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

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.

Results

Constructs for U. maydis Trojan horse experiments are cloned into the plasmid p123-P_Umpit2-Sp_Umpit2-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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2 mL syringe	B. Braun	4606027V
23 G 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 mm x 4 mm 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