This work was supported by the BMBF Forschungsinitiative &#8220;BioEnergie 2021 &#8211; Forschung f&#252;r die Nutzung von Biomasse&#8221; and the Cluster of Excellence &#8220;Tailor-made Fuels from Biomass&#8221;, which is funded through the Excellence Initiative by the German federal and state governments to promote science and research at German universities.

1. Growth of Wild Type N. tabacum&#160;Plants
<ol>
	<li>To grow N. tabacum L. cv. Petit Havana SR1 plants on soil, place tobacco seeds on appropriate pots filled with normal potting soil for germination. After 2 weeks, transfer seedlings to individual pots. Incubate plants in a greenhouse with constant 22 &#176;C (dark period) or 25 &#176;C (light period) and 70% relative air humidity. Provide illumination in a 16/8&#160;hr light/dark cycle (e.g. Philips IP65 400 W lamps; 180 &#956;mol&#183;sec-1&#183;m-2; &#955;= 400-700 nm) and water daily.</li>
</ol>
2. Transient Expression of TrCel5A and TrCel5A-mCherry Proteins
<ol>
	<li>Under sterile conditions, pick single colonies of Agarobacterium tumefaciens (GV3101::pMP90RK GmR, KmR, RifR) containing either pTRAkc-ER-TrCel5A or pTRAkc-ER -TrCel5A-mCherry and transfer to an Erlenmeyer flask containing 10 ml&#160;yeast extract broth (YEB, prepared as per manufacturers specifications) medium and kanamycin (50 mg/ml), rifampicin (50 mg/ml), and carbenicillin (100 mg/ml) for selection. Incubate for 36-40 hr at 26 &#176;C and 180 rpm. Note: The design of appropriate constructs for protein expression is outside the scope of this paper, but for guidance, the methods of Maclean et al.13 and Munro and Pellham14 can be followed for construct design.</li>
	<li>Take 200 &#181;l&#160;of each culture to inoculate 20 ml&#160;of YEB with the antibiotic concentrations mentioned above and incubate under the same conditions as before.</li>
	<li>After 36-40 hr, preinduce the cultures by adding 2 &#181;l&#160;acetosyringon (200 &#181;M final concentration), 100 &#181;l&#160;40% (w/v) glucose solution and 200 &#181;l&#160;of 1 M MES buffer pH 5.6. Incubate overnight.</li>
	<li>Prepare 100 ml&#160;of a 2x infiltration buffer (100 g/L sucrose, 3.6 g/L glucose, 8.6 g/L Murashige and Skoog basal salts, adjust pH to 5.6 using potassium hydroxide).</li>
	<li>Measure OD600 of the cultures with 1:5 dilutions until it reaches an OD600 of 5-6. Calculate culture volume needed for 30 ml&#160;infiltration medium at OD600. Add 15 ml&#160;of 2x infiltration buffer, then add deionized H2O to make the buffer up to 30 ml.</li>
	<li>Take plants from greenhouse and use a pipette tip to nick the abaxial side of the 3rd to 5th leaf from the top to ease infiltration.</li>
	<li>Use a pipette tip to nick the abaxial side of the leaves to ease infiltration.</li>
	<li>Fill a syringe with infiltration medium and put it (without needle) on one of the previously made nicks. Support syringe with a finger on the adaxial leaf side. Press the medium carefully into the tissue of interveinal leaf zones. Note: After infiltration, cultivate plants in a greenhouse under the same conditions as described above. Also, maintain an equal proportion of untreated, non-transient expression plants (NTEPs) as controls, under the same conditions as infiltrated plants.</li>
</ol>
3. Assessment of TrCel5A-mCherry Expression in Plant Leaves
<ol>
	<li>After 4-6 days, take plants infiltrated with A. tumefaciens containing pTRAkc-ER-TrCel5A-mCherry from the greenhouse and place them in a dark room for fluorescence analysis.</li>
	<li>To visualize the fluorescence in leaf sections expressing TrCel5A-mCherry, light plants using a portable light source emitting green light at 515&#160;nm with a red filter (620-750&#160;nm). Note: In depth characterization of protein expression in the plant leaves can be achieved by thin-sectioning leaves with a vibratome and examination under light and fluorescence microscopy. For this follow the steps below:
	<ol>
		<li>Cut small sections of approximately 5 x 5 mm size from an infiltrated leaf and embed them in 4% agarose dissolved in 50 mM Phosphate buffer (pH 5.7).</li>
		<li>Cut section of 80-90 &#181;m size from embedded leaves using a vibratome. Place them on a microscope slide, cover with buffer or a cover slip and examine them using a microscope at 10X&#160;magnification with a fluorescence detector, using the same wavelength and filter stated above.</li>
	</ol>
	</li>
</ol>
4. TrCel5A Extraction from Tobacco Leaves
<ol>
	<li>Cut pieces from tobacco leaves to an approximate weight of 1 g from plants&#160;infected with A. tumefaciens containing pTRAkc-ER-TrCel5A and place in a mortar precooled with liquid nitrogen. Add an appropriate amount of liquid nitrogen to the samples. Grind leaves to powder using a precooled pestle.</li>
	<li>Add 2 ml&#160;of phosphate buffered saline (PBS) containing 1 mM phenylmethylsulfonyl fluoride to the ground leaf matter and mix until the majority of leaf matter is in suspension. Centrifuge extract at 15,000 x g for 20 min at 4 &#176;C and take protein-containing supernatant.</li>
	<li>Centrifuge the extract at 15,000 x g for 20 min at 4&#160;&#176;C and take protein-containing supernatant being careful not to disrupt the pellet. Discard the pellet.</li>
	<li>Partially purify TrCel5A by incubating the protein-containing supernatant at 55 &#176;C for 10 min. Allow to rest at room temperature for a further 10 min, then centrifuge at 15,000 x&#160;g for 10 min at RT. Repeat partial purification process for non-infiltrated plants to obtain control samples. Use these samples for all following experiments. Note: Protein solutions can be stored at 4 &#176;C for up to 1 week or at -20 &#176;C for up to 3 months.</li>
	<li>Repeat the partial purification process for non-infiltrated plants to obtain control samples. Use these samples for all following experiments. Note: Protein solutions can be stored at 4&#160;&#176;C for up to 1 week or at -20&#160;&#176;C for up to 3 months.</li>
</ol>
5. SDS-PAGE, Western Blotting, and Azo-CMC Zymography of TrCel5A-mCherry
<ol>
	<li>Purchase or prepare 12% (w/v) SDS-PAGE gels as per established methods15.</li>
	<li>Dissolve CMC&#160;in water to obtain a 1.5% (w/v) solution. To make CMC-SDS-PAGE gels, substitute 1 ml&#160;of 1.5% CMC for 1 ml&#160;of water in a 10 ml&#160;SDS-PAGE gel mix solution, resulting in a 0.1% (w/v) CMC + 12% (w/v) SDS-PAGE gel mix. Pour gel normally. Note: Make certain that the CMC in the original solution is completely dissolved by a combination of stirring and heating to 40 &#176;C (repeat directly prior to preparing the gel solution).</li>
	<li>Denature samples by boiling in the presence of SDS, as per established methods12. As a positive control, use a 1:500 dilution of a commercially available cellulase mixture from T. reesei, such as T. reesei ATCC 26921. As a negative control use a similarly prepared protein solution from non-infiltrated leaves (NTEPs).</li>
	<li>Carry out the electrophoresis as per normal protocols15, e.g.&#160;use 200 V for ~50 min with the electrophoresis halted once the dye front reached the bottom of the gel.</li>
	<li>Stain one SDS-PAGE gel using 0.1% (w/v) Coomassie brilliant blue and destain using a methanol/glacial acetic acid mixture. Note: Rapid staining and destaining can be performed by gently heating the staining and destaining solutions, i.e. by using a microwave for ~15 sec to warm (but not boil) first the staining and then the destaining solutions, changing the destaining solution after 10 min, or when cooled, and repeat with new destaining solution.</li>
	<li>Perform Western blotting of one SDS-PAGE gel using established protocols16,17. Use the following antibodies: a polyclonal rabbit antibody against a&#160;His-tag (primary) and a polyclonal goat anti-rabbit antibody Fc region which has been conjugated to an alkaline phosphatase (secondary). Note: secondary antibodies should be selected to enable visualization using nitroblue tetrazolium chloride/5-bromo-4-chloro-3&#8242;-indolyphosphate p-toluidine salt (NBT/BCIP), as described18.</li>
	<li>Perform in-gel protein refolding on the 0.15% (w/v) CMC SDS-PAGE gel by incubating the gel in 1.5% (w/v) &#946;-cyclodextrin, 20 mM phosphate-citrate buffer, pH 6.5, at room temperature with gentle shaking for 15 min19.</li>
	<li>Discard the &#946;-cyclodextrin solution and briefly wash the gel with H2O. Incubate at RT in 50mM sodium acetate buffer (pH 4.8) for a further 15 min. Perform CMC zymography using the refolded 0.15% CMC SDS-PAGE gel by incubating the gel in 30 mM sodium acetate buffer pH 4.8 for 20 min at 50 &#176;C.</li>
	<li>Perform CMC zymography using the refolded 0.15% (w/v) CMC SDS-PAGE gel by incubating the gel in 30 mM sodium acetate buffer&#160;(pH 4.8) for 20 min at 50&#160;&#176;C.</li>
	<li>Stain the gel for 10&#160;min using a 0.1% w/v Congo red solution and destain for 30 min or until clear bands are observed using 1 M NaCl. Wash the gel with 0.3% (v/v) acetic acid for clearer imaging.</li>
</ol>
6. 4-MUC Activity Assay of TrCel5A
<ol>
	<li>Prepare a stock solution of 10 mM 4-MUC in 50% (v/v) methanol, and store it in the dark at RT</li>
	<li>Immediately prior to the assay, prepare a 4-MUC 2x working solution, composed of 1 mM 4-MUC and 60 mM sodium acetate, pH 4.8.</li>
	<li>Perform this test on samples containing 100 &#956;l of leaf protein (with or without TrCel5A inducibly expressed), 100 &#956;l a commercially available cellulase mixture from T. reesei 1:1,000, and pure H2O, respectively. Run each sample in triplicate.</li>
	<li>Pipette 100 &#956;l of the 4-MUC working solution into separate wells of a 96-well plate, then add 100 &#956;l of each sample.</li>
	<li>Determine 0 min timepoint of 4-MU fluorescence through fluorescent spectroscopy, using an excitation wavelength of 360 nm and an emission wavelength of 465 nm.</li>
	<li>Incubate plates for 20 min at 50 &#176;C, covered with adhesive lids to prevent evaporation. Stop the reaction by freezing (or by combining&#160;100 &#956;l 0.15 M glycine, pH 10.0, and 100 &#956;l of sample in a separate plate).</li>
	<li>Determine the endpoint of 4-MU fluorescence through fluorescent spectroscopy, using an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Subtract 0 min from endpoint (20 min) timepoint to obtain the change in fluorescence values.</li>
</ol>
7. Azo-carboxymethyl Cellulose Activity Assay of TrCel5A
<ol>
	<li>Prepare a stock of 1.5% (w/v) Azo-CMC which is stored at RT, and a precipitation solution containing 18 mM zinc acetate, 0.5 M sodium acetate, pH 5 and 80% ethanol (v/v), store at room temperature.</li>
	<li>Immediately prior to the assay, prepare an Azo-CMC 2x working solution, composed of 1% (w/v) Azo-CMC (in H2O) and 60 mM sodium acetate, pH 4.8.</li>
	<li>Combine 50 &#956;l of sample and 50 &#956;l of the working solution in 1.5 ml&#160;tubes.</li>
	<li>Test samples containing 50 &#956;l of leaf protein (with or without TrCel5A transiently expressed); 100 &#956;l of a commercially available cellulase mixture from T. reesei diluted 1:1,000; and pure H2O. Run samples and standards in triplicate.</li>
	<li>Incubate samples for 20 min at 50 &#176;C. Stop the reaction by adding 250 &#956;l of precipitation solution. Vortex each sample vigorously for 10 sec to ensure solutions were completely combined.</li>
	<li>Incubate samples at RT for 10 min and then vortex again. Centrifuge samples at 1,000 x g for 10 min. Remove 200 &#956;l&#160;of each sample supernatant to a 96-well plate, taking care not to disrupt the pellet.</li>
	<li>Assay using absorbance spectroscopy at a wavelength of 590 nm.</li>
</ol>
8. PAHBAH Assay of TrCel5A
<ol>
	<li>Use a hole-punch to cut a circle (~5.5 mm diameter) of filter paper. Add 100 &#956;l&#160;60 mM NaAc, pH 4.8, and 100 &#956;l&#160;of samples, either containing leaf protein (with or without TrCel5A inducibly expressed) or a commercial T. reesei cellulase mixture, diluted 1:1,000, and incubate with filter paper circles for 20 hr at 50 &#176;C with 300 rpm shaking. For each sample incubated with filter paper, also incubate a duplicate solution without filter paper as individual sample-controls.</li>
	<li>Prepare a 330 mM p-hydroxy benzoic acid hydrazide (PAHBAH) solution A by dissolving PAHBAH in H2O with the addition of 5 ml&#160;concentrated HCl for a total of 100 ml&#160;solution, and store at RT. Note: Best results are achieved by adding the HCl to a smaller volume of PAHBAH slurry, i.e. 30 ml, which is stirred under temperature to help dissolution before making the volume to 100 ml&#160;with H2O.</li>
	<li>Prepare PAHBAH solution B, containing 50 mM sodium citrate, 10 mM calcium chloride, and 500 mM NaOH and&#160;store&#160;at RT.</li>
	<li>Immediately prior to the assay, prepare a 1.5x working solution of 1 ml&#160;PAHBAH reagent to 9 ml&#160;buffer solution, store on ice until use (to be used within 4 hr).</li>
	<li>Prepare samples in thermostable 0.2 or 0.5 ml&#160;tubes. Combine 5 &#956;l of each solution incubated with filter paper (step 8.1) 45 &#956;l of H2O and 100 &#956;l of PAHBAH working solution. Sample controls (samples incubated without filter paper) should be run under the same concentrations (5 &#956;l sample, 45 &#956;l H2O, 100 &#956;l PAHBAH working solution). Also test 5 standards (5 &#956;l) containing between 0 and 0.2 g/L of glucose, and pure H2O. Run samples and standards in triplicate.</li>
	<li>Incubate samples for exactly 10 min at 100 &#176;C, then cool at RT for exactly 10 min. Pipette solutions into a 96 well plate and assay spectroscopically at a wavelength of 410 nm. Subtract individual sample-controls (incubated without filter paper) from samples to obtain final values.</li>
</ol>

<ol>
	<li>Garvey, M., Klose, H., Fischer, R., Lambertz, C., Commandeur, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulases+for+biomass+degradation:+comparing+recombinant+cellulase+expression+platforms.">Cellulases for biomass degradation: comparing recombinant cellulase expression platforms.</a> Trends in Biotechnology. 31, 581-593 (2013).</li><li>Klose, H., G&#252;nl, M., Usadel, B., Fischer, R., Commandeur, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+inducible+expression+of+a+mesophilic+cellulase+avoids+adverse+effects+on+plant+development.">Ethanol inducible expression of a mesophilic cellulase avoids adverse effects on plant development.</a> Biotechnology for Biofuels. 6, 53 (2013).</li><li>Klose, H., R&#246;der, J., Girfoglio, M., Fischer, R., Commandeur, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperthermophilic+endoglucanase+for+in+planta+lignocellulose+conversion.">Hyperthermophilic endoglucanase for in planta lignocellulose conversion.</a> Biotechnology for Biofuels. 5, 63 (2012).</li><li>Sharma, A. K., Sharma, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plants+as+bioreactors:+recent+developments+and+emerging+opportunities.">Plants as bioreactors: recent developments and emerging opportunities.</a> Biotechnology Advances. 27 (6), 811-832 (2009).</li><li>Tremblay, R., Wang, D., Jevnikar, A. M., Ma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco,+a+highly+efficient+green+bioreactor+for+production+of+therapeutic+proteins.">Tobacco, a highly efficient green bioreactor for production of therapeutic proteins.</a> Biotechnology Advances. 28 (2), 214-221 (2010).</li><li>Lee, T. M., Farrow, M. F., Arnold, F. H., Mayo, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+study+of+Hypocrea+jecorina+Cel5A.">A structural study of Hypocrea jecorina Cel5A.</a> Protein Science. 20, 1935-1940 (2011).</li><li>Saloheimo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGIII,+a+new+endoglucanase+from+Trichoderma+reesei:+the+characterization+of+both+gene+and+enzyme.">EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme.</a> Gene. 63, 11-21 (1988).</li><li>Davidson, M. W., Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+fluorescent+proteins:+innovations+and+applications.">Engineered fluorescent proteins: innovations and applications.</a> Nature Methods. 6, 713-717 (2009).</li><li>Pelham, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retention+signal+for+soluble+proteins+of+the+endoplasmic+reticulum.">The retention signal for soluble proteins of the endoplasmic reticulum.</a> Trends in Biochemical Sciences. 15, 483-486 (1990).</li><li>B&#233;guin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+cellulase+activity+in+polyacrylamide+gels+using+Congo+red-stained+agar+replicas.">Detection of cellulase activity in polyacrylamide gels using Congo red-stained agar replicas.</a> Analytical Biochemistry. 131, 333-336 (1983).</li><li>J&#248;rgensen, H., M&#248;rkeberg, A., Krogh, K. B. R., Olsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+enzyme+production+by+three+Penicillium+species+on+monosaccharides.">Growth and enzyme production by three Penicillium species on monosaccharides.</a> Journal of Biotechnology. 109, 295-299 (2004).</li><li>Lever, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+reaction+for+colorimetric+determination+of+carbohydrates.">A new reaction for colorimetric determination of carbohydrates.</a> Analytical Biochemistry. 47, 273-279 (1972).</li><li>Maclean, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+human+papillomavirus+type+16+(HPV-16)+L1+expression+in+plants:+comparison+of+the+suitability+of+different+HPV-16+L1+gene+variants+and+different+cell-compartment+localization.">Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization.</a> Journal of General Virology. 88, 1460-1469 (2007).</li><li>Munro, S., Pelham, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C-terminal+signal+prevents+secretion+of+luminal+ER+proteins.">A C-terminal signal prevents secretion of luminal ER proteins.</a> Cell. 48, 899-907 (1987).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Burnette, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#8220;Western+Blotting&#8221;:+Electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate-polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">&#8220;Western Blotting&#8221;: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112, 195-203 (1981).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elelctrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets+-+procedure+and+some+applications.">Elelctrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets - procedure and some applications.</a> Proceedings of the National Academy of Sciences of the United States of America. 76, 4350-4354 (1979).</li><li>Blake, M. S., Johnston, K. H., Russell-Jones, G. J., Gotschlich, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+sensitive+method+for+detection+of+alkaline+phosphatase-conjugated+anti-antibody+on+Western+blots.">A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots.</a> Analytical Biochemistry. 136, 175-179 (1984).</li><li>Yamamoto, E., Yamaguchi, S., Nagamune, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+&#946;-cyclodextrin+on+the+renaturation+of+enzymes+after+sodium+dodecyl+sulfate&#8211;polyacrylamide+gel+electrophoresis.">Effect of &#946;-cyclodextrin on the renaturation of enzymes after sodium dodecyl sulfate&#8211;polyacrylamide gel electrophoresis.</a> Analytical Biochemistry. 381, 273-275 (2008).</li><li>Fischer, R., Vaquero-Martin, C., Sack, M., Drossard, J., Emans, N., Commandeur, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+molecular+farming+in+the+future:+transient+protein+expression+in+plants.">Towards molecular farming in the future: transient protein expression in plants.</a> Biotechnology and Applied Biochemistry. 30 (2), 113-116 (1999).</li><li>Sammond, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulase+linkers+are+optimized+based+on+domain+type+and+function:+Insights+from+sequence+analysis,+biophysical+measurements,+and+molecular+simulation.">Cellulase linkers are optimized based on domain type and function: Insights from sequence analysis, biophysical measurements, and molecular simulation.</a> PLoS One. 7, (2012).</li><li>Boonvitthya, N., Bozonnet, S., Burapatana, V., O'Donohue, M. J., Chulalaksananukul, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+heterologous+expression+of+Trichoderma+reesei+endoglucanase+II+and+cellobiohydrolase+II+in+the+yeasts+Pichia+pastoris+and+Yarrowia+lipolytica.">Comparison of the heterologous expression of Trichoderma reesei endoglucanase II and cellobiohydrolase II in the yeasts Pichia pastoris and Yarrowia lipolytica.</a> Molecular Biotechnology. 54, 158-169 (2013).</li></ol>

The authors have nothing to disclose.

Recombinant expression of cellulases is a field of great interest, due to the desire for more efficient biofuel production systems1. Plants offer many possibilities for successful cellulase production, with features such as economic harvesting techniques and autohydrolysis methods being very desirable properties for large-scale biofuel production. Further, the use of different localizations for producing proteins allow, if required, posttranslational modifications20. Altering plants to enable consti...

Degradation of lignocellulosic biomass and its conversion into liquid fuel has been envisioned as a method to reduce reliance on fossil fuels. One significant hurdle in establishing economically feasible biomass processing systems is in the enzymatic degradation of cellulose and hemicellulose1. Plant expression systems show great potential for the production of enzymes on an industrial scale. Agricultural systems to harvest plants economically and in volume are already established, as they have been for thousands of years. The production of cellulases in plants is desirable due to factors like the ease of autohydrolysis2, and the potential for maximizing the use of lower enzyme levels by increasing the digestibility of plant cell walls1. Finally, plant systems allow the targeting of recombinant proteins to specific areas of plant cells and are able to posttranslationally modify enzymes where required3.

Nicotiana tabacum (tobacco) is very commonly used as a model organism for heterologous protein expression studies in plants, due to its rapid growth and biomass accumulation features4. Transient expression of recombinant proteins is a technique which enables protein production in short time periods5, while maintaining flexibility as to the localization and thus posttranslational modifications of the selected proteins, i.e. cellulases. This enables the production of the cellulase(s) for basic analysis, while also laying the groundwork for further expression strategies in plants. Using such a transient expression strategy, an endoglucanase from glycosyl hydrolase family 5, Cel5A, derived from the fungal host Trichoderma reesei (Hypocrea jecorina)6 is produced (hereafter referred to as TrCel5A). TrCel5A is a 42 kDa protein which is natively glycosylated and is highly active in hydroylzing cellulose chains7.

The transient expression technique described here is based on a relatively commonly used system, infiltrating the plant leaves with Agrobacteria carrying the gene of interest in an appropriate expression vector. To allow for rapid analysis of successful in planta expression, TrCel5A can also be expressed as a fusion protein with mCherry, a monomeric fluorescent protein originally derived from Discosoma sp. protein DsRed8, with an additional six Histidine residues (His-tag) fused to the C-terminus (TrCel5A-mCherry). Expression of the heterologous fusion protein, TrCel5A-mCherry, can thus be monitored within the growing plant by using green light to examine mCherry dispersal. If desired, thin sections of the plant material can be examined under green light microscopically to establish the specific localization of the protein. For this work, signal and transit peptides were incorporated in the construct, to localize the heterologous protein export to the endoplasmic reticulum9.

To analyze the activity of plant expressed cellulases, including TrCel5A, a number of cellulase activity tests can be run. After the extraction of the total soluble plant proteins, TrCel5A can be partially purified using a thermal incubation technique. Protein size is established using SDS-PAGE followed by Western blotting. Zymography can be used to analyze activity against substrates, e.g. cellulose which has been carboxy-methylated (making carboxymethyl cellulose: CMC) for solubility10. Cellulase and glucosidase activity can be monitored using the fluorophore 4-methylumbelliferyl (4-MU) associated with &#946;-D-cellobioside (combined: 4-MUC). Another method to assess endoglucanase activity involves the spectrometric analysis of CMC&#160;which has been associated with an azo-dye (Remazolbrilliant Blue R)11. In addition, protein activity of both endoglucanases and a range of cellulases can be monitored by the use of a sugar analysis test, such as the p-hydroxy benzoic acid hydrazide (PAHBAH) assay12. These techniques can be used to elucidate and quantify the activity of the expressed cellulase of interest.

<table border="0" cellpadding="0" cellspacing="0" width="823">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:308px;">4-methylumbelliferyl &#946;-D-cellobioside</td>
			<td style="width:127px;">SIGMA-ALDRICH</td>
			<td style="width:156px;">M6018</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>ROTH</td>
			<td>3738</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetosyringon (concentrated)</td>
			<td>ROTH</td>
			<td>6003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - kanamycin</td>
			<td>ROTH</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - rifampicin</td>
			<td>ROTH</td>
			<td>4163</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotic - carbenicillin</td>
			<td>ROTH</td>
			<td>6344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibody - rabbit anti His(6) pAb</td>
			<td>ROCKLAND</td>
			<td>600-401-382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibody - goat anti rabbit AP, FC pAb</td>
			<td>DIANOVA</td>
			<td>111-055-008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Azo-carboxymethyl cellulose</td>
			<td>MEGAZYME</td>
			<td>S-ACMC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-cyclodextrin</td>
			<td>SIGMA-ALDRICH</td>
			<td>C4805</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carboxymethyl cellulose</td>
			<td>ROTH</td>
			<td>3333.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellulase from Trichoderma reesei ATCC 26921</td>
			<td>SIGMA-ALDRICH</td>
			<td>C2730</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Congo red</td>
			<td>SIGMA-ALDRICH</td>
			<td>75768</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coomasie brilliant blue G 250</td>
			<td>ROTH</td>
			<td>9598.2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D(+)-glucose</td>
			<td>ROTH</td>
			<td>8337</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>ROTH</td>
			<td>K928</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter paper - Rotilabo blotting paper</td>
			<td>ROTH</td>
			<td>CL67</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NBT/BCIP</td>
			<td>SIGMA-ALDRICH</td>
			<td>72091</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES buffer</td>
			<td>ROTH</td>
			<td>4256</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol</td>
			<td>ROTH</td>
			<td>8388</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium citrate</td>
			<td>ROTH</td>
			<td>3580</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecylsulfate</td>
			<td>SERVA</td>
			<td>20765</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>ROTH</td>
			<td>6771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Soil, Einheitserde classic&#160;</td>
			<td>PATZER GMBH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectrometer - Infinite M200</td>
			<td>TECAN</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>SIGMA-ALDRICH</td>
			<td>S-5390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Hydroxy benzhydrazide&#160;</td>
			<td>SIGMA-ALDRICH</td>
			<td>H9882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>ROTH</td>
			<td>1058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV light source - BLAK RAY</td>
			<td>UVP</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vibratome - VT1000S</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

expression of recombinant cellulase cel5a from trichoderma reesei in tobacco plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

TrCel5A and TrCel5A-mCherry were expressed successfully in tobacco plants using the transient expression system (Figures 1A and 1B). Examination of the expression pattern of the TrCel5A-mCherry fusion protein revealed healthy leaves (Figure 1C), which showed widespread protein expression under green light (Figure 1D).
Extraction of the total soluble proteins was performed on tobacco plants which had leaves that contained the t...

Watch this Scientific Journal Video about Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants at JoVE.com

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

expression-recombinant-cellulase-cel5a-from-trichoderma-reesei

Research

JoVE Journal

Environment

12.7K Views.  RWTH Aachen University. Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.