1. Inducers of Plant Systemic Acquired Resistance Regulate NPR1 Function through Redox Changes By Tommy Grgic and Emily Allegretto-Smith Zhonglin Mou, Weihua Fan, and Xinnian Dong (2003)

2. OUTLINE Background Information Hypotheses Experiments Conclusions Future Directions

3. Plant Immunity: Acquired  Systemic acquired resistance (SAR):  Secondary response  After infection: elevated levels of SA at site of infection and in systemic tissues (= non-infected tissues)  Results in long lasting resistance to pathogen for the plant  SA is necessary and sufficient to induce SAR Stitcher et al. 1997

4. Previous experiments SA (or INA/BTH) Activation of SAR nahG suppressed SAR response

5. The NPR1 Mutant (npr1)  Fails to respond to SAR induction  Shows low levels of PR expression  Increased susceptibility to pathogen infection  NPR1 protein function: positive regulator of SAR, required for SA signal transduction to activate PR gene expression  NPR1 protein features: nuclear localization sequence (NLS), 2 protein interaction domains  Gene is highly conserved as shown by its ability to function in heterologous species

6. NPR1 Localization  NLS suggests localization to nucleus  Demonstrated by NPR1-GFP fusion experiments  Further suggests role in regulating transcription  Most likely NOT a transcription factor  Doesn’t have DNA binding domain  Does interact with TGA subclass of transcription factors  TGA subclass TF’s can bind as-1 element present in promoter sequence of PR1 gene promoter  Suggests indirect role of NPR1 in transcriptional regulation of PR genes Kinkema et al. 2000

7. NPR1 Mechanism of Regulation What do we know?  NPR1 is constitutively expressed  SA does not affect the transcription of NPR1 - perhaps it affects it at the protein level?  No binding activity between SA and NPR1  SAR induction leads to accumulation of NPR1 protein in the nucleus What DON’T we know?  The mechanism through which SA and NPR1 gene product interact

8. Plant Immunity: Innate Avirulence (avr) factors: specific pathogen derived molecules Resistance (R) proteins: recognize pathogen derived molecules R protein and avr factor interaction results in hypersensitive (HS) response HS response = cell death, production of reactive oxygen species, antimicrobial compounds

9. What does the previous literature tell us? Changes in SA concentration after pathogen infection can potentially affect the redox state of the cell (3 different studies showed this - Chen et al., 1993; Noctor et al., 2002; Vanacker et. al, 2000) NPR1 and NPR1-like proteins across four plant species have 10 conserved cysteines (unpublished data) - suggests these cysteines are important With these 2 pieces of information together we can logically infer that SA may mediate NPR1 through reduction of the sulfur bonds in the 10 conserved cysteines

10. Forming the Experimental Hypothesis: Using experimental observations and previous literature to develop the initial hypothesis: Hypothesis: the increase in SA mediates the localization of NPR1 to the nucleus through cellular redox changes that affect the cysteines in NPR1 protein

11. Experiment: Testing whether cysteines in NPR1 affect protein conformation in SAR induction  Extract proteins and then treat with (+) INA or without (-) INA  SDS PAGE and immunoblot analysis to detect which form of protein is present  Treat the same protein extract with DTT as control  SDS PAGE and immunoblot

12. Experimental Techniques  SDS-PAGE: method of protein separation by electrophoresis  Immunoblot (Western blot): detecting specific proteins on a gel with antibodies

13. Experiment: Testing whether cysteines in NPR1 affect protein conformation in SAR induction  Results suggest NPR1 exists in different conformation before and after SAR  Antibody against N terminal  Next step: detect activated and non activated protein forms (A) Total protein (100 g) extracted from wild-type Columbia (Col-0) plants treated with () or without () 0.5 mM INA for 2 days was subjected to SDS-PAGE with () or without () DTT (50 mM) in the sample buffer and analyzed by immunoblot using a polyclonal anti- NPR1 antibody. Numbers represent MW.

14. Experiment: Find inactivated and activated protein in 35S::NPR1-GFP npr1-1  INA or water treatment with GFP Ab  NPR1 complex formed through intermolecular disulfide bonds (B) Total protein extracted from 35S::NPR1-GFP transgenic plants (in npr1-1) was analyzed as in (A) using a monoclonal anti-GFP antibody. Both oligomeric (O) and monomeric (M) forms of NPR1-GFP were detected. The bottom of the wells and the top of the separating gel are shown for reference.

15. Experiment: Rule Out Crosslinking  Protein extractions with agents to block formation of new disulfide bonds  High MW complex confirmed to be present in plant cell before extraction (C) Tissues from 35S::NPR1-GFP transgenic plants (in npr1- 1) treated with () or without () 0.5 mM INA were homogenized in the presence () or absence () of 2 mM iodoacetamide (IOD) or 2 mM N-ethylmaleimide (NEM). The protein was denatured by adding an equal volume of sample buffer (125 mM Tris-HCl, pH 6.8, 5% SDS, 25% glycerol, and 0.4% bromophenol blue), incubating at room temperature for 30 min, and heating at 60C for 10 min. For the IOD or NEM treated samples, 2 mM IOD or 2 mM NEM was also included in the sample buffer. Immunoblot analysis was performed as in (B). (D

16. Experiment: Nature of High MW complex  High MW complex a homooligomer of NPR1 ( D) Tissues from 35S::NPR1-GFP transgenic plants (in npr1-1) treated with () or without () 0.5 mM INA were homogenized in the presence of a low concentration of DTT (0.5 mM). NPR1-GFP reduction intermediates, dimer (D) and trimer (T) as well as monomer (M) and oligomer (O), were detected using immunoblot. The bottom of the wells and the top of the separating gel are shown for reference. (E) Total protein was extracted from wild-type (lane 1) and 35S::NPR1-GFP transgenic (lane 2) plants. The extracts were immunoprecipitated using anti-GFP monoclonal antibody. The precipitated proteins were eluted and analyzed by immunoblot using the anti-NPR1 antibody.

17. Experiment: NPR1 oligomer reduced to monomer in vivo  Show that in the plant cell NPR1 changes conformation to become active  NPR1 monomer originated from reduction of oligomer (F) Total protein was extracted from 35S::NPR1-GFP plants treated with () or without () 0.5 mM INA (24 hr) and 100 M cycloheximide (CHX; 26 hr) and was subjected to SDS-PAGE with () or without () DTT (50 mM) in the sample buffer and analyzed by immunoblot analysis. A proteasome inhibitor MG132 (10 M) was included in all treatments to prevent protein degradation. 1D

18. Experiment #2 :Test the effect of SAR induction on cell redox state Hypothesis: if the INA-induced switch of NPR1 oligomer into NPR1 monomer is due to a reduction of sulfur-sulfur bonds, this suggests there might be a decrease in reduction potential of plants [following induction] How do we test this decrease in reduction potential? - measure the changes in glutathione pool (GSH + GSSG) in plant cells Glutathione = GSH: major thiol-disulfide redox buffer in plants

19. The Chemistry Behind GSH and GSSG Thiol groups: carbon bonded sulfylhydryl (R-SH) are reducing agents [they lose electrons and are oxidized] Glutathione reduces disulfide bonds to cysteines by serving as an electron donor. In the process, glutathione (GSH) gets oxidized into glutathione disulfide (GSSG) By measuring the ratio of GSH:GSSG we can determine the reduction potential in plant cells - GSH GSSG

20. Experimental Design: First they treated the plant cells with INA (INA = SAR inducer) Then they measured the total amount of glutathione as well as the GSH:GSSG ratio (at 0, 8, 16, 24 and 48 hrs) in the same plant cells (measuring the response in GSG:GSSH of only cells that were treated with the SAR inducer)

21. GSG:GSSH Ratio Results Following INA treatment: Similar changes were observed in both the total amount of GSH and the GSG:GSSH ratio - “Dramatic decrease 8 hr after INA treatment, followed by sharp increase reaching a plateau 24 hr after INA treatment” (A) 35S::NPR1-GFP transgenic plants (in npr1-1) were treated with 0.5 mM INA solution. Tissues were collected at the indicated time points after treatment. Total glutathione (GSHGSSG) and GSSG were measured and the GSH/GSSG ratio was calculated (Rao and Ormrod, 1995). The error bars represent standard deviation of three samples.

22. Is this redox reaction happening in systemic tissues? (= the non-infected tissues) Half leaves were infected with Pseudomonas syringae pv macuclicola ES4326 (bacterial pathogen) that carried the avirulence gene avrRpt2 Then the UNINFECTED half leaves were collected for measurements for the total GSH as well as GSH:GSSG ratio Infected Uninfected

23. Results: (in systemic tissues) (B) Half leaves were mock inoculated with MgCl2 (10 mM) or infected with Psm ES4326/ avrRpt2 (OD600 0.02) and the uninoculated halves were collected at the indicated time points for glutathione measurements. The error bars represent standard deviation of three samples. Total GSH and GSH:GSSG were “significantly” increased reaching peak levels at 24 hr after infection (0.34mM and 13.9:1 respectively)

24. Can the observed changes in GSH:GSSG ratio result in reduction of NPR1 oligomer in vitro How did they test this? Incubated uninduced protein extracts in defined concentrations of GSG:GSSH at 0 degrees celsius for 1 hr Then they performed SDS-PAGE and immunoblot analysis

25. Results: (C) Total protein (100 g) extracted from 35S::NPR1-GFP transgenic plants (in npr1-1) was incubated with the indicated amounts of GSH and GSSG at 0C for 1 hr and then subjected to SDS-PAGE with (+) or without (-) DTT (50 mM) in the sample buffer. Immunoblot was performed as described in Figure 1. These results indicate that the monomeric form of NPR1 only begins to appear on the gel when the ratio begins to exceed 15:1 (3 / 0.2 = 15)

26. Conclusions for this SAR induction experiment: The data shown previously only demonstrates a correlation between the GSH:GSSG ratio and NPR1 monomer, it doesn’t actually show that GSG and NPR1 are in a redox equilibrium (if they were in equilibrium it would be strong evidence for a causal relationship) Therefore the reducing agent that is directly involved in NPR1 oligomer → NPR1 monomer has not yet been determined The data does however suggest that SAR induction leads to a change in cellular reduction potential as measured by the total GSH and GSH:GSSG ratio

27. Experiment: is NPR1 reduction required for PR gene expression?  Does NPR1 monomerization allow SA to turn on NPR1-dependent genes?  Time course experiment following NPR1 reduction and subsequent expression of PR genes  NPR1 monomerization required for PR gene expression (A) 35S::NPR1-GFP transgenic plants (in npr1-1) were treated with 0.5 mM INA solution. Tissues were collected at the indicated time points after INA treatment. Total protein (100 g) was extracted and analyzed using immunoblots as in Figure 1. Expression of the PR1 gene was examined using RNA gel blot analysis. Loading of total RNA (20 g) for each lane on the blot was represented by ethidium bromide staining of rRNA prior to blotting

28. Experiment: NPR1 reduction required for PR gene expression  Confirm NPR1 reduction associated with SAR  NPR1 monomer-PR1 gene expression correlation in system tissues after inoculation (B ) Half leaves of 3-week-old 35S::NPR1-GFP transgenic plant (in npr1-1) were inoculated with Psm ES4326/avrRpt2 (OD600 0.02). The uninfected halves (adjacent) and distal leaves (distal) were collected 24 hr and three days after infection, respectively, and used for immunoblot and RNA gel blot analyses.

29. Experiment: NPR1 reduction required for PR gene expression  Establish causative relationship  Prevent NPR1 reduction with 6-AN  Plant treatment with INA and then 6- AN  NPR1 reduction likely required for PR gene expression (A) Plant tissues (0.2 g) were used to measure the total glutathione (GSHGSSG) pool and the GSH/GSSG ratios. Error bars represent standard deviation of three samples. (B) Total protein (100 g) was extracted and the effects of 6-AN on INA- induced NPR1-GFP monomerization and PR1 gene expression were examined using immunoblot and RNA blot analysis, respectively.

30. Experiment: NPR1 monomer sufficient for PR gene expression  Rule out possibility that 6-AN might prevent reduction of unknown factors in PR gene regulation pathway  Mutations introduced into 35S::NPR1-GFP construct, transformed into npr1-1 background→ conserved cysteines mutated  npr1cys-GFP protein measured

31. Experiment: NPR1 monomer sufficient for PR gene expression  Cysteine to tyrosine: C150Y, C155Y  Cysteine to serine: C223S  Cysteine to alanine: C82A, C160A, C212A, C216A, C306A, C394A, C511A  Cysteine to alanine mutants (C82A and C216A) NPR1 monomer constitutive → constitutive PR1 gene expression  Monomeric NPR1 is active

32. Experiment: NPR1 monomerization causes nuclear accumulation  Further investigating NPR1 activation through monomerization of the oligomeric form  Performed nuclear fractionation followed by protein extract and analysis → only monomeric form of NPR1 present in INA treated plant nuclei (A) Total protein (T) and the nuclear-fractionated protein (N) were extracted from 35S::NPR1-GFP transgenic plants (in npr1-1) treated with (+) or without (-) 0.5 mM INA. The total protein (100μg) and the nuclear fractionated protein (12μg) were subjected to SDS-PAGE with (+) and without (-) DTT (50mM) in the sample buffer, respectively. NPR1-GFP was detected using immunoblot with the anti-GFP antibody.

33. Experiment: NPR1 monomerization causes nuclear accumulation  If monomerization is sufficient for nuclear accumulation we would expect the mutants that constitutively expressed the monomeric form of NPR1 to show accumulation in the nucleus without INA treatment  Mutants show enhanced nuclear fluorescence (B) Leaf tissues from the transgenic lines carrying 35S::NPR1-GFP treated with (+) or without (-) INA (0.5 mM) for 24 hr and untreated 35S::npr1C82A-GFP or 35S::npr1C216A-GFP were mounted in water and viewed with a fluorescence microscope.

34. Experiment: NPR1 monomerization causes nuclear accumulation  Nuclear fractionation, SDS-PAGE, immunoblot to confirm monomer nuclear localization  In absence of INA, only mutants that expressed constitutively monomerized proteins were found in nucleus  Conclude: monomerization of NPR1 leads to nuclear accumulation (C) Total protein (T; 100 g) and the nuclear-fractionated protein (N; 12 μ g) from 35S::NPR1-GFP, 35S::npr1C82A-GFP and 35S::npr1C216A-GFP, transgenic lines (in npr1-1) were extracted, subjected to SDS-PAGE with DTT (50 mM) in the sample buffer, and immunoblotted using the anti-GFP antibody.

35. Conclusions Initial hypothesis: NPR1 protein conformation may be sensitive to cellular redox changes that are mediated by changes in SA concentration Testing: Examine NPR1 protein conformation under different redox conditions with and without SAR inducers

36. Conclusions Findings:  In the absence of SAR induction, NPR1 exists as oligomer  Reductive environment after SAR induction can cause NPR1 conversion from oligomer to monomer, its active form  NPR1 monomer moves into nucleus to control SAR gene expression

37. Future Research  Identification of mediators involved in SA signaling of NPR1  Find more mutants

38. Current Research  NPR1 is the receptor for SA (Wu et al. 2012)  SA binds NPR1 through Cu cofactor (Wu et al. 2012)  C-terminal region has TAD with 2 cysteines that are crucial for function (Wu et al. 2012)  Transgenic cotton plants constitutively expressing Arabidopsis NPR1 show resistance to nematodes (Parkhi et al. 2010) Wu et al. 2012

39. Questions?