Lecture 3-4. Identification of Positive Regulators downstream of SA

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1 Lecture 3-4 Identification of Positive Regulators downstream of SA

2 Positive regulators between SA and resistance/pr Systemic resistance PR gene expression? SA Local infection Necrosis SA What could they be?

3 Hunt for regulators of SAR Genetic screens for positive regulators downstream of SA How do you find mutants non-responsive to SA?

4 Hunt for regulators of SAR Case 1: John Ryals Group Host plant: Arabidopsis Ws ecotype Pathogen: oomycete Peronospora parasitica Emwa2, causes downy mildew Treatment: INA or SA spray followed by Emwa2 inoculation 280,000 M2 plants from EMS mutagenesis and 80,000 T4 plants (from T-DNA transformation) identified 75 lines that were defective in SAR. nim1 compromised the most.

5 WT nim1 H 2 O INA SA

6 Where to place NIM1?

7 NIM1 Systemic resistance PR gene expression SA? Local infection Necrosis? SA

8 SA SA UDP-Glycosyltransferase SAG (SA+SAG) What does the figure tell us about NIM1 function?

9 Systemic resistance PR gene expression NIM1 SA? Necrosis SA Local infection

10 What genetic tests need to be done with nim1? - Heritable? - Dominant/recessive? - Single gene? - Allelism test

11 nim1 x WT Conclusions?

12 Conclusions?

13 Conclusions?

14 Induction of PR-1 mrna by pathogen infection or chemicals Pathogens? SA?? NIM1 nim1-5 PR1 PR1? - Not allelic to nim1-1 - unusual allele of nim1 - Allelic to nim1-1, other mutations affect the phenotype

15 How would you clone nim1? nim1-1 x Ler F1 F2 Crude mapping fine mapping

16 Crude mapping Fine mapping Cosmid contig

17 Which clones contain the NIM1 gene?

18 Sequenced the overlapping region and a mutation found in nim1-1

19 Is nim1 cloned? What else should we do? - Sequence other alleles - Single gene complementation - Test T-DNA knockout alleles

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22 nim1-5 - Not allelic to nim1-1 - unusual allele of nim1 - Allelic to nim1-1, other mutations affect the phenotype? Which agrees with the sequencing data?

23 Systemic resistance PR gene expression NIM1 SA? Necrosis SA Local infection

24 Hunt for regulators of SAR Forward genetic screens conducted to search for positive regulators downstream of SA Case 2: Xinnian Dong s Group at Duke University Screen for mutants in which PR genes can not be induced by SA How?

25 BGL2 (PR2) Promoter-GUS reporter BGL2 promoter GUS Pathogen SA or INA GUS: β-glucuronidase

26 WT SA or INA npr1-1 SA or INA EMS, 13,500 M2 plants screened, 76 putative mutants identified npr: non-expresser of PR genes

27 What kinds of mutations can abolish PR2 promoter-gus expression? - Genes required for induction of PR2 by SA - GUS reporter gene

28 How would you confirm your mutants? Systemic resistance PR gene expression SA? Necrosis SA Local infection

29 npr1-1

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31 NPR1 Systemic resistance PR gene expression SA? Necrosis SA Local infection

32 Map of the NPR1 Locus on Chromosome I 1 2 3

33 H 2 O INA SA Clone 1 Clone 2 Clone 3 WT npr1-2

34 Map of the NPR1 Locus on Chromosome I 1 2 3

35 Summary of positional cloning Generate a mapping population Pick plants with the mutant phenotype in F2 Crude mapping Generate additional markers Fine mapping of the mutation Generate cosmid contigs Perform complementation analysis Sequence overlapping region to identify the mutation PCR Seq Whole genome Seq

36 Predict the function of NPR1

37 Predict the function of NPR1 1. Blast search 2. Identification of protein domains SMART (Simple Modular Architecture Research Tool) 3. Prediction of protein subcellular localization PSORT, WoLF PSORT 4. Analysis of gene expression pattern Arabidopsis efp Browser Where were the data used to build the efp Browser coming from?

38 NPR1 is a protein with ankyrin repeats What does it tell us about how NPR1 functions?

39 NIM1/NPR1 Systemic resistance PR gene expression SA? Necrosis SA Local infection

40 Forward genetic screens conducted to search for positive regulators downstream of SA Lab Screens Mutants obtained Ryals, Novartis Non-immunity (NIM) nim1-1 to nim1-6 Klessig, Rutgers Dong, Duke Ausubel, Harvard SA insensitive (SAI) No PR-gene expression (NPR) Enhanced disease susceptibility (EDS) sai1-1 and sai1-2 npr1-1 npr1-2 to npr1-4

41 Why all the SAR screens only yielded mutants carrying mutations in one gene --- NPR1? Why other things such as TF were not found?

42 NPR1 is a positive regulator of SAR. Can we use NPR1 to regulate the SAR pathway? How would you enhance SAR with NPR1?

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44 Disease symptoms caused by Psm ES4326

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49 Growth of the oomycete pathogen Peronospora parasitica Noco2 on wild type and NPR1 transgenic lines

50 What can we conclude from the data?

51 35S-NPR1-H snc1 WT

52 References: 1. Buchanan et al Biochemistry and molecular biology of plants. ASPP, Rockville, MD. Chapter Cao H, Bowling SA, Gordon AS, Dong X. Characterization of an Arabidopsis Mutant That Is Nonresponsive to Inducers of Systemic Acquired Resistance. Plant Cell Nov;6(11): Cao H, Glazebrook J, Clarke JD, Volko S, Dong X. The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell ;88(1): Cao H, Li X, Dong X. Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proc Natl Acad Sci U S A May 26;95(11): Delaney TP, Friedrich L, Ryals JA. Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance. Proc Natl Acad Sci U S A Jul 3;92(14): Ryals J, Weymann K, Lawton K, Friedrich L, Ellis D, Steiner HY, Johnson J, Delaney TP, Jesse T, Vos P, Uknes S. The Arabidopsis NIM1 protein shows homology to the mammalian transcription factor inhibitor I kappa B. Plant Cell Mar;9(3):