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 GENEPRODUCTDETECTIONPROSCONS GUS (uidA)  -glucuronidaseColour reaction, fluorescence Extremely well characterised Excellent assay systems available Qualitative, quantitative Many reports of endogenous GUS-like activity Problems with cell autonomy Quenching LUC (luc) Luciferase (firefly/ bacterial) Luminescence (light emission) Extremely sensitive Qualitative, quantitative Very expensive Very labile Cannot increase sensitivity antRCRegulatory proteins Anthocyanin pigment Cell-autonomous No substrate required Non-destructive Not for quantification Toxicity problems GFP (gfp) Green fluorescent protein FluorescenceCell-autonomous Non-destructive No substrate required Pictures look great! Problems with quantification Chlorophyll autofluorescence

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

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

11. Focus Counting: Threshold Effect Detection threshold Saturation 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: 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 Xylanase

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 Actin OG OG X OG S OG M OG D3 OG D1 0 bp 300 900 1200 bp 600 GFP XEBMS TSS UTR D1 D3 Actin OG OG D1 OG D3 OG X OG S OG M Barley Wheat

20. Deletion Analysis – GUS:XYN

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 Assays in Plant Tissues Claudia 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.claudia.vickers@csiro.au Introduction Reporter Genes Used Detailed Method Solutions References Print Friendly Version (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