1 and tga2-1 tga5-1 tga6 -1
tga2-1 tga5-1 double mutant and tga6-1 knockout mutant shows similar response to WT
Figure 2

27. What does this suggest about the relationship of the TGAs with NPR1?
QUESTION
What does this suggest about the relationship of the TGAs with NPR1?
Indicate TGAs have similar role to NPR1 in regulating tolerance to SA

28. PR-1 Expression in Triple Mutant
Objective: to determine if TGA2/5/6 share similar functions with NPR1
Analyzed PR-1 expression (INA treated and untreated)
Triple mutant is highly sensitive to SA so INA was used instead.
Suggests SAR is compromised when all TGA2/5/6 are mutated
(+) = treated with INA
(-) = not treated
npr1 blocks PR-1 expression even when SA or INA are applied.
Wild Type shows a high accumulation of PR-1
tga6-1 knockout mutant and tga2-1 tga5-1 double mutant show a similar build up as the WT.
tga6-1 tga2-1 tga5-1 triple mutant did not show a build up of PR-1 when INA
Figure 3

29. Non-induced
Triple and tga2-1 tga5-1 mutant → increased basal PR-1 expression
Triple mutant → 50 fold increase vs WT.
tga2-1 tga5-1 mutant → 10 fold increase vs WT.
tga6-1 knockout mutant did not affect the accumulation levels of PR-1 when compared to WT.
Figure 3

30. QUESTION
What could be one of the reasons that triple and double mutant show increased basal levels?
TGAs may repress basal levels of PR-1 → TGA6 is partially responsible for the negative regulation of basal PR-1 levels

31. SAR Abolished in Triple Mutant
Objective: determine whether INA-induced pathogen resistance was affected in triple mutant
Suggests SAR is compromised in TGA triple mutant
2 week old plants→ treated with INA → virulant oomycete after 3 days. (peronospora parasitica Noco2 )
WT, tga6-1 and tga2-1 tga5-1 double mutant all showed resistance.
No conidiophores after 7 days.
Triple mutant showed conidiophores after 7 days
Figure 4

32. Reduced Avirulent Pathogen Resistance in Triple mutant
Objective: determine if SAR can be induced by avirulent pathogen (rather than INA) in triple mutant
Triple mutant = reduced
resistance to virulent pathogen
compared to WT after avirulent
pathogen exposure
Suggests that SAR is
compromised in the triple
mutant
infiltrated with P.s.t (avirulent pathogen) or buffer solution 3 days before inoculation with virulent P.s.m
induced systemic resistance to Pseudomonas syringae pv maculicola ES4326 in WT.
The triple mutant did not show similar results as the WT.
Figure 5

33. QUESTION Next, researchers wanted to analyze the role of TGA6 in SAR.
How do we check to see if the reduced SAR is related to tga6-1?
co-segregation analysis

34. Co-segregation of reduced SAR with tga6-1
Objective: to see if the loss of resistance co-segregated with the tga6-1 deletion.
F2 heterozygous for tga6-1 and homozygous for tga2-1 tga5-1 (4 independent lines)
F3 were analysed with susceptibility to P.parasitica Noco2 after INA treatment.
In all 4 lines that were tested ¼ were susceptible.
All susceptible were homozygous for tga6-1.
Loss of INA-induced resistance co-segregated with the tga6-1 deletion.
~ ¼ lost INA-induced Resistance

35. TGA2 TGA5 rescue
Objective: to determine if TGA2 or TGA5 can complement triple mutant phenotypes
Designed TGA2 and TGA5 WT vectors with glufosinate herbicide resistance
Floral Dip Transformation
Selected Transformants (T1) with glufosinate spray
T2 Plants → phenotypic analysis
What would be the result of a rescue analysis if either of the triple mutants were used.

36. QUESTION
How did the researchers confirm that the phenotypes they observed in the TGA triple mutant plants are caused by mutations in all three TGA transcription factors?
Presence/absence of tga6-1 mutant and complementation test with TGA2 and TGA5

37. TGA2 TGA5 Rescue INA-induced PR-1 expression → restored with TGA2/5
SA Tolerance → restored with TGA2/5
Figure 6: Complementation of PR-1 expression and triple mutant phenotype

38. Pathogen resistance → Complemented with TGA2 and TGA5 constructs
TGA2 and TGA5 Rescue
Pathogen resistance → Complemented with TGA2 and TGA5 constructs
Figure 6: Complementation of INA-induced pathogen resistance

39. Discussion Summary
TGA2/5/6 serve as essential positive regulators of SAR
Triple mutant → INA-induced PR gene expression/resistance and avirulent pathogen resistance cannot be induced
(Figures 3-5)
TGA2 or TGA5 sufficient to restore INA PR-1 expression and pathogen resistance
TGA2 and TGA5 clones can rescue triple mutant phenotype (Figure 6 A-D)
INA-induced resistance co-segregated with tga6-1
SAR phenotype only observed in triple mutant not double mutant (Table 1).
These results suggest that the three TGAs have redundant functions
How do we know if the TGA factors act as redundancies?
What does the increased basal levels of the PR tell us about the TGA factors?

40. Figure 3: INA-induced PR-1 Gene Expression

41. Figure 4: INA-Induced Resistance

42. Figure 5: Avirulent pathogen resistance

43. Discussion
Due to the increased basal levels of PR-1 in the tga2-1 tga5-1 and tga6-1 tga2-1 tga5-1 mutants → TGA factors may function as suppressors of basal expression of PR-1 (TGA6 being partially responsible) (Figure 3)
TGA2 or TGA5 complementation sufficient to suppress basal PR-1 expression (Figure 6A)
Hypothesis: Increased basal PR-1 expression → loss of binding of TGA factors to a negative element on the PR-1 promoter.
Supported by presence of an as-1 related TGACG element (weak silencer) in the PR-1 promoter.
While the triple mutant does show higher sensitivity to SA (like npr1) it does not show higher susceptibility to P.s.m ED4326.
This can be due to other TGA factors regulate resistance to P.s.m ES4326.
Y2H show other TGAs that bind to NPR1 (Després et al., 2000; Zhou et al.,2000) → Do not share TGA2/5/6 function

44. npr1 vs triple mutant npr1-1 tga2 tga5 tga6
Increased basal level PR-1 expression
No enhanced susceptibility P.s.m ES4326
Negligible basal level PR-1 expression (like WT)
enhanced susceptibility to P.s.m ES4326
Reduced SAR phenotypes
sensitivity to SA toxicity (Figure 1)
Reduced INA-induced PR-1 expression
What are the similarities and differences in phenotype between npr1-1 and triple mutant?

45. QUESTIONS?

46. References
Conrath, U. (2006). Systemic Acquired Resistance. Plant Signaling & Behavior, 1(4), 179–184.
Cao, H., Bowling, S. A., Gordon, A. S., & Dong, X. (1994). Characterization of an Arabidopsis Mutant That 1s Nonresponsive to lnducers of Systemic Acquired Resistance. The Plant Cell, 6, 1583–1592. Retrieved from
Li, X., Song, Y., Century, K., Straight, S., Ronald, P., Dong, X.,Lassner, M., and Zhang, Y. (2001). A fast neutron deletion mutagenesis-based reverse genetics system for plants. Plant J. 27, 235–242.
Li, X., Lassner, M., & Zhang, Y. (2002). Deleteagene: A Fast Neutron Deletion Mutagenesis-Based Gene Knockout System for Plants. Comparative and Functional Genomics,3(2), doi: /cfg.148
Nair, S., Hagberg, H., Krishnamurthy, R., Thornton, C., & Mallard, C. (2013). Death Associated Protein Kinases: Molecular Structure and Brain Injury. International Journal of Molecular Sciences, 14(7), 13858–