Knockout Analysis of Arabidopsis Transcription Factors TGA2, TGA5, and TGA6 Reveals Their Redundant and Essential Roles in Systemic Acquired Resistance Yuelin Zhang, Mark J. Tessaro, Michael Lassner, and Xin Li Plant cell Volume 15(11): November 3, 2003 Paul Kapos, Wanwan Liang, Jianhua Huang

Outline  Introduction --- Systemic acquired resistance (SAR) --- SA --- NPR1 and TGAs  Objective and hypothesis  Results and conclusions  Perspective

Systemic acquired resistance (SAR) Pieterse ea al., 2009  a pathogen-induced defense response against broad-spectrum of pathogens  PR genes are highly induced  SA level increases locally and systemically

Salicylic Acid (SA)  A key signal molecule in SAR  SA is known to be both necessary and sufficient to induce SAR  Both SA and its analogs are able to induce SAR INA SA BTH

The importance of SA in plant defence SA degradation occurs in nahG transgenic plants OR SA synthesis is affected in the mutants eds5 and sid2 Plants are more susceptible to pathogens!

NPR1 (Non-expresser of PR genes) npr1-1 phenotype:  Compromised basal resistance  Insensitive to all known SAR inducers  Abolished SAR Cao et al., 1994 BLG2= PR2 Cao et al., 1994

Question: How does NPR1 exert its function? BTB/POZ Ankyrin-repeat domain NLS NPR1 Kinkema et al., 2000 GFPNPR1-GFP BTB/POZ domain: homomeric dimerization and heteromeric dimerization; ANK domian ： protein–protein interaction What are the possible types of protein that NPR1 would associate with?

TGA transcription factors  Belong to a group of bZIP transcription factors in all eukaryotes  Bind specifically to variants of the palindrome TGACGTCA  10 members in Arabidopsis factors-/ html

NPR1 interactors----TGA2, TGA5, TGA6 identified in Yeast-two hybrid screening Yeast two-hybrid assay of interactions between NPR1 and bZIP transcription factors Bait: tomato NPR1 homolog (Tom NPR1) Prey: a tomato cDNA library Identify tomato NIF1 (NPR1-interacting factor 1) Sequence tomato NIF1 and do Blast analysis Identified three A. thaliana genes TGA2, TGA5,TGA6 Confirmation of the interactions

 Objective: To provide genetic evidence for the roles of these TGAs in SAR promoter TGAs NPR1 PR-1 Nucleus

A fast neutron deletion mutagenesis-based reverse genetics system for plants ---- Isolation of tga6-1 and tga2-1 tga5-1 double mutant Li et al., 2001

Construction of tga2-1 tga5-1 tga6-1 triple mutant tga2-1 tga5-1 tga6-1 Crossing tga2-1 tga5-1 tga6-1 triple mutant Li et al., 2001

Question: For the tga6 tga2 tga5 triple mutant what ratio of genotypes would you expect in the F2 generation?

Results

The TGA Triple Knockout Mutant Is More Sensitive to the Toxicity of SA These TGA transcription factors play roles similar to that of NPR1 in the regulation of tolerance to SA

Induction of PR-1 by INA Is Blocked in the TGA Triple Mutant pathogen HR SA chemical (INA, BTH) NPR1 PR1 Question: What might it suggest that the basal level of PR1 was higher in tga2 tag5 double mutant and tga6 tga2 tga5 triple mutant?

Disease rating scores: 0: no conidiophores on the plants; 1: no more than 5 conidiophores per infected leaf; 2: 6 to 20 conidiophores on a few of the infected leaves; 3: 6 to 20 conidiophores on most of the infected leaves; 4: 5 or more conidiophores on all infected leaves; 5: 20 or more conidiophores on all infected leaves. SAR Analysis 1. Treat plants with an SA analog or a pathogen in order to elicit SAR 2. Then treat the plants with a virulent pathogen (Peronospora parasitica Noco2 or P.s.m. ES4326) 3. Measure the growth of the pathogen

SAR Is Abolished in TGA Triple Knockout Mutants What would happen with an avirulent pathogen?

Plant immune system Defense Responses Pathogen-associated molecular patterns (PAMPs) PAMP triggered immunity(PTI) Pattern recognition receptor MAPKKK MAPK Resistance protein Effector triggered immunity(ETI) Effectors

Growth of P.s.m. ES4326 in plants preinoculated with avirulent P.s.t. DC3000 avrRpt2 SAR Is Abolished in TGA Triple Knockout Mutants What is a possible reason that SAR is abolished in both npr1 and the tga triple mutant while basal resistance is only compromised in npr1?

Conclusion In the TGA triple mutant:  Sensitivity to SA  Induction of PR-1 is blocked  SAR is abolished

Confirmation that the TGA genes are actually causing the SAR phenotype Question: What are some methods of connecting a phenotype to specific genes?

F1tga6-1/+ tga2-1/+ tga5-1/+ ↓ F2tga6-1/+ tga2-1 tga5-1 F3 ¼ +/+ ½ tga6-1/+ ¼ tga6-1/tga6-1 What would we expect the F3 generation to look like?

The plants which were susceptible were homozygous for the tga6-1 mutation

TGA2 and TGA5 are tightly linked, such that the tga2-1 tga5-1 double mutant is caused by a single mutation Co-segregation with each of these genes is not possible What can be done to show that these genes are essential for SAR? Transgene Complementation

Both TGA2 and TGA5 can rescue the phenotype of tga triple mutant

TGA2 and TGA5 Complementation Either Transgene is capable of: Restoring PR gene expression in response to INA Eliminating SA sensitivity in seedlings Restoring acquired resistance in response to SA Question: What does this information tell us?

Summary In the TGA triple mutant:  More sensitive to SA  Induction of PR-1 is blocked  SAR is abolished  Compromised SAR cosegregates with tga6-1 and either transgenic TGA2 or TGA5 can complement these phenotypes. These TGA’s are redundant and play an essential role in SAR!

Future Perspectives  In what way do TGA transcription factors interact with NPR1?  How do TGA transcription factors lead to PR gene expression and enhanced defense?  Do the other TGA transcription factors have a role in resistance?

The Roles of the Various TGA Transcription Factors  TGA2 TGA5 TGA6 are involved SAR, jasmonic acid/ethylene-dependent immune response and detoxification program upon treatment with toxic chemicals.(Zhang et al., 2003; Zander et al., 2010; Fode et al., 2008)  TGA1 and TGA4 are involved in nitrate response in Arabidopsis roots(Alvarez et al., 2014)  TGA9 and TGA10 are required for Anther Development(Boyle et al., 2010)

Boyle et al. Plant Cell 2009;21: NPR1 and TGA2 TGA2 repress transcription by forming an oligomer capable of binding to its cognate TGACG sequence in the promoter of target genes. After a rise in SA, the NPR1 POZ domain would either assist in disassembling the TGA2 oligomer or assist in recruiting TGA2 dimers to cognate DNA

Meenu Kesarwani et al. Plant Physiol. 2007;144: TGA3 is a positive regulator of PR1 expression and basal resistance

Meenu Kesarwani et al. Plant Physiol. 2007;144: Overexpression of TGA6 leads to activation of PR1 expression in tga2-2

Questions?

Thanks for your attention!