Plant Defense: A Glimpse By Wisuwat Songnuan

Outline Background Systemic Acquired Resistance NPR1-TGAs That’s not all… Future

Background

Background Outline Why study plant resistance? Pathogen Recognition Gene-for-gene interactions Hypersensitive Response (HR) Systemic Acquired Resistance (SAR)

Why study plant resistance? 80% of total calories consumed by human population come from only six crops: wheat, rice, maize, potatoes, sweet potatoes, and manioc (Raven, P.H. et al, 1999). We lose 12% of total crop yields to pathogen infection– equivalent to nine hundred million tons worldwide annually (Krimsky S. and Wrubel R., 1996).

Plants under attack Microorganisms: viruses, bacteria, fungi Nematodes Insects & a few others Us?

What will YOU do? Lots of enemies, attacking from all sides Huge body Cannot escape No “patrol” (no NIH grant)

How THEY do it Right after plants are dead, they are rotten No wasting energy for ‘just in case’ immunity All through “signaling”

Pathogen recognition Gene-for-gene hypothesis: Upon infection by a particular avirulent pathogen, a corresponding R gene recognizes the avr product and triggers the defense mechanism. Why do pathogens still possess avr genes? Non-host resistance: Resistance of all members of a host species against all members of pathogen species

Resistance (R) Genes Dominant Many ID so far 5 classes recognized NBS: Nucleotide binding site Leucine-zipper and leucine-rich repeat (LRR) Toll/IL-1R (TIR) Protein kinase (PK), receptor-like kinase (RLKs)

The popular ones… Maize Hm1 (1992): toxin reductase Tomato Pto (1993): Ser/Thr kinase Arabidopsis RPS2: Tobacco N: Tomato Cf9 Flax L6 Rice Xa21

Hypersensitive Response (HR) Burst of oxygen reactive species around infection site Synthesis of antimicrobial phytoalexins Accumulation of Salicylic Acid (SA) Directly kill and damage pathogens Strengthen cell walls, and triggers apoptosis Restrict pathogen from spreading Rapid and local

Systemic Acquire Resistance (SAR) Secondary response Systemic Broad-range resistance Leads to Pathogenesis-Related (PR) gene expression Signals: SA, JA, ethylene

Systemic Acquired Resistance (SAR)

Salicylic Acid (SA) COOH OH Accumulates in both local and systemic tissues (not the systemic signal) Removal of SA (as in nahG plants) prevents induction of SAR Analogs: INA or BTH

Mutants affecting SA synthesis Elevated SA accumulation dnd1 (defense, no death 1): increased SA, but reduced HR, DND1 gene encodes cyclic-nucleotide-gated ion channel mpk4: constitutive SA accumulation edr1 (enhanced disease resistance 1): defective MAPKKK

Mutants affecting SA synthesis reduced SA accumulation eds1 (enhanced disease susceptibility 1): lipase homolog pad4 (phytoalexin deficient 4): another lipase homolog sid1 and sid2 (salicylic acid induction-deficient): defects in chorismate pathway

Mutant Screen Aimed at identifying regulatory genes of SAR Strategy: Transform Arabidopsis with GUS reporter driven by SA- and INA-responsive promotor from BGL2 gene npr1 (non-expresser of PR genes) mutant: reduced induction of reporter gene with or without SA, INA cpr (constitutive expresser of PR genes) mutants: constitutively express reporter genes

NPR1: non-expresser of PR genes Also known as NIM1 or SAI1 Positive regulator of SAR Downstream of SA, upstream of PR genes npr1 mutants are susceptible to various pathogens Overexpression of NPR1 generates broad-spectrum resistance Unique, but similar to Iκ-B (negative regulator of immunity in animals)

NPR1 overexpression

Pathogen-Related (PR) Genes Antimicrobial properties Many identified Categorized according to activity Examples PR-2 : beta-1,3-glucanase PR-3 : chitinase PR-12: defensin

SAR Avr R gene SA NPR1 PR-1 PR-2 PR-5 SAR

Structural features of NPR1 nim 1-2 npr 1-1 NLS S S BTB ARD 593 amino acids, 67 kD Two protein-protein interaction domains: BTB/POZ and Ankyrin repeats Contains NLS Multiple phosphorylation sites No DNA binding domain

NPR1-GFP localizes in nucleus upon SAR induction MS MS-INA NPR1-GFP GFP

TGA Factors Found to interact with NPR1 through yeast-two hybrid bZIP transcription factors Six members in Arabidopsis (TGA1-6) Might be redundant Bind to as-1 element

NPR1-TGA2 interaction Direct visualisation

TGA2 C-term interacts with NPR1

PR-1 expression reduced in TGA2CT lines Figure 2A, 2B

Reduced resistance to P.parasitica and tolerance to SA Figure 2C, D

DN effects depends on NPR1 Figure 3A, B

SA affects NPR1-TGA2 interaction Figure 3C, D

Chimera Reporter System Figure 4

TGA2-GAL4 is SA-responsive Figure 5A,B

TGA2-GAL4 as an activator Figure 5C

DNA binding dependent on NPR1 and enhanced by SA Figure 5D

Current model Figure 6

SAR Avr R gene SA TGA2 NPR1 PR-1 PR-2 PR-5 SAR

NPR1-TGA5

Yeast-two hybrid Figure 1 a-d

Co-purification

TGA2 mRNA accumulation untreated P.parasitica INA Figure 2

TGA5 mRNA accumulation untreated P.parasitica INA Figure 3a

Surprising accumulation of TGA5 in antisense lines untreated P.parasitica INA Figure 3b

PR-1 induction in TGA2 transformants Figure 4

Reduced PR-1 expression in lines with high TGA5 mRNA Figure 5

TGA5-antisense lines resistant to infection WT AS15 AS16 Figure 6

TGA5-antisense lines resistant to infection

AS15 resistance is independent of NIM1

SAR Avr R gene SA TGA2 NPR1 TGA5 PR-1 PR-2 PR-5 SAR SAR independent resistance

That’s not all…

A few others Ethylene-mediated response Jasmonic acid-mediated response Induced systemic resistance (ISR) MAPK cascades

The future Still a lot to learn 2010 project The golden era

Thank you!