The Xylanase Transient Quantification System

1. A u s t r a l i a ’ s G r o w i n g F u t u r e The Xylanase Transient Quantification System Claudia Vickers

2. Transient vs. Stable Transformation • Transient transformation • DNA extra-chromosomal • Seen over several days following transformation • Stable transformation • DNA integrated into chromosome • Demonstrated by transferral of transgene to offspring • Segregation occurs in offspring

3. Transient Transformation as a Tool • Quick and easy (days compared to months) • Not prone to position-of-integration effects • Cheap and less labour-intensive than producing transgenics (esp. cereals) • Measure tissue specificity and expression strength • But – expression patterns not always reflected in stable transgenics

4. Reporter Genes • Genes which allow easy detection of expression to confirm occurrence of transformation • Different reporters useful for different experiments • Properties of the reporter gene must be tuned to the experiment

5. Desirable Features • Short coding sequence (easy to manipulate) • Low (preferably no) endogenous activity in plants • No endogenous substrates in plants • Easy, quantitative assay • Cell autonomous • Tolerate terminal fusions (purification) • Active and stable under a range of cellular conditions • Cheap!!!

6. Reporter Genes

7. Transient Analysis • Tissue specificity • How many tissues?? • Limited to tissues that can be transformed • Tissue specificity may require chromosomal integration • Most reporters will suffice • Quantification • Assess promoter strength • Test effect of introns, enhancers, 5  and 3 UTRs etc. • Not all reporters are suitable

8. Expression Strength • Focus counting • Co-transform with GFP and GUS plasmids • Count foci (GFP and GUS) • Expression strength = ratio of GFP:GUS • Problems with threshold effects

9. Focus Counting • Quick, cheap, easy Plasmids containing construct and reporter 1m gold particles

10. Focus Counting: Threshold Effect Barley Wheat Actin E X S M B OG OG X OG S OG M OG D1 OG D3 GFP D3 D1 UTR TSS 300 900 600 0 bp 1200 bp

11. Focus Counting: Threshold Effect Saturation threshold Detection threshold

12. Focus Counting – Threshold Effect

13. Expression Strength • Focus counting • Co-transform with GFP and GUS plasmids • Count foci (GFP and GUS) • Expression strength = ratio of GFP:GUS • Problems with threshold effects • Protein extraction and reporter quantification • Shoot with GUS and LUC plasmids • Extract protein from tissues • Quantitative reporter gene assays • Expression strength = ratio of reporter activity • Problems…. Schledzewski & Mendel 1994

14. Reporters for Quantitative Assay • GUS: Fluorometric, time-response assay • Very sensitive (can increase incubation time) • Slope over time – good statistical significance • Problems with endogenous activity and/or quenching (particularly in leaf extracts) • LUC: Luminometric assay • Extremely labile • No option to increase sensitivity • Expensive (substrate, co-factors, detection equipment)

15. Solution: Xylanase • Thermostable XYN developed by gene shuffling • Codon optimised • Stable transformants generated • Time-response assays possible (increase sensitivity) • Substrate:AZCL-xylan • Minor problem: insoluble • Other potential substrates: 4-MU-xylan (fluorometric), soluble colorimetric substitute • Cheap Cheap Cheap (like the birdie)! • Used in concert with GUSPlus greatly incr. sensitivity

16. Physical Optima • pH = 4.5 • Temp. = 40C • Linear response to enzyme concentration • Substrate saturates at 0.5%

17. Time Response • Linear over time • Up to 48 hr • Allows accurate quantification of weak promoters

18. High-Throughput • Colour intensity for each well depends on: • Transformation efficiency • Concentration of sample • Time of incubation • Standardised to internal control (GUS)

19. Deletion Analysis- Focus Counting Barley Wheat Actin E X S M B OG OG X OG S OG M OG D1 OG D3 GFP D3 D1 UTR TSS 300 900 600 0 bp 1200 bp Actin OG OG X OG S OG M OG D1 OG D3

20. Deletion Analysis – GUS:XYN Hordein Globulin Glo X Glo S Glo M Glo D1 Glo D2 Glo D3 Negative GUSPlus GUSPlus :Xylanase :Xylanase 100 100 200 200 300 300 400 400 500 500 300 900 600 0 bp 1200 bp 300 300 900 900 600 600 0 0 bp bp 1200 1200 bp bp

21. Summary: XYN/GUSPlus • Quick (transient vs. stable) • User-friendly • Accurate • Cheap • Sensitive • Vickers, C.E.; Xue, G.-P.; Gresshoff, P.M. (2003) A synthetic xylanase as a novel reporter in plants. Plant Cell Rep. 22(2):135-140

22. Xylanase Assaysin Plant TissuesClaudia Vickers [claudia.vickers@csiro.au]Caveat: It is assumed that the readers have read the paper entitled, ”A synthetic xylanase as a novel reporter in plants” (Plant Cell Reports 22(2):135-140, 2003) before reading these notes. IntroductionReporter Genes UsedDetailed MethodSolutionsReferences Print FriendlyVersion (PDF)click here Introduction Quantification of the effect of promoter regions on reporter gene expression in transient assays can be used to gain information about how a particular section of DNA drives gene expression in isolation from a chromosomal context. There are advantages and disadvantages associated with using transient analysis. The obvious disadvantage is that promoters do not always behave in the same fashion when integrated into plant DNA. Conversely, one can obtain data that is not affected by position-of-integration effects. In addition, the method is quick and simple compared to the production of transgenic plants. As noted previously, this is particularly useful when investigating promoter activity in species for which the transformation efficiency is relatively low. Accurate quantification of transient expression requires extraction of protein and quantitative analysis of reporter gene activity. Extremely high sensitivity of reporter gene assays is required because of the dilution of the gene product during protein extraction (the vast majority of cells in bombarded tissues are untransformed). This is generally achieved through extreme sensitivity of detection of the reporter gene product or of the product of the reporter gene’s activity. Enzymatic activity is a practical necessity for this kind of detection. In practice, this results in amplification of the signal. Enzymatic systems that allow extension of incubation periods and consequent accumulation of the cleavage product are preferable, as this allows for further increases in assay sensitivity. http://www.pi.csiro.au/XylanaseAssays/index.htm

23. Ordering the XYN Assay Kit

24. Xylanase Assay Kit

25. Thanks • Supervisors • Gangping Xue (CSIRO) • Peter Gresshoff (UQ) • Scholarship • Grains Research and Development Corporation