Investigating processes on genetic and protein levels is crucial in crop research. Our approach enables inspection of secreted maze proteins with highest spatial temporal resolution at different infection sides and tissues. The Trojan Horse Method explores useful automatous secretory capabilities.
It circumvents time consuming and challenging maze transformations, and facilitates detailed studies on secreted proteins within the maze appelplatz. Detailed information on proper infection of added maze leafs, tassels and ear. And shows the successful study of infection progression and protein function in the target tissue.
Demonstrating the procedure will be Isabell Fiedler, a grad student from the laboratory. To begin the experiment, scratch Eumadous from a pediatr plate using a sterile pasteur pipette. Inoculate 5 milliliters of YEPS Light Medium with Eumadous.
Grow the culture at 28 degrees Celsius, with constant shaking at 200 rpm for 16 hours. The next day mix 900 microliters of fresh YEPS Light with 100 microliters of the overnight culture. Measure the OD-600 using YEPS Light Medium as a blank in the spectrophotometer analysis.
Dilute the overnight culture with fresh YEPS Light Medium to an OD-600 of 2. Let the culture grow at 28 degree Celsius with constant shaking at 200 rpm, until it reaches the mid log growth phase, indicated by an OD-600 of 8 to 1.0. After incubation, harvest the cells by spinning them at 3000 times chi for 10 minutes.
Discard the supernatant. Next, wash the cell pilette one time with 1 culture volume of DDH2O. Spin the cells at 3000 time chi for 10 minutes and discard the supernatant.
Then, re-suspend the cell pilette carefully in DDH2O, using a 20 milliliter glass pipette. Thereby adjusting the final OD-600 TO 3.0 for a Trojan Horse assay or 1.0 for disease rating. Cultivate the maze plants to a stage of adult leafs, where at least leaf 7 grows within the stalk.
Next, transfer the Eumadous culture into a 3 milliliter syringe with a 20 gauge bi one hypodermic needle. Press the stalk carefully to localize the Mirror Stem Tissue. The base of the Mirror Stem can be distinguished by a transition from harder stalk to softer tissue.
Mark the Mirror Stem on the stalk using a pen. Inject 1.5 milliliters of Eumadous culture 1 centimeter above the Shoot Mirror Stem or Inflorescence Mirror Stem. Once the maze plants reach the tassel stage, press the stalk carefully to localize the tassel in the stem.
Mark the tip and the base of the tassel on the stem using a pen. Transfer the Eumadous culture into a 3 milliliter syringe with a 20 gauge bi one hypodermic needle to inject a total of 1.5 milliliters of the inoculum around the tassel. To insure equal distribution of the inoculum, slowly place 5 milliliters at the tip, the middle, and the base of the tassel marked with the pen.
Transfer the Eumadous culture into a 3 milliliter syringe with 20 gauge bi one hypodermic needle. Inject the inoculation needle into the space between the husk leafs as deeply as possible without injuring the ear, then release 1.5 milliliters of the inoculum around the ear. Next, remove the syringe and needle and carefully massage the cob to distribute the Eumadous solution equally.
Drop 10 microliters of inoculum on a PD charcoal auger plate. Then incubate at room temperature for 2 days. Hyphae secreting ZM Mach1 are surrounded by fluorescent and cherry signal.
In contrast, nonsecreting hyphae only show fluorescent signal in the fungal cytoplasm. Improper tassel localization or unequal distribution of the inoculum can result in noninfected tassel or only partial infection of the tassel compared to even distribution. The solo pathogenic Trojan Horse Progenester strain SG-200, cultured on PD charcoal auger, forms white fluff filaments on the plate as shown.
While the Eumadous strain FB1 requires mating before infectious filament formation. Following infection, the possible response of signs the Trojan Horse liver protein can be analyzed using various methods including disease ratings or microscopic imaging. For many pathogens, transformation and secretion are well understood and can be explored in a similar manner.
Therefore, they enable the study of the host, which are not susceptible to transformation.
