ABA-dependant signalling of PR genes and potential involvement in the defence of lentil to Ascochyta lentis

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1 ABA-dependant signalling of PR genes and potential involvement in the defence of lentil to Ascochyta lentis Barkat Mustafa, David Tan, Paul WJ Taylor and Rebecca Ford Melbourne School of Land and Environment The University of Melbourne Australia

2 Ascochyta lentis Host specific to lentil Necrotroph Foliar lesions (reduced photosynthesis) and stem girdling Flower and pod abortion Seed discoloration, decreased size and marketability

3 Host response Genotype (R) Tissue Genetic model for resistance Reference Indian head Foliar 2 additive recessive Ye et al, 2001 Laird Seed 1 recessive Tay and Slinkard, 1989 Laird Foliar 1 recessive Ye et al, 2001 Northfield Foliar 1 dominantt Ford et al, 1999 Northfield Seed 1 dominant Vakulabharanam, 1997 Northfield Foliar 1 dominant + minor Ye et al, 2003 ILL5684 Seed 1 dominant Tay and Slinkard, 1989 ILL5684 Foliar 1 dominant Ye et al, 2001 ILL7537 Foliar 2 complimentary dominant Nguyen et al, 2001 ILL6002 Foliar/seed Highly susceptible Ford et al, 1999; Nguyen et al, 2001

4 Genotypes Disease reactions to isolate pathotypes* I II III IV V VI VII Digger S S S S S S MR ILL6002 S S S S S S S Indianhead MR MR MR S MR S R ILL5588 MR S MR MR S S R ILL7537 R R R R R R R Nipper MR MR R R MR MR R Prabhakaran et al. unpub

5 Molecular response - PulseChip microarray 565 chickpea/a. rabiei ESTs 156 lathyrus/a. pinodes ESTs DNA/RNA synthesis/turnover 1% Structural 3% Metabolism 8% Homeostasis 4% Defence 3% Signalling/Transport 5% Homeostasis 4% Metabolism 7% Structural 3% Photosynthesis 14% Protein synthesis/turnover 5% DNA/RNA synthesis/turnover 2% Defence 5% Photosynthesis 13% Unknown 44% Protein synthesis/turnover 22% Signalling/Transport 2% Unknown 55%

6 Evidence of a time-dependant response 0.9 Superoxide dismutase IlLL7537 (R) h 24h 48h 72h 96h ILL6002 (S) Evidence of an oxidative burst Detoxifies cells of superoxide to hydrogen peroxide Mustafa et al, 2009

7 Carbonic anhydrase IlLL7537 (R) ILL6002 (S) 6h 24h 48h 72h 96h Local antioxidant activity (SA binding)

8 6 hpi in ILL7537 (R) Spore germination and appressoria development serine/threonine protein kinase magnesium chelatase β-1,3-glucanase peptidyl-prolyl isomerase

9 24 hpi in ILL7537 (R) Penetration and host cell cytoplasm disturbance PR4 protein carbonic anhydrase nucleotide-sugar dehydratase superoxide dismutase

10 48-96 hpi in ILL7537 (R) Host cell cytoplasm and nucleus disintegrates leading to symptoms of disease (HR) PR10a protein amine oxidase ubiquinol-cytochrome C reductase magnesium chelatase lipid-transfer protein precursor serine:glyoxylate aminotransferase

11 PR gene families Family Biochemical properties PR1 antifungal PR2 β- 1,3-glucanse PR3 chitinase type I,II,IV,V,VI,VII PR4 chitinase type I,II PR5 thaumatin- like PR6 proteinase-inhibitor PR7 endoproteinase PR8 chitinase type III PR9 peroxidase PR10 ribonuclease- like PR11 chitinase type I PR12 defensin PR13 thionin PR14 lipid-transfer protein PR15 oxalate oxidase PR16 oxalate-oxidase-like PR17 unknown

12 Aims of research Study the effects of global signalling molecules on the transcription of genes encoding two previously identified Ascochyta lentis-responsive PR proteins; PR4 (DY396388) PR10a (DY396377) Better understand the defence pathways involved in resistance to A. lentis in ILL7537 Determine potential to use a signalling molecule as a protectorate spray against A. lentis and as part of an IPM program on susceptible lentil lines (ILL6002)

13 Global signaling defence molecules Signaling molecules activate signal transduction cascades for local and SAR responses: Salicylic Acid (SA) regulates oxidative burst, phytoalexins, HR and PR1, PR2 and PR5 expression Methyl Jasmonate/Jasmonic Acid (MeJA/JA) regulates developmental processes (embryo, seed etc) and PR3, PR4 and PR12 expression Ethylene and immediate precursor (ET/ACC) regulates metabolic processes (germination, senescence etc) and PR2, PR3, PR4 and PR12 expression Abscisic Acid (ABA) regulates development and stress responses. Role in plant defence is unclear

14 Defence signaling in plants (Anderson et al 2005)

15 PR4 gene from lentil Single copy chitinase gene revealed by Southern blot Isolated using Lathyrus sativus PR4 sequence (DY396388; Skiba et al 2005) Full-length cdna of 636 bp, predicted 146 amino acid protein. mw = 15.8 kda CGCG cis-element in the promoter region indicates involved in defence signaling Nuclear export signal in the signal peptide indicates extracelluler localisation.

16 PR10a gene from lentil Single copy ribonuclease-like cytoplasmic protein gene revealed by Southern blot Isolated using Lathyrus sativus PR10 sequence (DY396344, Skiba et al 2005) Full-length cdna of 783 bp, predicted 156 amino acid protein. mw = 17.9 kda 99 pr10a lentil ACJ83999 Medicago truncatula 74 AAQ72568 Pisum sativum 100 AAW80931 Astragalus membranaceus CAG34224 Cicer arietinum AAD50376 Glycine max 33 EEF36378 Ricinus communis CAA11844 Rubus idaeus CAA04770 Fragaria vesca 99 XP Populus trichocarpa CAB85634 Vitis vinifera CAC43292 Beta vulgaris CAC83580 Arabidopsis ABA01325 Gossypium hirsutum ABV89615 Brassica rapa 99 CAA96536 Malus x domestica CAA71619 Catharanthus roseus BAD29692 Oryza sativa

17 Approach 1 14-day-old ILL7537 seedlings Sprayed until run-off with 100 µm of ABA, ACC, MeJA or SA Controls were sprayed with H 2 O only Foliage harvested 0, 6, 24, 48, 72, 96 h after treatment Total RNA extracted and cdna synthesized Triplicate qrt-pcr with optimised PR4 and PR10a primers quantified transcribed fold changes relative to actin via the ΔΔC t method

18 Relative PR-4 expression Relative PR-4 expression Relative PR-4 expression Relative PR-4 expression PR4 relative transcript fold changes in response to signal compounds alone A 10 B Cont ABA ACC MeJA SA 6 h post treatments 0.1 Cont ABA ACC MeJA SA 24 h post treatments C D Cont ABA ACC MeJA SA 48 h post treatments 0.1 Cont ABA ACC MeJA SA 96 h post treatments

19 Relative PR-10a expression Relative PR-10a expression Relative PR-10a expression Relative PR-10a expression PR10a relative transcript fold changes in response to signal compounds alone A 10 B Cont ABA ACC MeJA SA 6 h post treatment 0.1 Cont ABA ACC MeJA SA 24 h post treatment C D Cont ABA ACC MeJA SA 48 h post treatment 0.1 Cont ABA ACC MeJA SA 96 h post treatment

20 Approach 2 14-day-old ILL7537 seedlings Sprayed until run-off with 100 µm of ABA, ACC, MeJA or SA, dried and then 1 x 10 5 spores ml -1 A. lentis (AL4) applied Or sprayed with H 2 O then A. lentis spore suspension

21 PR4 transcription patterns: Signaling molecules vs A. lentis in ILL7537 ABA sustained PR4 expression

22 PR10a transcription patterns: Signaling molecules vs A. lentis in ILL7537

23 Percent leaf disease Protectorate activity of ABA on ILL6002 (S - missing the R-gene?) A. lentis +/-10 M ABA A. lentis +/-100 M ABA Control 20 Control 10 ABA 100 ABA Day 11 Day 18 Day 25 Day 32 0 Day 11 Day 18 Day 25 Day 32 Days after inoculation

24 Hypothetical model of lentil defence hpi Elicitor 6 ß -1,3 glucanase R gene? S/T kinase chitinase 24 O 2 - ABA PR-4 SOD H 2 O 2 SAR 48 Amine oxidase ribonuclease HPR glycoprotein PR10a Cell wall reinforcement

25 Conclusions Both PR4 and PR10a were up-regulated in response to A. lentis and ABA (between 24 and 96 hpi) Initial screening shows ABA to provide some level of protection against A. lentis disease on leaves of the susceptible ILL6002 Propose that ABA plays a role in the signal transduction of defence responses against A. lentis in lentil Further functional validation of ABA modulation of defence is required