1. Slide 1 © CSIR 2006 www.csir.co.za The salicylic signalling pathway confers tolerance to a biotrophic rust pathogen in pearl millet Bridget Crampton Dave Berger

2. Slide 2 © CSIR 2006 www.csir.co.za Aims of the study  Elucidate defence response mechanisms in pearl millet  Evaluate pearl millet defence signalling pathways involved in leaf rust (Puccinia substriata) tolerance

3. Slide 3 © CSIR 2006 www.csir.co.za Pearl millet  Sixth most important cereal crop  Staple food in Africa and Asia  Cultivated for forage and grain  High temperature and drought tolerance  Abundant natural diversity  Resistance to diseases, insects and parasitic weeds  Defence response mechanisms poorly understood and not exploited

4. Slide 4 © CSIR 2006 www.csir.co.za Plant defence response mechanisms Compatible vs incompatible reaction PathogenHR SAR Salicylic acid = essential molecule in SAR induction Jasmonic acid and ethylene Salicylic acid

5. Slide 5 © CSIR 2006 www.csir.co.za Plant defence response mechanisms

6. Slide 6 © CSIR 2006 www.csir.co.za a a b waterwater + rustMeJa + rust SA + rust Defence signalling molecule treatment and subsequent rust infection  Arabidopsis – resistance to biotrophic pathogens = SA defence signalling

7. Slide 7 © CSIR 2006 www.csir.co.za Microarray preparation Chitin, flg, woundWater ID SSH/cDNA library Target amplification Array Spotter Probe preparation and hybridisation SA, MeJA,treatment 0, 12, 24 and 48 hpt RNA extraction Oh treatment12 h treatment Cy dye incorporation Hybridisation & scanning Data analysis Image analysis in Genepix Normalisation Average within slide replicates Average replicates between slides Up- and downregulated genes over time R//LimmaGUI/Excel

8. Slide 8 © CSIR 2006 www.csir.co.za A. Induced B. Repressed MeJASA 75 189 108 MeJASA 01642 Regulatory relationship between SA and MeJA signalling pathways Relative to untreated control P<0.05

9. Slide 9 © CSIR 2006 www.csir.co.za SA responsive genes involved in resistance to rust

10. Slide 10 © CSIR 2006 www.csir.co.za qRT-PCR validation of microarray data

11. Slide 11 © CSIR 2006 www.csir.co.za Concluding remarks Pretreatment of pearl millet with salicylic acid conferred tolerance to subsequent rust infection. Gene expression analysis revealed substantial overlap in pearl millet’s gene expression responses to SA and MeJA treatments. Large scale gene expression profiling identified transcripts that were uniquely responsive to SA treatment.

12. Slide 12 © CSIR 2006 www.csir.co.za The way forward MicroArray Data Interface for Biological Annotation (MADIBA) Virus induced gene silencing (VIGS) Transgenic plant studies Promoter mining studies

13. Slide 13 © CSIR 2006 www.csir.co.za Publications Crampton BG, I Hein and DK Berger. 2007. The salicylic acid signalling pathway confers tolerance to a biotrophic rust pathogen in pearl millet. In preparation. Berger DK, B Crampton, I Hein, and W Vos. 2006. Screening cDNA libraries on glass slide microarrays, In: J. M. Walker (ed.), DNA Arrays. Humana Press, Totowa, New Jersey, USA. Van den Berg N, BG Crampton, I Hein, PRJ Birch and DK Berger. 2004. High- throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis. Biotechniques 37:818-824. O’Kennedy MM, B Crampton, and P Ozias-Akins P 2007. Pearl millet, In: “The Blackwell Handbook of Transgenic and Biotech Plants”. Wiley Blackwell, In Press.

14. Thank you