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 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.
1. Construction of an U. maydis Trojan Horse
NOTE: See Figure 1.
2. Culture Media
3. Plant Infection
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 infection21. To direct secretion of the protein of interest peptide into the biotrophic interaction zone, the coding region is fused to the signal peptide sequence of U. maydis Umpit221 (Figure 1). Upon U. maydis transformation, the transgene is inserted into the SG200 ip-locus by homologous recombination, and targeted genome insertion can be verified by Southern blot analysis. Secretion of the fusion protein is confirmed by confocal laser scanning microscopic imaging in seedling leaves infected with a Trojan horse strain (Figure 3). As an example, seedlings infected with either the U. maydis Trojan horse strain SG200Zmmac1 secreting a ZmMAC1-mCHERRY or a non-Trojan horse control strain expressing noSP-Zmmac1-mCHERRY that lacks the Umpit2-SP, and subsequently does not secret the ZmMAC1-mCHERRY-HA protein, are shown in Figure 315. Hyphae secreting ZmMAC1 are surrounded by fluorescent mCherry signal (Figure 3A). In contrast, non-secreting hyphae only show fluorescence signal in the fungal cytoplasm (Figure 3B).
In particular, tassel infection with U. maydis relies on proper tissue inoculation, as described in step 3.5. Improper tassel localization or unequal distribution of the inoculum can result in non-infected tassel (Figure 4A) or only partial infection of the tassel (Figure 4B). To ensure even distribution, the inoculum needs to be slowly released from the inoculation needle to acquire entire tissue infection (Figure 4C). Viability of the inoculum can be verified by placing a droplet on PD-Charcoal agar. Infectious strains form filaments appearing as write fluff on the plate as shown for the solopathogenic Trojan Horse progenitor strain SG200 (Figure 5A) while the U. maydis strain FB1 requires mating before infectious filament formation (Figure 5B).
Name | Primer addition | Sequence (5´→3´) |
Forward | XbaI-maize gene | GCTCTAGA… |
Reverse | NcoI-RSIATA-maize gene | CATGCCATGGAGGCGGTGGCGATCGAGCG…. |
Table 1: Sequences of primer additions to add restriction sides and the RSIATA motif coding sequence to the maize gene of interest.
Figure 1: Schematic overview of the U. maydis Trojan horse plasmid cloning strategy. Zmmac1 (light grey) is released by double digest from p123-PUmpit2–SpUmpit2-Zmmac1-mCherry–Ha. In parallel, the gene of interest (yellow) is amplified by PCR. For cloning purpose, forward and reverse primers are designed which include XbaI and NcoI cloning sites and a RSIATA linker (purple). The PCR product is digested with XbaI and NcoI. After ligation, the Trojan horse plasmid contains the following elements: Driven by the Umpit2 promoter, an Umpit2-SP (blue) is fused N-terminally to a maize gene of interest ORF (yellow). At the C-terminus, a RSIATA linker, a mCHERRY reporter gene (red) and an HA epitope tag (green) are fused followed by a stop codon. Prior to U. maydis transformation, the Trojan horse plasmid is digested with SspI to allow homologous integration into the U. maydisip-locus (grey). Please click here to view a larger version of this figure.
Figure 2: Light-microscopic examination of U. maydis inoculation culture. Several cigar-shaped U. maydis sporidia are visible, some of which undergo budding (indicated by asterisks). No further cells are present which would indicate a contamination of the culture. Scale bar = 20 µm. Please click here to view a larger version of this figure.
Figure 3: In planta confocal laser scanning microscopic imaging of an Ustilago Trojan horse strain. Imaging of the mCherry-fused maize protein ZmMAC1 after maize seedling infection using the Trojan horse strain SG200Zmmac1 (A) or a non-Trojan horse strain SG200Zmmac1-noSP lacking the Umpit2-SP (B). Secreted ZmMAC1-mCHERRY-HA fusion protein is located on the surface of U. maydis hyphae (A)15, indicated by the arrow heads. In SG200Zmmac1-noSP only cytoplasmic localization of ZmMAC1 is visible (B), indicated by the asterisk 15. Scale bars = 5 µm. Please click here to view a larger version of this figure.
Figure 4: Tassel infection with U. maydis. Unsuccessful infection of tassel with U. maydis (A), partial tassel infection (B) and complete tassel infection (C) 12 days after infection are shown. Please click here to view a larger version of this figure.
Figure 5: Inoculum viability assay on PD-Charcoal agar. The solopathogenic strain SG2001 was used for Trojan horse generation. SG200 is self-stimulating and forms infectious filaments on a PD-Charcoal agar plate (A). The haploid U. maydis strain FB1 requires mating with a compatible strain prior to filamentous growth (B)22. Scale bars = 2 mm. Please click here to view a larger version of this figure.
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 proteins into tissues. However, despite advances in heterologous protein expression, targeted infiltration into crop tissues remains difficult and often inefficient for protein functional analysis. The Trojan horse strategy is an alternative approach that does not require transformation or protein infiltration. By employing the secretion apparatus of the maize pathogen U. maydis, delivery of theoretically any protein of interest into the plant apoplast of infected tissue can be achieved. Regarding the size of a protein of interest, the limits of this technique have yet to be explored. In former assays, the 290 amino acids U. maydis effector Cmu1 fused to mCherry was successfully secreted7. However, the applicability of the Trojan horse method to bigger proteins remains to be tested. If additions of posttranslational modifications to the protein of interest are undesirable, unconventional secretion may be used as an alternative route to classical secretion via SP14.
U. maydis is employed as a standard tool in protein biotechnology because of reliable protein folding, posttranslational modification, and secretion efficiencies11,12,13. Nevertheless, protein secretion for each new Trojan horse strain needs to be carefully analyzed as described in the Step 3. It is recommended to perform an initial testing of every newly generated Trojan horse strain by infecting seedlings that are easy to infect and to inspect by microscopic imaging.
SG200 is a solopathogenic strain that does not require mating prior to the Trojan horse experiments and is thus easier to handle. Although the infection efficiency of compatible strains like FB1 and FB2 is higher23, the efficiency of SG200 is sufficient for Trojan horse experiments. Some U. maydis effector proteins are taken up by host cells, while others remain in the apoplast. The differentiation between both groups seems to be a controlled and specific process; however, the underlying mechanisms remain elusive8 and cannot be taken into account when designing an experiment. Therefore, proteins of interest that have to be integrated into the cell wall or that have to act intracellularly are no suitable candidates for the Trojan horse approach.
Since U. maydis is omnipotent in infecting diverse aerial maize tissues, the Trojan horse method can be applied for multiple proteins, in distinct tissues and at different plant developmental stages, such as seedling leaves, adult leaves, tassels, and ears. To name just a few useful applications, the Trojan horse allows testing local overdosage, offside protein effects, or functional characterization of distinct protein domains.
Maintaining an uncontaminated U. maydis culture is crucial for all described experiments since co-infection with a large amount of bacteria triggers the plant's immune response and alters its reaction towards the protein of interest, thus rendering any results inconclusive. Trojan horse studies in adult leaves, tassels and ears have to be performed with maximally 1.5 mL infection culture as higher volumes may result in tissue damage. Tassel and ear infections can be trained using food color-stained water instead of Ustilago inoculum.
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.
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 |