TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol modified for <em>Puccinia striiformis</em> f. sp <em>tritici</em> (<em>Pst</em>), based on:</p>
<p>Michielse, C. B., Hooykaas, P. J., van den Hondel, C. A., &amp; Ram, A. F. (2008). Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. <em>Nature protocols</em>, <em>3</em>(10), 1671-1678. </p>
<p> </p>
<p>I am grateful for critical suggestions from Prof. John Rathjen and Dr. Benjamin Schwessinger.</p>
<p> </p>

[BEFORE_START]
<ol>
<li>Grow 12 pots of wheat seedlings (15 seeds/pot) and infect them with 50 uL of <em>Pst</em> spores to reproduce fresh spores for co-culture.</li>
<li>Plant certain numbers of wheat (depend on each experiment, I do 6 to 8 pots) 7 days before the experiment start (day 1)</li>
<li>Streak out <em>A. tumefaciens</em> contains the T-DNA in binary vector onto a LB-Agar plate and grow at 28<sup>o</sup>C for 48 h. Pick up one colony of the <em>A. tumefaciens</em> into 5 ml liquid LB media with proper selection and grow overnight. The co-culture experiment need to start from a nice overnight <em>A. tumefaciens</em> culture.</li>
</ol>

[GUIDELINES]
<p><strong>Chemical solutions:</strong></p>
<p>All chemicals used were from Sigma-Aldrich of the highest obtainable grades.</p>
<p>Buffers are best when fresh and not older than 3-6 months</p>
<ul>
<li>0.2 M Acetosyringone (AS): Dissolve 785 mg of AS in DMSO to make up a total volume of 20 ml; aliquot and store in the dark at -20 <sup>o</sup>C. Do not thaw and use an aliquot more than twice, as activity of AS decreases during thawing and freezing</li>
<li>1% CaCl<sub>2</sub> (wt/vol): Dissolve 10 g of CaCl<sub>2</sub>•2H<sub>2</sub>O in water to make up a total volume of 1 litter; autoclave.</li>
<li>0.01% FeSO<sub>4</sub> (wt/vol): Dissolve 0.1 g of FeSO<sub>4</sub>•7H<sub>2</sub>O in water to make up a total volume of 1 liter; filter-sterilize.</li>
<li>20% glucose (wt/vol): Dissolve 200 g of glucose in water to make up a total volume of 1 litter; autoclave.</li>
<li>50% glycerol: Add 50 ml of glycerol to 50 ml of water to make up a total volume of 100 ml; autoclave.</li>
<li>1.25 M KH<sub>2</sub>PO<sub>4</sub>: Dissolve 170.1 g of KH<sub>2</sub>PO<sub>4</sub> in water to make up a total volume of 1 litter; autoclave.</li>
<li>1.25 M K<sub>2</sub>HPO<sub>4</sub>: Dissolve 217.7 g of K<sub>2</sub>HPO<sub>4</sub> in water to make up a total volume of 1 litter; autoclave.</li>
<li>1 M MES: Dissolve 195.24 g of MES in water to make up a total volume of 1 litter; adjust to pH 5.5 by adding NaOH; filter-sterilize. Solution can be stored for a month in the dark or alternatively aliquot and frozen at -20<sup>o</sup>C.</li>
<li>K-buffer: Add solution 1.25 M KH<sub>2</sub>PO<sub>4</sub> to 1.25 M K<sub>2</sub>HPO<sub>4</sub> until pH 4.8 is reached.</li>
<li>MN buffer: Dissolve 30 g of MgSO<sub>4</sub>•7H2O and 15 g of NaCl in water to make up a total volume of 1 litter; autoclave.</li>
<li>20% NH<sub>4</sub>NO<sub>3</sub> (wt/vol): Dissolve 200 g of NH<sub>4</sub>NO<sub>3</sub> in water to make up a total volume of 1 litter; autoclave.</li>
<li>Trace elements for IM medium: Dissolve 100 mg of ZnSO<sub>4</sub>•7H<sub>2</sub>O, 100 mg of CuSO<sub>4</sub>•5H<sub>2</sub>O, 100 mg of H<sub>3</sub>BO<sub>3</sub>, 100 mg of MnSO<sub>4</sub>•H<sub>2</sub>O and 100 mg of Na<sub>2</sub>MoO<sub>4</sub>•2H<sub>2</sub>O in water to make up a total volume of 1 litter; autoclave.</li>
</ul>
<p> </p>
<p> </p>
<p><strong>Reagent set up:</strong></p>
<ul>
<li>Hybond N+ filter (Amersham) need to be cut into small stripes at 5 mm in width. Do not autoclave.</li>
<li>Induction medium (IM) (liquid): Add 0.8 ml of K-buffer, 20 ml of MN buffer, 1 ml of 1% (wt/vol) CaCl<sub>2</sub>•2H<sub>2</sub>O, 10 ml of 0.01% (wt/vol) FeSO<sub>4</sub>, 5 ml of trace elements for IM medium, 2.5 ml of 20% (wt/vol) NH<sub>4</sub>NO<sub>3</sub>, 10 ml of 50% (vol/vol) glycerol, 40 ml of 1 M MES, pH 5.5, and 10 ml of 20% (wt/vol) glucose to 900.7 ml of sterilized water to make up 1 litter of liquid IM. No glucose IM just substitute 10 ml 20% glucose by 10 ml H<sub>2</sub>O.</li>
<li>IM (solid): Dissolve 15 g of bacto agar in water to make up a total volume of 905.7 ml; autoclave. Add 0.8 ml of K-buffer, 20 ml of MN buffer, 1 ml of 1% (wt/vol) CaCl<sub>2</sub>•2H<sub>2</sub>O, 10 ml of 0.01% (wt/vol) FeSO<sub>4</sub>, 5 ml of trace elements for IM medium, 2.5 ml of 20% (wt/vol) NH<sub>4</sub>NO<sub>3</sub>, 10 ml of 50% (vol/vol) glycerol, 40 ml of 1 M MES, pH 5.5, and 5 ml of 20% (wt/vol) glucose to make up 1 litter of solid IM.</li>
</ul>
<p> </p>
<p>Wheat cultivar used: <em>Morocco</em></p>
<p><em>A. tumefaciens </em>strain: GV3101 pM90</p>
<p><em>Pst</em> strain used: <em>P. striiformis </em>f.sp. <em>tritici </em>strain 104E137A-</p>
<p> </p>
<p><strong><em>Pst</em> spore reproduction and collection:</strong></p>
<p>Spores are resuspended in 3M<sup>TM</sup> Novec<sup>TM</sup> 7100 engineered fluids immediately before inoculation. 7-day old seedlings were inoculated with fresh urediniospores of <em>Pst</em> using flat brush and incubated for 24 h in 100% humidity at 8<sup>o</sup>C in the dark. Subsequently plants were transferred to a growth chamber at 17<sup>o</sup>C with a 16:8 light cycle. Spores can be harvested after 14 dai, with 2 days intervals, by dusting the heavily infected leaves on aluminium foil or a weighing paper.</p>
<p> </p>
<p><strong>GUS staining of <em>Pst </em>spores:</strong></p>
<p>substrate sollution: 0.05M sodium phosphate buffer, pH7.4; 1mM EDTA; 0.1% (v/v) TritonX-100; 0.1mM K<sub>3</sub>Fe(CN)<sub>6</sub>; 0.5mg X-Gluc/ml</p>
<p>Freshly harvested <em>Pst</em> spores are germinated on the surface of water for 6 h, before replacing all water by the substrate sollution using glass Pasteur pipette. Spores in the substrate sollution are then incubated at 37 oC for 40 h, before moving to a glass slice using glass Pasteur pipette for imaging.</p>
<p> </p>
<p><strong>DNA extraction from <em>Pst</em> spores</strong> is according to Yiheng Hu: High quality DNA extraction from Fungi_small scale. protocols.io https://www.protocols.io/view/high-quality-dna-extraction-from-fungi-small-scale-exmbfk6</p>
<p> </p>
<p><strong>Critical steps to obtain high transformation efficiency:</strong></p>
<ul>
<li>Must use fresh spores/wheat seedlings/Acetosyringone/<em>Agrobacterium</em></li>
<li>Must use glass plates/beakers to germinate spores</li>
<li>Make sure the filter contain co-cultured spores attached tightly to the wheat leaf to maximize the infection.</li>
</ul>

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