Name: using ustilago maydis as a trojan horse for in situ delivery of maize proteins
Uploaded: 2019-02-08T13:00:00.000+00:00
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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. 
	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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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

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 ...

using-ustilago-maydis-as-trojan-horse-for-situ-delivery-maize

Research

JoVE Journal

Genetics

11.3K Views.  Regensburg University. 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.

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

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

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

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

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.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

Environment

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

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 replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

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

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.

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 ...

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

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

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of &#945;-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. &#945;-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus &#945;-amylase enzyme and belongs to the lectin&#8211;arcelin&#8211;&#945;-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus &#945;-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values.&#160; The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%&#8211;72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%&#8211;88% reduction in aflatoxin levels.

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

Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.&#160; 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.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are ...

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

Bioengineering

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Herein are protocols for collecting and microinjecting precellular corn planthopper embryos for the purpose of modifying their genome via CRISPR/Cas9-based genome editing or for the addition of marked transposable elements through germline transformation.

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the ...

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

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

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

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.

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

Biology

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

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22)&#160;or&#160;phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.

An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus&#160;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.

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the ...

Scalable Transfection of Maize Mesophyll Protoplasts

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation or polyethylene glycol (PEG). Previous methods were developed to produce tens of thousands of transfected protoplasts at once. Here,&#160;we describe a straightforward method to isolate and transfect millions of leaf mesophyll protoplasts in maize (Zea mays L.). This streamlined process removes certain common protoplasting steps, such as washing in W5. Additionally, steps such as centrifugation, PEG-mediated transfection, and incubation have been modified to work with a greater number of protoplasts. The ability to express large libraries of plasmid constructs enables genome-scale experiments, such as massively parallel reporter assays in maize.

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

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation ...

Enabling High-throughput Transgene Expression Studies Using Automated Liquid Handling for Etiolated Maize Leaf Protoplasts

The field of plant biotechnology has witnessed remarkable advancements in recent years, revolutionizing the ability to manipulate and engineer plants for various purposes. However, as research in this field increases in diversity and becomes increasingly sophisticated, the need for early, efficient, dependable, and high-throughput transient screening solutions to narrow down strategies proceeding to stable transformation is more apparent. One method that has re-emerged in recent years is the utilization of plant protoplast, for which methods of isolation and transfection are available in numerous species, tissues, and developmental stages. This work describes a simple automated protocol for the randomized preparation of plasmid within a 96-well plate, a method for the isolation of etiolated maize leaf protoplast, and an automated transfection procedure. The adoption of automated solutions in plant biotechnology, exemplified by these novel liquid handling protocols for plant protoplast transfection, represents a significant advancement over manual methods. By leveraging automation, researchers can easily overcome the limitations of traditional methods, enhance efficiency, and accelerate scientific progress.

This protocol describes the creation of a randomized transfection layout using an automated liquid handler, a protoplast isolation protocol for etiolated maize leaf, and a 96-well transfection procedure using a liquid handler.

The field of plant biotechnology has witnessed remarkable advancements in recent years, revolutionizing the ability to manipulate and engineer plants for ...

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

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

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

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

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

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

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

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

Scalable Transfection of Maize Mesophyll Protoplasts

Enabling High-throughput Transgene Expression Studies Using Automated Liquid Handling for Etiolated Maize Leaf Protoplasts