Name: using ustilago maydis as a trojan horse for in situ delivery of maize proteins
Uploaded: 2019-02-08T13:00:00
Description: Watch this Scientific Journal Video about Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins at JoVE.com

The authors would like to thank Thomas Dresselhaus, Martin Parniske, Noureddine Djella, and Armin Hildebrand for providing lab space and plant material. The original work on the Trojan horse method was supported by a Leopoldina postdoc fellowship and NSF project IOS13-39229. The work presented in this article was supported by SFB924 (projects A14 and B14) of the DFG.

1. Construction of an U. maydis Trojan Horse
NOTE: See Figure 1.
<ol>
	<li>Amplify a gene of interest from maize cDNA using gene-specific primers and a proofreading DNA polymerase. Clone the primary PCR product and transform the construct into E. coli following the plasmid vendor&#39;s instructions. Verify the correct gene of interest sequence by Sanger sequencing prior to use for the next cloning steps. 
	<strong...

<ol>
	<li>K&#228;mper, 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=Insights+from+the+genome+of+the+biotrophic+fungal+plant+pathogen+Ustilago+maydis.">Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.</a> Nature. 444, 97-101 (2006).</li><li>Doehlemann, G., 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=Establishment+of+compatibility+in+the+Ustilago+maydis/maize+pathosystem.">Establishment of compatibility in the Ustilago maydis/maize pathosystem.</a> Journal of Plant Physiology. 165, 29-40 (2008).</li><li>Matei, A., 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=How+to+make+a+tumour:+cell+type+specific+dissection+of+Ustilago+maydis-induced+tumour+development+in+maize+leaves.">How to make a tumour: cell type specific dissection of Ustilago maydis-induced tumour development in maize leaves.</a> New Phytologist. , (2018).</li><li>Doehlemann, G., 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=Reprogramming+a+maize+plant:+transcriptional+and+metabolic+changes+induced+by+the+fungal+biotroph+Ustilago+maydis.">Reprogramming a maize plant: transcriptional and metabolic changes induced by the fungal biotroph Ustilago maydis.</a> The Plant Journal. 56, 181-195 (2008).</li><li>Doehlemann, G., 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=Pep1,+a+secreted+effector+protein+of+Ustilago+maydis.,+is+required+for+successful+invasion+of+plant+cells.">Pep1, a secreted effector protein of Ustilago maydis., is required for successful invasion of plant cells.</a> PLOS Pathogens. 5, e1000290 (2009).</li><li>Redkar, A., 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=A+secreted+effector+protein+of+Ustilago+maydis+guides+maize+leaf+cells+to+form+tumors.">A secreted effector protein of Ustilago maydis guides maize leaf cells to form tumors.</a> The Plant Cell. 27, 1332-1351 (2015).</li><li>Djamei, A., 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=Metabolic+priming+by+a+secreted+fungal+effector.">Metabolic priming by a secreted fungal effector.</a> Nature. 478, 395-398 (2011).</li><li>Tanaka, S., 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=A+secreted+Ustilago+maydis+effector+promotes+virulence+by+targeting+anthocyanin+biosynthesis+in+maize.">A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize.</a> eLife. 3, e01355 (2014).</li><li>Mueller, A. N., Ziemann, S., Treitschke, S., Assmann, D., Doehlemann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compatibility+in+the+Ustilago+maydis-maize+interaction+requires+inhibition+of+host+cysteine+proteases+by+the+fungal+effector+Pit2.">Compatibility in the Ustilago maydis-maize interaction requires inhibition of host cysteine proteases by the fungal effector Pit2.</a> PLOS Pathogens. 9, e1003177 (2013).</li><li>Ziemann, S., 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=An+apoplastic+peptide+activates+salicylic+acid+signalling+in+maize.">An apoplastic peptide activates salicylic acid signalling in maize.</a> Nature Plants. 4, 172-180 (2018).</li><li>Ju&#225;rez-Montiel, 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=The+corn+smut+('Huitlacoche')+as+a+new+platform+for+oral+vaccines.">The corn smut ('Huitlacoche') as a new platform for oral vaccines.</a> PLoS One. 10, e0133535 (2015).</li><li>Sarkari, P., Feldbr&#252;gge, M., Schipper, K., Schmoll, M., Dattenb&#246;ck, 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> Gene Expression Systems in Fungi: Advancements and Applications. , 183-200 (2016).</li><li>Monreal-Escalante, E., 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=The+corn+smut-made+cholera+oral+vaccine+is+thermostable+and+induces+long-lasting+immunity+in+mouse.">The corn smut-made cholera oral vaccine is thermostable and induces long-lasting immunity in mouse.</a> Journal of Biotechnology. 234, 1-6 (2016).</li><li>Stock, 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=Applying+unconventional+secretion+of+the+endochitinase+Cts1+to+export+heterologous+proteins+in+Ustilago+maydis.">Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis.</a> Journal of Biotechnology. 161, 80-91 (2012).</li><li>van der Linde, K., 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=Pathogen+Trojan+horse+delivers+bioactive+host+protein+to+alter+maize+(Zea+mays)+anther+cell+behavior+in+situ.">Pathogen Trojan horse delivers bioactive host protein to alter maize (Zea mays) anther cell behavior in situ.</a> The Plant Cell. 30, 528-542 (2018).</li><li>B&#246;sch, K., 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=Genetic+manipulation+of+the+plant+pathogen+Ustilago+maydis+to+study+fungal+biology+and+plant+microbe+interactions.">Genetic manipulation of the plant pathogen Ustilago maydis to study fungal biology and plant microbe interactions.</a> Journal of Visualized Experiments. , e54522 (2016).</li><li>Chavan, S., Smith, S. 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+rapid+and+efficient+method+for+assessing+pathogenicity+of+Ustilago+maydis+on+maize+and+teosinte+lines.">A rapid and efficient method for assessing pathogenicity of Ustilago maydis on maize and teosinte lines.</a> Journal of Visualized Experiments. 50712, (2014).</li><li>Kelliher, T., Walbot, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+and+patterning+of+the+five+cell+types+of+the+Zea+mays+anther+locule.">Emergence and patterning of the five cell types of the Zea mays anther locule.</a> Developmental Biology. 350, 32-49 (2011).</li><li>Egger, R. L., Walbot, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Zea+mays.+tassel+development+and+correlation+with+anther+developmental+stages+as+a+guide+for+experimental+studies.">Quantifying Zea mays. tassel development and correlation with anther developmental stages as a guide for experimental studies.</a> Maydica. 60, M34 (2015).</li><li>Holliday, R., King, R. 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> Bacteria, Bacteriophages, and Fungi: Volume 1. , 575-595 (1974).</li><li>Doehlemann, G., Reissmann, S., A&#223;mann, D., Fleckenstein, M., Kahmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+linked+genes+encoding+a+secreted+effector+and+a+membrane+protein+are+essential+for+Ustilago+maydis-induced+tumour+formation.">Two linked genes encoding a secreted effector and a membrane protein are essential for Ustilago maydis-induced tumour formation.</a> Molecular Microbiology. 81, 751-766 (2011).</li><li>Banuett, F., Herskowitz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+a+alleles+of+Ustilago+maydis+are+necessary+for+maintenance+of+filamentous+growth+but+not+for+meiosis.">Different a alleles of Ustilago maydis are necessary for maintenance of filamentous growth but not for meiosis.</a> Proceedings of the National Academy of Sciences. 86, 5878-5882 (1989).</li><li>Bortfeld, M., Auffarth, K., Kahmann, R., Basse, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ustilago+maydis+a2+mating-type+locus+genes+lga2+and+rga2+compromise+pathogenicity+in+the+absence+of+the+mitochondrial+p32+family+protein+Mrb1.">The Ustilago maydis a2 mating-type locus genes lga2 and rga2 compromise pathogenicity in the absence of the mitochondrial p32 family protein Mrb1.</a> The Plant Cell. 16, 2233-2248 (2004).</li></ol>

Modern crop research demands protocols for molecular analysis on genetic and protein levels. Genetic accessibility via transformation is not available or inefficient and time-consuming for most crop species such as maize. Moreover, reliable genetic tools such as promoter reporter systems are scarce, which makes it difficult to study in situ protein function with high spatiotemporal resolution at distinct tissue sites. Apoplastic proteins can be studied by infiltration of heterologously expressed and purified pro...

The biotrophic pathogen Ustilago maydis is the causative agent of the corn smut disease1. It infects all aerial parts of maize resulting in large tumors that contain melanized, black spores. On the global level, U. maydis is estimated to cause an annual loss of around 2% of corn yield, while tumors are appreciated as a gastronomical delicacy in Mexico. Plant infection is initiated by an appressorium that secretes cell-wall lysing enzymes to penetrate the first layer of maize epidermal cells. From a primary infection site, U. maydis grows intracellularly and intercellularly, invading one to two cell layers every day1,2. Successful infection results in plant hypertrophy that turns into visible tumors upon five days post infection1,3,4. During all infection stages, fungal hyphae invaginate the plant cytoplasm membrane without any direct contact to the host cytoplasm1,2. The tight apoplasmic space between the infecting hyphae and the plant plasma membrane is considered to be the host/pathogen interactive site, called the biotrophic interaction zone. In order to overcome the plant innate immune system, U. maydis secretes an array of effector proteins into the biotrophic interaction zone1. Some effectors are taken up by plant cells, while others remain in the biotrophic interaction zone5,6,7,8. One apoplastic effector is UmPit2, which interacts with apoplastic maize proteases to prevent the release of the signaling peptide ZmZIP1 from ZmPROZIP by apoplastic protease activity9,10.
Over the last decades, U. maydis became not only a model for fungal genetics in plant-pathogen interaction, but also a valuable tool in biotechnology due to a well-understood life cycle, easy genetic accessibility and heterologous expression of secreted proteins11,12,13. Signals for both conventional and unconventional protein secretion have been determined allowing the control of posttranslational modifications14. Recently, U. maydis was employed as a Trojan horse tool to study small, secreted maize proteins in situ15. The Trojan horse approach was successfully used to analyze the function of the small, secreted protein ZmMAC1 that is involved in anther development. ZmMAC1 induces the periclinal division of pluripotent cells and cell fate specification of the newly formed cells15. By the same method, the biological function of the maize damage-associated peptide ZmZIP1 was revealed. U. maydis secreting the maize ZmZIP1 resulted in impaired tumor formation10. Thus, the Trojan horse approach represents a valuable alternative route to protein in situ studies with high spatiotemporal resolution that does neither require generation of stable maize transformation lines nor tissue infiltration with heterologously expressed and purified proteins. In particular, the Trojan horse strategy enables the secretion of any heterologous protein into the maize apoplast and direct comparison of infected versus non-infected plant cells within the same tissue.
This protocol illustrates the major steps for generating an U. maydis Trojan horse strain to study a protein of interest. It further includes precise information on infection procedures of three different maize tissue types (adult leaves, tassels and ears) with U. maydis, which is a prerequisite for studying the spatiotemporal infection progression and protein function in these target tissues. No further specifications are given on maize gene amplification and microscopic imaging techniques, since these steps are target-specific and instrument-dependent. Thus, this protocol is addressed to experienced users of standard molecular biology techniques.

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">2 mL syringe&#160;</td>
			<td style="width:119px;">B. Braun</td>
			<td style="width:89px;">4606027V</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">23G x 1 1/4 hypodermic needle</td>
			<td style="width:119px;">B. Braun</td>
			<td>4657640</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Peptone&#160;</td>
			<td style="width:119px;">BD</td>
			<td>211677</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">cDNA from maize</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">from maize tissue expressing the gene of interrest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Charcoal</td>
			<td>Sigma-Aldrich</td>
			<td style="width:89px;">05105</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal laser scanning microscope</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">use locally available equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cuvette (10 x 4 x 45 mm)</td>
			<td style="width:119px;">Sarstedt</td>
			<td style="width:89px;">67742</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator-shaker set to 28 &#176;C, 200 rpm</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">use locally available equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Light microscope with 400-fold magnification</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">use locally available equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Nco I</td>
			<td style="width:119px;">NEB</td>
			<td style="width:89px;">R0193</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;">p123-PUmpit2-SpUmpit2-Zmmac1-mCherry-Ha</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">please contact the corresponding author&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Pasteur pipet (glass, long tip)</td>
			<td style="width:119px;">VWR</td>
			<td>14673-043</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">pCR-Blunt-II-TOPO</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td>K280002</td>
			<td style="width:311px;">can be exchanged for other basic cloning vectors like pENTR or pJET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potato Dextrose Agar&#160;</td>
			<td style="width:119px;">VWR</td>
			<td style="width:89px;">90000-745</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Sharpie pen</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">use locally available equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Spectrophotometer</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">use locally available equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Ssp I</td>
			<td style="width:119px;">NEB</td>
			<td style="width:89px;">R0132</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td style="width:89px;">S0389</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">T4 DNA ligase</td>
			<td style="width:119px;">NEB</td>
			<td style="width:89px;">M0202</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">TRIS</td>
			<td>Sigma-Aldrich</td>
			<td style="width:89px;">TRIS-RO</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Xba I</td>
			<td style="width:119px;">NEB</td>
			<td style="width:89px;">R0145</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract&#160;</td>
			<td style="width:119px;">BD</td>
			<td>212750</td>
			<td style="width:311px;"></td>
		</tr>
	</tbody>
</table>

using ustilago maydis as a trojan horse for in situ delivery of maize proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Constructs for U. maydis Trojan horse experiments are cloned into the plasmid p123-PUmpit2-SpUmpit2-gene of interest-mCherry-Ha. The maize gene of interest is fused to a mCherry fluorescence reporter and an epitope HA-tag. The expression of the fusion protein is under control of the U. maydis Umpit2 promoter which is specifically activated during infection<...

Watch this Scientific Journal Video about Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins at JoVE.com

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

Investigating processes on genetic and protein levels is crucial in crop research. Our approach enables inspection of secreted maze proteins with highest spatial temporal resolution at different infection sides and tissues. The Trojan Horse Method explores useful automatous secretory capabilities.It circumvents time consuming and challenging maze transformations, and facilitates detailed studies on secreted proteins within the maze appelplatz. Detailed information on proper infection of added maze leafs, tassels and ear. And shows the successful study of infection progression and protein function in the target tissue.Demonstrating the procedure will be Isabell Fiedler, a grad student from the laboratory. To begin the experiment, scratch Eumadous from a pediatr plate using a sterile pasteur pipette. Inoculate 5 milliliters of YEPS Light Medium with Eumadous.Grow the culture at 28 degrees Celsius, with constant shaking at 200 rpm for 16 hours. The next day mix 900 microliters of fresh YEPS Light with 100 microliters of the overnight culture. Measure the OD-600 using YEPS Light Medium as a blank in the spectrophotometer analysis.Dilute the overnight culture with fresh YEPS Light Medium to an OD-600 of 2. Let the culture grow at 28 degree Celsius with constant shaking at 200 rpm, until it reaches the mid log growth phase, indicated by an OD-600 of 8 to 1.0. After incubation, harvest the cells by spinning them at 3000 times chi for 10 minutes.Discard the supernatant. Next, wash the cell pilette one time with 1 culture volume of DDH2O. Spin the cells at 3000 time chi for 10 minutes and discard the supernatant.Then, re-suspend the cell pilette carefully in DDH2O, using a 20 milliliter glass pipette. Thereby adjusting the final OD-600 TO 3.0 for a Trojan Horse assay or 1.0 for disease rating. Cultivate the maze plants to a stage of adult leafs, where at least leaf 7 grows within the stalk.Next, transfer the Eumadous culture into a 3 milliliter syringe with a 20 gauge bi one hypodermic needle. Press the stalk carefully to localize the Mirror Stem Tissue. The base of the Mirror Stem can be distinguished by a transition from harder stalk to softer tissue.Mark the Mirror Stem on the stalk using a pen. Inject 1.5 milliliters of Eumadous culture 1 centimeter above the Shoot Mirror Stem or Inflorescence Mirror Stem. Once the maze plants reach the tassel stage, press the stalk carefully to localize the tassel in the stem.Mark the tip and the base of the tassel on the stem using a pen. Transfer the Eumadous culture into a 3 milliliter syringe with a 20 gauge bi one hypodermic needle to inject a total of 1.5 milliliters of the inoculum around the tassel. To insure equal distribution of the inoculum, slowly place 5 milliliters at the tip, the middle, and the base of the tassel marked with the pen.Transfer the Eumadous culture into a 3 milliliter syringe with 20 gauge bi one hypodermic needle. Inject the inoculation needle into the space between the husk leafs as deeply as possible without injuring the ear, then release 1.5 milliliters of the inoculum around the ear. Next, remove the syringe and needle and carefully massage the cob to distribute the Eumadous solution equally.Drop 10 microliters of inoculum on a PD charcoal auger plate. Then incubate at room temperature for 2 days. Hyphae secreting ZM Mach1 are surrounded by fluorescent and cherry signal.In contrast, nonsecreting hyphae only show fluorescent signal in the fungal cytoplasm. Improper tassel localization or unequal distribution of the inoculum can result in noninfected tassel or only partial infection of the tassel compared to even distribution. The solo pathogenic Trojan Horse Progenester strain SG-200, cultured on PD charcoal auger, forms white fluff filaments on the plate as shown.While the Eumadous strain FB1 requires mating before infectious filament formation. Following infection, the possible response of signs the Trojan Horse liver protein can be analyzed using various methods including disease ratings or microscopic imaging. For many pathogens, transformation and secretion are well understood and can be explored in a similar manner.Therefore, they enable the study of the host, which are not susceptible to transformation.

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Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated ...