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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´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.
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
1. Construction of an U. maydis Trojan Horse
NOTE: See Figure 1.
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<.......
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 authors have nothing to disclose.
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.
....Name | Company | Catalog Number | Comments |
2 mL syringe | B. Braun | 4606027V | |
23G x 1 1/4 hypodermic needle | B. Braun | 4657640 | |
Bacto Peptone | BD | 211677 | |
cDNA from maize | from maize tissue expressing the gene of interrest | ||
Charcoal | Sigma-Aldrich | 05105 | |
Confocal laser scanning microscope | use locally available equipment | ||
Cuvette (10 x 4 x 45 mm) | Sarstedt | 67742 | |
Incubator-shaker set to 28 °C, 200 rpm | use locally available equipment | ||
Light microscope with 400-fold magnification | use locally available equipment | ||
Nco I | NEB | R0193 | |
p123-PUmpit2-SpUmpit2-Zmmac1-mCherry-Ha | please contact the corresponding author | ||
Pasteur pipet (glass, long tip) | VWR | 14673-043 | |
pCR-Blunt-II-TOPO | Thermo Fisher Scientific | K280002 | can be exchanged for other basic cloning vectors like pENTR or pJET |
Potato Dextrose Agar | VWR | 90000-745 | |
Sharpie pen | use locally available equipment | ||
Spectrophotometer | use locally available equipment | ||
Ssp I | NEB | R0132 | |
Sucrose | Sigma-Aldrich | S0389 | |
T4 DNA ligase | NEB | M0202 | |
TRIS | Sigma-Aldrich | TRIS-RO | |
Xba I | NEB | R0145 | |
Yeast extract | BD | 212750 |
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