Name: Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins (Video) | JoVE Journal
Uploaded: 2019-02-08T13:00:00.000+00:00
Duration: 5 min 38 s
Description: Discover the Trojan Horse Method for in situ delivery of maize proteins using Ustilago maydis. This approach enables detailed studies of secreted proteins in leaves, tassels, and ears with high spatial resolution.

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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. 
	NOTE: PCR specifications need to be optimized due to primer sequence specificities and optimal DNA polymerase reaction conditions.</li>
	<li>Design primers to amplify the maize gene of interest without the sequence encoding a signal peptide (SP).</li>
	<li>Extend the 5&#180; end of the reverse primer with the RSIATA motif and an NcoI cutting site (Table 1).</li>
	<li>Amplify the maize gene of interest with a proofreading DNA polymerase using the PCR construct generated in 1.1 as the PCR template.</li>
	<li>Double-digest the PCR product and the U. maydis transformation plasmid, p123-PUmpit2-SpUmpit2-Zmmac1-mCherry-Ha15, with XbaI and NcoI. Purify the digested PCR product and plasmid.</li>
	<li>Ligate the digested PCR product into the p123-PUmpit2-SpUmpit2-Zmmac1-mCherry-Ha template using T4 DNA ligase following the manufacturer&#180;s instructions. Transform the ligation product into E. coli and verify the correct gene of interest sequence by Sanger sequencing.</li>
	<li>Linearize the p123-PUmpit2-SpUmpit2-Zmgene of interest-mCherry-Ha with the restriction enzyme SspI and transform DNA into the solopathogenic U. maydis strain SG20016. Isolate U. maydis transformants by carboxin selection and confirm isolated transformants by Southern blot analysis16. 
	NOTE: For each protein of interest, at least three independent U. maydis transformants should be isolated and analyzed to estimate any phenotype effects of random background mutations.</li>
</ol>
2. Culture Media
<ol>
	<li>Prepare YEPSlight liquid medium16: 1.0% (w/v) yeast extract, 0.4% (w/v) Bacto-Peptone, and 0.4% (w/v) sucrose. Dissolve all components in ddH2O and autoclave at 121 &#176;C for 15 min; autoclaving at a higher temperature, for a longer period of time or repeatedly would reduce the quality of the medium.</li>
	<li>Prepare potato-dextrose-agar (PD-agar)20: 3.9% (w/v) potato dextrose agar, and 1.0% (w/v) 1 M Tris-HCl pH 8.0 (f.c. 0.01 M). Mix all components directly in the bottle for autoclaving and add ddH2O plus a magnetic stir bar. Autoclave at 121 &#176;C for 15 min; autoclaving at a higher temperature, for a longer period of time or repeatedly would reduce the quality of the medium.</li>
	<li>Prepare PD-Charcoal agar20: 3.9% (w/v) potato dextrose agar, 1% (w/v) charcoal, and 1.0% (v/v) 1 M Tris-HCl pH 8.0 (f.c. 0.01 M). Mix all components directly in the bottle for autoclaving and add ddH2O plus a magnetic stir bar. Autoclave at 121 &#176;C for 15 min; autoclaving at a higher temperature, for a longer period of time or repeatedly would reduce the quality of the medium.</li>
</ol>
3. Plant Infection
<ol>
	<li>Perform analysis of maize cell division in response to Trojan horse delivered protein (e.g., microscopy-based cell counting)&#160;beforehand. U. maydis-induced maize cell proliferation and subsequent tumor formation starts around 4-5 days post infection. Quantitative disease assessments are tissue-dependent and should be performed from 6 to 14 days post infection. 
	NOTE: Distinct maize cultivars show different levels of susceptibility to U. maydis infection. Maize cvs. W23, A188, Gaspe flint, Early Golden Bantam or Va35 show susceptibility towards this pathogen and are thus suitable cultivars for Trojan horse studies.</li>
	<li>Preparation of the U. maydis inoculum
	<ol>
		<li>Include the progenitor strain SG200 as a negative control in all Trojan horse experiments in order to estimate side effects on the infection by the transgenic strain. Here, use an U. maydis strain expressing a non-secreted version of the protein of interest as a negative control. However, for reasons of practicability (e.g., larger screenings), the progenitor strain may be the easier choice of control.</li>
		<li>Before starting the experiment, estimate what amounts of infection culture are needed. Keep in mind that infection of each plant requires 1 - 1.5 mL of U. maydis suspension dependent on the maize tissue type. 
		NOTE: Approximately 1 mL of an overnight culture is sufficient for dilution to an OD600 of 0.2 in 20 mL of YEPSlight medium, and 25 mL of an U. maydis culture with an OD600 of 0.8-1.0 are sufficient for infection of 13-16 plants.</li>
		<li>Scratch U. maydis from a PD agar plate using a sterile Pasteur pipette, inoculate in 5 mL of YEPSlight medium and let the culture grow at 28 &#176;C with constant shaking at 200 rpm for 16 h.</li>
		<li>Prior to infection, examine the U. maydis inoculation culture by standard light microscopy for proper growth and bacterial contamination at 400X magnification. 
		NOTE: In a suitable culture, only the cigar-shaped fungus is visible (Figure 2).</li>
		<li>Mix 900 &#181;L of fresh YEPSlight with 100 &#181;L of the overnight culture and measure the OD600 using YEPSlight medium as a blank in the spectrophotometer analysis.</li>
		<li>Dilute the overnight culture with fresh YEPSlight medium to an OD600 of 0.2 and let the culture grow at 28 &#176;C with constant shaking at 200 rpm until reaching the mid-log growth phase indicated by an OD600 nm of 0.8-1.0. 
		NOTE: U. maydis cells duplicate every 2 h under these conditions, thus the desired OD600 is reached after 4-5 h of cultivation.</li>
		<li>Harvest the cells at OD600 of 0.8-1.0 by spinning at 3,000 x g for 10 min and discard the supernatant.</li>
		<li>Wash the cell pellet one time with ddH2O. For this purpose, add one culture volume of ddH2O, spin with 3000 x g for 10 min and discard the supernatant.</li>
		<li>Resuspend the cell pellet carefully in ddH2O using a 20-mL glass pipette, thereby adjusting the final OD600 to 3.0 (for a Trojan horse assay) or 1.0 (for disease rating).</li>
	</ol>
	</li>
	<li>Verification of the Trojan horse: in planta secretion of the maize fusion protein
	<ol>
		<li>Infect maize seedlings with a Trojan horse strain17.</li>
		<li>Perform microscopic imaging of infected seedlings at 2 - 3 days post infection using a confocal laser-scanning microscope. To this end, excise a rectangular piece of the leaf 1 cm below the point of injection, place the sample onto a microscope slide and add a drop of ddH2O. To visualize mCherry fusion protein, excite specimen at &#955;= 561 nm and record emission at &#955;= 580 - 630 nm.</li>
	</ol>
	</li>
	<li>Infection of adult maize leaves
	<ol>
		<li>Cultivate maize plants to the stage of adult leaves (when at least leaf 7 grows within the stalk). 
		NOTE: This stage is reached after four weeks upon sowing under greenhouse conditions of 14 h, 28 &#176;C day/10 h, 22 &#176;C night rhythm using cv. W23. Duration may vary with the maize cultivar and greenhouse conditions.</li>
		<li>Transfer the U. maydis culture (see 3.2.9) into a 3-mL syringe with a 20G x 1 hypodermic needle.</li>
		<li>Press the stalk carefully to localize the meristem tissue in the stalk. The base of the meristem can be distinguished by a transition from harder stalk to softer tissue.</li>
		<li>Mark the meristem on the stalk using a pen.</li>
		<li>Inject 1.5 mL of U. maydis culture 1 cm above the shoot meristem or inflorescence meristem.</li>
		<li>Rate disease symptoms at 6 and 12 days post infection.</li>
	</ol>
	</li>
	<li>Infection of tassels
	<ol>
		<li>Grow maize plants until reaching the tassel stage. 
		NOTE: A detailed timeline on anther and tassel development in maize cv. W23 was previously described18,19. Tassels containing pre-meiotic anthers are highly susceptible to U. maydis infection; in the maize cv. W23 tassels are the size of 4 - 7 cm.</li>
		<li>Press the stalk carefully to localize the tassel in the stem.</li>
		<li>Mark the tip and the base of the tassel on the stem using a pen.</li>
		<li>Transfer the U. maydis culture (see 3.2.9) into a 3-mL syringe with a 20G x 1 hypodermic needle.</li>
		<li>Inject 1.5 mL of the inoculum around the tassel. To ensure equal distribution of the inoculum, slowly place 0.5 mL each at the tip, the middle part, and the base of the tassel marked with the pen.</li>
		<li>Rate disease symptoms at 10 days post infection.</li>
	</ol>
	</li>
	<li>Infection of ears 
	NOTE: The ear tissue development differs in distinct maize cultivars and greenhouse conditions and must be carefully observed prior to inoculation. Ears starting to outgrow silks are highly susceptible to U. maydis infection.
	<ol>
		<li>Transfer the U. maydis culture (see 3.2.9) into a 3 mL syringe with a 20 G x 1 hypodermic needle.</li>
		<li>Inject the inoculation needle into the space between the husk leaves as deeply as possible without injuring the ear.</li>
		<li>Release 1.5 mL of the inoculum around the ear.</li>
		<li>Remove the syringe plus needle and carefully massage the cob to distribute the U. maydis solution equally.</li>
		<li>Rate disease symptoms at 14 days post infection.</li>
	</ol>
	</li>
	<li>Confirm viability of U. maydis inoculum
	<ol>
		<li>Drop 10 &#181;L of the inoculum on a PD charcoal agar plate and incubate at room temperature for 2 days. 
		NOTE: If the respective U. maydis culture is able to form filaments, fluffy, white mycelium becomes visible (Figure 5)</li>
	</ol>
	</li>
</ol>

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<li>Matei, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+to+make+a+tumour%3A+cell+type+specific+dissection+of+Ustilago+maydis-induced+tumour+development+in+maize+leaves%5BTitle%5D)+AND+%22New+Phytologist%22%5BJournal%5D)">How to make a tumour: cell type specific dissection of Ustilago maydis-induced tumour development in maize leaves.</a> New Phytologist. , (2018).</li>
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<li>Doehlemann, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pep1%2C+a+secreted+effector+protein+of+Ustilago+maydis.%2C+is+required+for+successful+invasion+of+plant+cells%5BTitle%5D)+AND+%22PLOS+Pathogens%22%5BJournal%5D)">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/pubmed?term=((A+secreted+effector+protein+of+Ustilago+maydis+guides+maize+leaf+cells+to+form+tumors%5BTitle%5D)+AND+%22The+Plant+Cell%22%5BJournal%5D)">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/pubmed?term=((Metabolic+priming+by+a+secreted+fungal+effector%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((A+secreted+Ustilago+maydis+effector+promotes+virulence+by+targeting+anthocyanin+biosynthesis+in+maize%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">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/pubmed?term=((Compatibility+in+the+Ustilago+maydis-maize+interaction+requires+inhibition+of+host+cysteine+proteases+by+the+fungal+effector+Pit2%5BTitle%5D)+AND+%22PLOS+Pathogens%22%5BJournal%5D)">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/pubmed?term=((An+apoplastic+peptide+activates+salicylic+acid+signalling+in+maize%5BTitle%5D)+AND+%22Nature+Plants%22%5BJournal%5D)">An apoplastic peptide activates salicylic acid signalling in maize.</a> Nature Plants. 4, 172-180 (2018).</li>
<li>Juárez-Montiel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+corn+smut+%28%27Huitlacoche%27%29+as+a+new+platform+for+oral+vaccines%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">The corn smut ('Huitlacoche') as a new platform for oral vaccines.</a> PLoS One. 10, e0133535(2015).</li>
<li>Sarkari, P., Feldbrügge, M., Schipper, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSarkari%2C+P+%22Gene+Expression+Systems+in+Fungi%3A+Advancements+and+Applications%22">Gene Expression Systems in Fungi: Advancements and Applications</a>. Schmoll, M., Dattenböck, C. , Springer International Publishing. 183-200 (2016).</li>
<li>Monreal-Escalante, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+corn+smut-made+cholera+oral+vaccine+is+thermostable+and+induces+long-lasting+immunity+in+mouse%5BTitle%5D)+AND+%22Journal+of+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Applying+unconventional+secretion+of+the+endochitinase+Cts1+to+export+heterologous+proteins+in+Ustilago+maydis%5BTitle%5D)+AND+%22Journal+of+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Pathogen+Trojan+horse+delivers+bioactive+host+protein+to+alter+maize+%28Zea+mays%29+anther+cell+behavior+in+situ%5BTitle%5D)+AND+%22The+Plant+Cell%22%5BJournal%5D)">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>Bösch, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+manipulation+of+the+plant+pathogen+Ustilago+maydis+to+study+fungal+biology+and+plant+microbe+interactions%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((A+rapid+and+efficient+method+for+assessing+pathogenicity+of+Ustilago+maydis+on+maize+and+teosinte+lines%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Emergence+and+patterning+of+the+five+cell+types+of+the+Zea+mays+anther+locule%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">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/pubmed?term=((Quantifying+Zea+mays.+tassel+development+and+correlation+with+anther+developmental+stages+as+a+guide+for+experimental+studies%5BTitle%5D)+AND+%22Maydica%22%5BJournal%5D)">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. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHolliday%2C+R+%22Bacteria%2C+Bacteriophages%2C+and+Fungi%3A+Volume+1%22">Bacteria, Bacteriophages, and Fungi: Volume 1</a>. King, R. C. , Springer US. 575-595 (1974).</li>
<li>Doehlemann, G., Reissmann, S., Aßmann, D., Fleckenstein, M., Kahmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+linked+genes+encoding+a+secreted+effector+and+a+membrane+protein+are+essential+for+Ustilago+maydis-induced+tumour+formation%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">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/pubmed?term=((Different+a+alleles+of+Ustilago+maydis+are+necessary+for+maintenance+of+filamentous+growth+but+not+for+meiosis%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">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>
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</ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>2 mL syringe&amp;nbsp;</td><td>B. Braun</td><td>4606027V</td><td></td></tr><tr><td>23 G x 1 1/4 hypodermic needle</td><td>B. Braun</td><td>4657640</td><td></td></tr><tr><td>Bacto Peptone&amp;nbsp;</td><td>BD</td><td>211677</td><td></td></tr><tr><td>cDNA from maize</td><td></td><td></td><td>from maize tissue expressing the gene of interrest</td></tr><tr><td>Charcoal</td><td>Sigma-Aldrich</td><td>05105</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td></td><td></td><td>use locally available equipment</td></tr><tr><td>Cuvette (10 mm x 4 mm x 45 mm)</td><td>Sarstedt</td><td>67742</td><td></td></tr><tr><td>Incubator-shaker set to 28 &amp;deg;C, 200 rpm</td><td></td><td></td><td>use locally available equipment</td></tr><tr><td>Light microscope with 400-fold magnification</td><td></td><td></td><td>use locally available equipment</td></tr><tr><td>&lt;em&gt;Nco&lt;/em&gt; I</td><td>NEB</td><td>R0193</td><td></td></tr><tr><td>p123-P&lt;sub&gt;Um&lt;/sub&gt;&lt;em&gt;&lt;sub&gt;pit2&lt;/sub&gt;&lt;/em&gt;-Sp&lt;sub&gt;Um&lt;/sub&gt;&lt;em&gt;&lt;sub&gt;pit2&lt;/sub&gt;&lt;/em&gt;-Zm&lt;em&gt;mac1&lt;/em&gt;-&lt;em&gt;mCherry-Ha&lt;/em&gt;</td><td></td><td></td><td>please contact the corresponding author&amp;nbsp;</td></tr><tr><td>Pasteur pipet (glass, long tip)</td><td>VWR</td><td>14673-043</td><td></td></tr><tr><td>pCR-Blunt-II-TOPO</td><td>Thermo Fisher Scientific</td><td>K280002</td><td>can be exchanged for other basic cloning vectors like pENTR or pJET</td></tr><tr><td>Potato Dextrose Agar&amp;nbsp;</td><td>VWR</td><td>90000-745</td><td></td></tr><tr><td>Sharpie pen</td><td></td><td></td><td>use locally available equipment</td></tr><tr><td>Spectrophotometer</td><td></td><td></td><td>use locally available equipment</td></tr><tr><td>&lt;em&gt;Ssp&lt;/em&gt; I</td><td>NEB</td><td>R0132</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202</td><td></td></tr><tr><td>TRIS</td><td>Sigma-Aldrich</td><td>TRIS-RO</td><td></td></tr><tr><td>&lt;em&gt;Xba&lt;/em&gt; I</td><td>NEB</td><td>R0145</td><td></td></tr><tr><td>Yeast extract&amp;nbsp;</td><td>BD</td><td>212750</td><td></td></tr></tbody></table>

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

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<...

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.

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

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Research

JoVE Journal

Genetics

Source: Beernink, B. M. et al. Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones. J. Vis. Exp. (2021)This video demonstrates the Agrobacterium-mediated delivery of recombinant viral clones into maize seedlings for gene silencing. It outlines the process of Agrobacterium infection, systemic viral spread, and RNA interference-driven phenotypic changes.

Agrobacterium-Mediated Delivery of Recombinant Viral Clones into Maize Seedlings for Gene Silencing

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described.  The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.  Using a GFP-expressing A. flavus strain we monitored the infection and spread of the fungus in mature kernels in real time. The assay is rapid, reliable, and reproducible.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus and sugarcane mosaic virusclones into maize seedlings. Inoculation in this manner leads to viral infection, virus-induced gene silencing of marker genes, and viral overexpression of GFP.

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells.

Scalable Transfection of Maize Mesophyll Protoplasts

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of Copy Number Alterations Using Single Cell Sequencing

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

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

Discover the Trojan Horse Method for in situ delivery of maize proteins using Ustilago maydis. This approach enables detailed studies of secreted proteins in leaves, tassels, and ears with high spatial resolution.

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

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