Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene

Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene YUELIN ZHANG, WEIHUA FAN, MARK KINKEMA, XIN LI, AND XINNIAN DONG

SAR, SA, PR Genes, and NPR-1 SAR: Systemic Acquired Resistance = General plant immune response after local infection SA: Salicylic Acid = Released endogenously by plant after pathogen detection PR Genes: Pathogenesis-related genes= induced as part of SAR (some express antimicrobial proteins, others induce more signalling pathways) NPR1/NIM1: Key component of the SA-regulated PR gene expression and disease resistance

Objective How does NPR1 transduce the SA signal and up-regulate the PR genes??

Background Mutant screen for impaired PR gene expression in SA/INA induced conditions 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 http://www.plantcell.org/content/plantcell/6/11/1583.full.pdf

npr-1 phenotype - No expression of PR-1, PR-2, and PR-5 - Loss of resistance to infection even after treatment with SA or INA 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 http://www.plantcell.org/content/plantcell/6/11/1583.full.pdf

Ankyrin Repeats - 33-residue motif in proteins consisting of two alpha helices separated by loops Mediate protein protein interactions NPR-1 contains at least 4 ankyrin repeats (Cao H., et al. 1997) 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 13872. https://doi.org/10.3390/ijms140713858

HYPOTHESIS Hypothesis: NPR-1 regulates PR gene expression through interaction with other proteins How can you test for protein-protein interaction? Yeast Two Hybrid System! https://www.sciencenews.org/article/yeast-life-span-calorie-restriction-may-be-wash

http://technologyinscience.blogspot.ca/2014/07/yeast-two-hybrid-system-for-protein.html

(Nuclear Localization Signal) TomNPR-1 (High Copy) (Low Copy) 1.8kb cdna cdna Library NPR-1 LexA ADH1 Promotor: Constitutive yeast promotor B42 AD 107 independent clones PGAL1 Promotor: Conditional yeast promotor to presence of galactose

Question? Why use tomato? Quality of cdna library crucial for Y2H success Y2H system is known for having false positives Could use well characterized PTO-PTI interaction as positive control Can understand general significance of the gene if investigated in 2 different plants

R-1 NP peg202 Transformation EGY84 Selection Genotype: MATa, his3, trp1, ura3, LexAop(x6)-LEU2 Amp+ His3+ Culture EGY84 with peg202:tomnpr-1 Media with no Histidine NA cd 2.5x107 cells PJG4-5 EGY84 Transfer Leucine Drop out Plates Selection Media with High ph Transformation Genotype: MATa, his3, trp1, ura3, LexAop(x6)-LEU2, peg202:tomnpr-1 Amp+ Trp-1+

PREY: Tomato cdna Library BAIT: Tomato NPR-1 EGY84 cdna B42 AD NPR-1 X LexA LexAop LEU2 lacz Media: Has galactose, no Leucine Genotype: MATa, his3, trp1, ura3, LexAop(x6)-LEU2, PEG202:TomNPR-1, PFJ4-5:cDNA cdna NPR-1 B42 AD LexA LexAop LEU2 lacz Media: Has galactose, no Leucine Media: Has galactose and X-gal, no Leucine

Discovery of NIF1 Found 7 classes of tomato genes that interacted with the NPR-1 bait NIF1 (NPR1-interacting factor 1) had the highest reporter activity Sequencing and BLAST search for biochem function NIF1 clone identified encodes carboxyl ⅔ of bzip transcription factor Did not include DNA-binding or leucine zipper domains Llorca, C. M., Potschin, M., & Zentgraf, U. (2014). bzips and WRKYs: two large transcription factor families executing two different functional strategies. Frontiers in Plant Science, 5, 169. https://doi.org/10.3389/fpls.2014.00169

Basic Leucine Transcription Factors (bzip TFs) 2 conserved structural domains: DNA-binding and leucine zipper DNA-binding domain contains many basic (positively charged) residues Binds major groove

Basic Leucine Transcription Factors (bzip TFs) 2 conserved structural domains: DNA-binding and leucine zipper Leucine zipper involved in dimerization: leucine residue in every 7th position so they are directly in line with each other Form hydrophobic core

Discovery of NIF1 Found 7 classes of tomato genes that interacted with the NPR-1 bait NIF1 (NPR1-interacting factor 1) had the highest reporter activity Sequencing and BLAST search for biochem function NIF1 clone identified encodes carboxyl ⅔ of bzip transcription factor Did not include DNA-binding or leucine zipper domains Llorca, C. M., Potschin, M., & Zentgraf, U. (2014). bzips and WRKYs: two large transcription factor families executing two different functional strategies. Frontiers in Plant Science, 5, 169. https://doi.org/10.3389/fpls.2014.00169

Question? What is the relationship between NIF1 and AHBP-1b, TGA6, OBF5?

FIG 2 - Bioinformatics After seeing tomato NIF1 interaction with tomato NPR1 in Y2H system via reporter expression... Used GenBank to identify Arabidopsis bzip transcription factors with homology: AHBP-1b, TGA6, OBF5 Aligned using MULTALIN: red is high consensus residues, blue is low consensus, black is no consensus MULTALIN

FIG 1 - Yeast Two-Hybrid Assay Y2H assay showing interactions of tomato and Arabidopsis NPR1 with Arabidopsis bzip transcription factors After growth seen with leucine drop-out plates, used -gal activity as quantitative assay Strong interaction of TomNPR1 with NIF1 Significantly lower interaction of Arabidopsis NPR1 with OBF5 than with AHBP-1b and TGA6

Question Hypothesis: Arabidopsis NPR-1 interacts with the Tomato NIF1 homologs TGA6, OBF5, and AHBP-1b How can you further confirm NPR-1 interacts with these three bzip proteins? His-tag Ni-NTA Column Co-purification

Column Purification New constructs made tagging the AHBP-1b and OBF5 proteins with 6 histidine residues then recombinant proteins were expressed in E.coli His has high affinity for Ni so tagged proteins will stay in column While untagged proteins and cell debris will be washed through Then tagged proteins eluted from column

FIG 3 - NPR1 co-purification with bzip TFs +ve AHBP-1b and OBF5 bind to NPR1 Purified with His-tagged AHBP-1b and OBF5 in Ni-NTA column Antibodies against NPR1 used to blot for co-purification What was the -ve control? What was the +ve control? Purified NPR-1 AHBP-1b + NPR-1 OBF5 + NPR-1 AHBP-1b + NPR-1 -ve NPR-1 Immunoblot Assay using NPR-1 antiserum Lanes 2-5 were run through the Ni-NTA Resin

Question Hypothesis: The bzip transcription factors interact with the ankyrin repeat domain on NPR-1. How can you confirm NPR-1 interacts with bzip TFs through the ankyrin repeat domain? Yeast Two Hybrid using: a) b) Smaller segments of NPR-1 as bait NPR-1 with mutations in ankyrin domain as bait

FIG 4A - Interaction Domain Determination Determining region of NPR1 protein that associates with AHBP-1b NPR1 gene fragments cloned into bait vector Exon 2: ankyrin repeat region X-Gal Reporter Gene Assay

FIG 4A - Interaction Domain Determination N terminal and anykrin repeat regions show similar -gal expression to full NPR1 gene N terminal appears to have intrinsic transactivation ability because -gal activity detected without prey N terminal likely stabilizes ankyrin repeat region X-Gal Reporter Gene Assay

Question? What is an ankyrin repeat? Conserved motif involved in protein-protein interactions Highly conserved histidine residues form H-bonds to stabilize the 3D structure of the domain Cao, H., Glazebrook, J., Clarke, J. D., Volko, S., & Dong, X. (1997). The Arabidopsis NPR1 Gene That Controls Systemic Acquired Resistance Encodes a Novel Protein Containing Ankyrin Repeats. Cell, 88(1), 57-63. doi:10.1016/s0092-8674(00)81858-9

FIG 4B - Point mutations abolish bzip TF binding Y2H assay using npr1-1 and npr1-2 proteins as bait npr1-1: His-334 Tyr-334 (ankyrin domain) npr1-2: Cys-150 Tyr-150 (N terminus) Growth on leucine drop-out seen only in WT NPR1 interactions with AHBP-1b and TGA6 Same results also seen with X-gal assay Yeast Two Hybrid Assay

FIG 4C - Point mutations do not cause improper protein expression Wanted to ensure that lack of reporter expression was due to mutation in npr proteins... Vs poor expression of the npr1-1 and npr1-2 proteins themselves Immunoblot shows similar expression levels to WT NPR1 Meaning point mutations impaired association with AHBP-1b and TGA6 Immunoblot Assay

Question Hypothesis: NPR-1 interacts with the bzip TFs (TGA6 and AHBP-1b), which bind to PR-genes and promote expression. How can you confirm bzip TFs interact with PR-gene regulatory sequences? Gel Mobility Shift Assay

Gel Mobility Shift Assay Lane 1: no protein added Lane 2: protein bound to probe runs slower than free probe so band seen on top of gel Lane 3: unlabelled probe binds all protein so only unbound labelled probe seen Unlabelled protein-bound probe will be at top of gel but cannot visualize Radiolabelled probe

as-1-like element in PR-1prom SA responsive as-1 element in CaMV 35S promoter Previously identified as binding motif for bzip TFs Previously shown to be essential for SA and INA-induced PR gene expression Pape, S., Thurow, C., & Gatz, C. (2010). Exchanging the as-1-like element of the PR-1 promoter by the as-1 element of the CaMV 35S promoter abolishes salicylic acid responsiveness and regulation by NPR1 and SNI1. Plant Signaling & Behavior, 5(12), 1669 1671. http://doi.org/10.4161/psb.5.12.14033

FIG 5 - Determining specificity of AHBP-1b binding to PR-1 Competitor probes to ensure specificity of AHBP-1b binding At 40x and 100x molar excess of unlabelled probe, no band shifting seen Ensures specificity of AHBP-1b to as-1 probe vs non-specific protein binding as-1m (non-competitor probe) shows band shifting so no AHBP-1b binding to unlabelled as-1m Gel Mobility Shift Assay

Redundancy of the bzip TFs Possible redundancy of AHBP-1b and TGA6 but only AHBP-1b was shown to bind to as-1-like element in gel mobility shift assay Strong AHBP-1b binding to as-1-like element requires 2 TGACG motifs but 1 appears sufficient for TGA6 Interaction of OBF5 and NRP1 significantly weaker than interaction of either AHBP-1b or TGA6 and NRP1 NIF1 less similar to OBF5 than AHBP-1b and TGA6

Question? What were some of the next steps proposed in the paper? Examine NPR1-bZIP TF interaction in vivo (Arabidopsis) Examine TGA6 and AHBP-1b expression patterns under SAR conditions Figure out role of bzip TF in PR gene regulation Knockout mutants Dominant-negative mutants Overexpression transgenic plants Role of bzip TF dimerization in the context of NPR1 binding Gel mobility with TGA6 and OBF5

NO SAR (uninduced conditions) Repressor? AHBP -1b TGA6 NPR-1 X PR GENE CYTOPLASM NUCLEUS

SAR (induced conditions) Repressor? AHBP -1b SA TGA6 NPR-1 AHBP -1b AHBP -1b PR GENE as-1 like element NPR-1 NPR-1 NPR-1 TGA6 NPR-1 TGA6 NPR-1 PR GENE as-1 like element CYTOPLASM NUCLEUS

Summary 1. 2. 3. NPR-1 interacts with bzip proteins TGA6, OBF5, AHBP-1b in vivo (yeast) and In vitro (Y2H and Ni-NTA column co-purification) The N-terminal region and the ankyrin repeat domain are necessary for bzip protein binding (Y2H with NPR-1 fragments and Y2H with npr1-1 and npr1-2 mutants) The bzip ABHP-1b protein bind specifically to the as-1 like element on PR-1. (Gel Mobility Shift Assay)

Review How does NPR1 transduce the SA signal and up-regulate the PR genes? Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for induction of the PR-1 genes?