Pathogen Trojan Horse Delivers Bioactive Host Protein to Alter Maize Anther Cell Behavior in Situ

Abstract

Small proteins are crucial signals during development, host defense, and physiology. The highly spatiotemporal restricted functions of signaling proteins remain challenging to study in planta. The several month span required to assess transgene expression, particularly in flowers, combined with the uncertainties from transgene position effects and ubiquitous or overexpression, makes monitoring of spatiotemporally restricted signaling proteins lengthy and difficult. This situation could be rectified with a transient assay in which protein deployment is tightly controlled spatially and temporally in planta to assess protein functions, timing, and cellular targets as well as to facilitate rapid mutagenesis to define functional protein domains. In maize (), secreted ZmMAC1 (MULTIPLE ARCHESPORIAL CELLS1) was proposed to trigger somatic niche formation during anther development by participating in a ligand-receptor module. Inspired by Homer's Trojan horse myth, we engineered a protein delivery system that exploits the secretory capabilities of the maize smut fungus , to allow protein delivery to individual cells in certain cell layers at precise time points. Pathogen-supplied ZmMAC1 cell-autonomously corrected both somatic cell division and differentiation defects in mutant Zm anthers. These results suggest that exploiting host-pathogen interactions may become a generally useful method for targeting host proteins to cell and tissue types to clarify cellular autonomy and to analyze steps in cell responses.

Application of the pathogen Trojan horse approach in maize (Zea mays)

“…To overcome these drawbacks, we developed an alternative, the Trojan horse method. 6,7 The biotrophic corn smut fungus U. maydis infects all aerial plant organs. During infection the hyphae never contact the plant cytoplasm; instead the plant plasma membrane invaginates resulting in a narrow apoplastic space between hyphae and plant cells.…”

“…This approach permits single-cell resolution by tracking the Trojan horse hyphae during plant infection and allows comparison of maize cells receiving secreted protein to non-receiving cells in the same tissue. 7 Expression of foreign proteins by Trojan horse strains is driven by a strong in planta active promoter. 6,7,17 This allows protein delivery to all infected tissue layers.…”

“…7 Expression of foreign proteins by Trojan horse strains is driven by a strong in planta active promoter. 6,7,17 This allows protein delivery to all infected tissue layers. To permit expression at specific time points or locations, alternative promoters may be used in the future.…”

“…After 3 dpi the entire organ is colonized. [7][8][9][10]24 In seedling leaves, U. maydis strain SG200-induced cell proliferation occurs 4-5 dpi. 27 In SG200-infected anthers, no significant formation of additional cells was observed 3 dpi.…”

“…Subsequently, developmental studies using the Trojan horse approach were performed 3-4 dpi. 7 To analyze the impact of the small secreted maize protein ZmMAC1 on Layer2-derived and archesporial cells present in 120-300 µm anthers, tassels with 50-125 µm anthers were infected. Three dpi, anthers were now 400--700 µm and were harvested for confocal imaging.…”