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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • תוצאות
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

Inspired by Homer´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.

Introduction

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 day

Protocol

1. Construction of an U. maydis Trojan Horse

NOTE: See Figure 1.

  1. 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'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.

תוצאות

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<.......

Discussion

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.......

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
2 mL syringe B. Braun4606027V
23G x 1 1/4 hypodermic needleB. Braun4657640
Bacto Peptone BD211677
cDNA from maizefrom maize tissue expressing the gene of interrest
CharcoalSigma-Aldrich05105
Confocal laser scanning microscopeuse locally available equipment
Cuvette (10 x 4 x 45 mm)Sarstedt67742
Incubator-shaker set to 28 °C, 200 rpmuse locally available equipment
Light microscope with 400-fold magnificationuse locally available equipment
Nco INEBR0193
p123-PUmpit2-SpUmpit2-Zmmac1-mCherry-Haplease contact the corresponding author 
Pasteur pipet (glass, long tip)VWR14673-043
pCR-Blunt-II-TOPOThermo Fisher ScientificK280002can be exchanged for other basic cloning vectors like pENTR or pJET
Potato Dextrose Agar VWR90000-745
Sharpie penuse locally available equipment
Spectrophotometeruse locally available equipment
Ssp INEBR0132
SucroseSigma-AldrichS0389
T4 DNA ligaseNEBM0202
TRISSigma-AldrichTRIS-RO
Xba INEBR0145
Yeast extract BD212750

References

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Ustilago MaydisTrojan HorseMaize ProteinsGenetic AnalysisProtein SecretionInfectionTissue AnalysisCell CulturePlant CultivationInjection

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