Background:

Plasmodesmata (PD) are intercellular channels that mediate the transport of small and macromolecules between plant cells, including metabolites, proteins and RNAs, but also plant viruses. Although PD play a central role in development, signaling, and pathogen spread, the molecular mechanism governing selective transport of proteinaceous cargo remains unresolved. 

Transport through PD and the nuclear pore complex (NPC) share common features, namely both, NPC and PD, transport a similar spectrum of cargo species (Lee et al., 2000; Ejike et al., 2025); For both NPC and PD, the ability for pore dilation has been described (Kragler et al., 1998; Zimmerli et al., 2021). Both NPC and PD constitute selective barriers, allowing passive passage of molecules with an apparent size exclusion limit and facilitated translocation of specific macromolecules. In contrast to PD, the molecular mechanisms underlying facilitated translocation of cargo has been resolved for the NPC. Engineered GFP surface variants with different surface residue compositions allowed to reveal that cargo transport is governed by surface-exposed residues on the cargo which interact with key components of the NPC (Frey et al., 2018). Subtle changes in surface chemistry on the cargo, such as changes in aromatic or arginine residue density, can dramatically alter mobility.

The Project:

The aim of this project is to determine whether cell-to-cell transport of cargo through PD is also dependent on certain surface residues on the cargo. Using plant cell-to-cell transport assays such as transient infiltration, fluorescence recovery after photobleaching (FRAP), and particle bombardment experiments, combined with advanced confocal microscopy, you will study the cell-to-cell transport through PD of several aforementioned engineered GFP surface variants with similar molecular masses but different surface residue compositions. This approach will enable you to show whether other biophysical features of the cargo, in addition to size, play a role in the cell-to-cell transport through PD. Moreover, this approach could help to pinpoint which residues may play a role in the transport selectivity. Understanding PD transport mechanisms is of fundamental importance and could lead to the discovery of new molecular targets for engineering virus-resistant crops by interfering with viral movement.

If you are interested, please send your application to:  imp.application1(at)hhu.de

References:

Ejike JO, Davis GV, Restrepo-Escobar A, Dalal A, Nakamura M, Frommer WB, Schladt TM. 2025. The role of phase separation for RNA and protein transport through the nuclear pore complex. Journal of Experimental Botany, eraf271.

Frey S, Rees R, Schünemann J, Ng SC, Fünfgeld K, Huyton T, Görlich D. 2018. Surface properties determining passage rates of proteins through nuclear pores. Cell 174, 202-217.e9.

Kragler F, Monzer J, Shash K, Xoconostle-Cázares B, Lucas WJ. 1998. Cell-to-cell transport of proteins: Requirement for unfolding and characterization of binding to a putative plasmodesmal receptor. Plant Journal 15, 367–381.

Lee JY, Yoo BC, Lucas WJ. 2000. Parallels between nuclear-pore and plasmodesmal trafficking of information molecules. Planta 210, 177–187.

Zimmerli CE, Allegretti M, Rantos V, et al. 2021. Nuclear pores dilate and constrict in cellulo. Science 374, eabd9776.

Supervision: Prof. Dr. Wolf B Frommer, Nora Zöllner Institute for Molecular Physiology

Our lab pioneered the development of genetically encoded biosensors for metabolites and used them in microbes, plants and animal cells to study transport and metabolic processes with minimal invasion.

Several HHU groups including ours recently developed a new set of sensors for xylose, glucose, sucrose, ATP, iron and cyclic-di-GMP on the basis of our Matryoshka sensor design concept (Ast et al., 2015, 2017; De Michele et al., 2013; Ejike et al., 2024). These sensor are not yet published.

We are exploring the virulence of a gram-negative bacterium, Xanthomonas oryzae pv. oryzae that cause massive yield losses in rice in Asia and Africa. We have succeeded in unraveling key aspects of the virulence mechanism and have used it to generate broad spectrum resistance against bacterial blight in several elite varieties (Eom et al., 2019; Huguet-Tapia et al., 2025; Loo et al., 2025; Oliva et al., 2019; Schepler-Luu et al., 2023). We now want to explore the colonization of the rice xylem and implement the sensors in the bacteria to study their physiology in vitro and in planta (Redzich et al., 2025; Zöllner et al., 2025).

In this thesis project, the sensors will be expressed in Xanthomonas oryzae pv. oryzae, and intra- and extracellular dynamic of the metabolized and signaling molecules will be characterized. Mutants will be generated using genome editing in Xanthomonas oryzae pv. oryzae to learn more about the metabolism and signaling processes, both as a basis for subsequently exploring the dynamics in the plant and to anticipate novel virulence mechanisms the bacteria my develop.

If you are interested, please send your application to:  imp.application1(at)hhu.de

References:

Ast, C., De Michele, R., Kumke, M.U., and Frommer, W.B. (2015) Single-fluorophore membrane transport activity sensors with dual-emission read-out. eLife, 4, e07113.

Ast, C., Foret, J., Oltrogge, L.M., Michele, R.D., Kleist, T.J., Ho, C.-H., and Frommer, W.B. (2017) Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins. Nat. Commun., 8, 431.

De Michele, R., Ast, C., Loque, D., Ho, C.H., Andrade, S.L., Lanquar, V., et al. (2013) Fluorescent sensors reporting the activity of ammonium transceptors in live cells. Elife, 2, e00800.

Ejike, J.O., Sadoine, M., Shen, Y., Ishikawa, Y., Sunal, E., Hänsch, S., et al. (2024) A monochromatically excitable green–red dual-fluorophore fusion incorporating a new large Stokes shift fluorescent protein. Biochemistry, 63, 171–180.

Eom, J.-S., Luo, D., Atienza-Grande, G., Yang, J., Ji, C., Luu, V.T., et al. (2019) Diagnostic kit for rice blight resistance. Nat. Biotechnol., 37, 1372–1379.

Huguet-Tapia, J.C., Loo, E.P.I., Buchholzer, M., Hartwig, T., Yang, B., White, F.F., and Frommer, W.B. (2025) Ensuring effective removal of transgenes before release of genome-edited crops. Nat. Biotechnol., 43, 1603–1605.

Loo, E.P.-I., Huguet‐Tapia, J.C., Selvaraj, M.G., Stiebner, M., Killing, B., Buchholzer, M., et al. (2025) Removal of transgenes and evaluation of yield penalties in genome edited bacterial blight resistant rice varieties. Plant Biotechnol. J., accepted.

Oliva, R., Ji, C., Atienza-Grande, G., Huguet-Tapia, J.C., Pérez-Quintéro, A., Li, T., et al. (2019) Broad-spectrum resistance to bacterial blight in rice using genome-editing. Nat. Biotechnol., 37, 1344–1350.

Redzich, L., Ma, Z., Restrepo-Escobar, A., Bäumers, M., Schepler-Luu, V., Loo, E.P.I., and Frommer, W.B. (2025) Differentiation of Xanthomonas oryzae pv. oryzae in vitro and during rice leaf infection. 2025.10.12.680524.

Schepler-Luu, V., Sciallano, C., Stiebner, M., Ji, C., Boulard, G., Diallo, A., et al. (2023) Genome editing of an African elite rice variety confers resistance against endemic and emerging Xanthomonas oryzae pv. oryzae strains. eLife, 12, e84864.

Zöllner, N.R., Long, J., Song, C., Sharkey, J., Wudick, M.M., Loo, E.P.I., et al. (2025) A critical role of sux cistron-mediated sucrose uptake for virulence of the rice blight pathogen Xanthomonas oryzae pv. oryzae. 2025.06.02.657373.

Supervision: Prof. Dr. Wolf B Frommer, Dr. Eliza Loo; Institute for Molecular Physiology

Multicellularity is often considered to be a feature unique to eukaryotes. However already Archaea can form multicellular assemblies and can communicate between neighboring cells. Many pathogenic bacteria can filament (incomplete cell division leading to either cells containing multiple nucleoids, or they can divide without cell separation. In mammalian pathogens, filamentation has been associated with stress conditions, however it is likely that the filamentation is developmentally controlled and serves specific functions such as improved adhesion or penetration of host tissues. 

We recently found that the gram-negative bacterium, Xanthomonas oryzae pv. oryzae which causes bacterial blight of rice can filament in vitro and in planta (Redzich et al., 2025). 

The aim of this project is to determine the differences in gene expression during the transition using RNAseq. The project will involve induction of the filamentation in vitro (e.g. by high salt, high density, etc.) and the analysis of changes in the transcript profiles during the transition to pinpoint both mechanism and regulation. 

We have already obtained dual RNAseq data during the infection of rice leaves and plan on comparing the changes in transcript abundance in vitro and in planta.

If possible, we will also try to generate Xanthomonas oryzae pv. oryzae mutants that are defective in filamentation and characterize their virulence, in particular using bacteria expressing fluorescent proteins.

Notably, filamentation has been found in other key plant-pathogenic bacteria such as Xylella, however only so far in vitro. The work may thus also be suitable to develop new strategies to protect plants against Xylella infections. The project is expected to have relevance beyond the plant filed since bacterial differentiation is not well understood. Moreover, we hope to identify new ways to generate resistance.

If you are interested, please send your application to:  imp.application1(at)hhu.de

References:

Redzich, L., Ma, Z., Restrepo-Escobar, A., Bäumers, M., Schepler-Luu, V., Loo, E.P.I., and Frommer, W.B. (2025) Differentiation of Xanthomonas oryzae pv. oryzae in vitro and during rice leaf infection. 2025.10.12.680524. biorXiv. BMC Microbiol., submitted.