Intercellular Macromolecular Transport
The main focus of Dr. Fritz Kragler's group is the characterization of the transport mechanism and function of proteins and RNA molecules moving between cells. Pores named plasmodesmata connect adjacent plant cells and facilitate the exchange of proteins and RNA molecules between cells. A plasmodesma forms a channel through the cellulosic cell wall and viruses use the connection to spread from cell to cell. We are especially interested in the mechanism of intercellular transport and the function of the transported molecules such as RNA and transcription factors as morphogenic and metabolic signals.
The transport mechanism and the transported macromolecules are addressed in the model plants A. thaliana, C. maximus (pumpkin), N. tabacum, and Cuscuta (dodder) plants using genomic and proteomic approaches as well as molecular in vivo and in vitro methods.
In plants homeotic proteins involved in meristem maintenance such as KNOTTED1 (KN1) are thought to move like viral movement proteins to adjacent cells via intercellular pores formed by plasmodesmata. Cell-to-cell transport of proteins and RNA molecules seems to be regulated depending on a number of cellular components. Homeotic transcription factors function within the nucleus, thus, a decision between non-cell-autonomy versus nuclear import has to be made within the cytoplasm. Next, actively transported transcription factors have to interact with specific receptors regulating access to the intercellular transport machinery established by plasmodesmata, which in turn transfers the non-cell-autonomous proteins to neighboring cells via the plasmodesmatal pores. However, limited knowledge is available regarding interaction partners of non-cell-autonomous transcription factors such as KN1-binding proteins and their functional role(s) in cellular distribution.
Recent results indicate that a novel microtubules-associated protein, named MPB2C, can negatively regulate entry into the plasmodesmal transport pathway. Structurally and functionally distinct proteins such as the Tobacco mosaic virus movement protein (TMV-MP), KNOTTED1 (KN1) as well as the A. thaliana KN1 homologes SHOOT MERISTEMLESS (STM) or KNAT1 interact with MPB2C. Ample presence of MPB2C protein prevents cell-to-cell movement of the homeobox transcription factors KN1 and TMV-MP. In addition, we isolated a novel KN1/STM/KNAT1 binding protein, KNB36, which is also transported from cell to cell and binds to MPB2C. In microinjection experiments and transient expression assays KNB36 has no effect on homeobox protein cell-to-cell transport. However, KNB36 seems to be a novel factor involved in triggering with the help of MPB2C degradation of homeobox transcription factors.
Plants constitutively expressing MPB2C exhibit increased viral resistance to tobamoviruses and a limited homeodomain transcription factor transport activity (Link to: Winter et al. 2007). We use such plants in genetic and proteomic approaches to identify further factors regulating intercellular transport of proteins and viruses. In combination with advanced confocal microscopy techniques this should allow us to find answers to the following questions:
- What are the cellular components involved in facilitating or regulating transport between cells?
- What are the morphogenic function(s) of mobile proteins and mRNAs?
The general aim is to characterize the biogenesis and function of non-coding RNAs allocated via the vasculature of flowering plants. In plants it is well established that the phloem serves as system-wide delivery pathway for nutrients such as amino acids and sugars and growth regulating hormones. In the last decade this simple view was challenged by the discovery that via the phloem also specific RNA molecules are transported to distant tissues. Such phloem-delivered RNAs include messenger RNA (mRNA), silencing-induced RNA (siRNA) and micro RNA (miRNA) and were shown to act as long-distance signals regulating growth.
In course of our studies we uncovered the presence of a considerable number of small non-coding RNAs (ncRNA) such as ribosomal and RNA (rRNA) and transfer RNA (tRNA) and fragments thereof in the phloem sap (PS). Although our analysis of the PS ncRNAs is incomplete with respect to the number of identified transcripts, we observed an interesting bias of the classes of tRNAs present in the PS. Some specific tRNAs could not be detected, which suggests a selective tRNA delivery into and via the phloem.
Emphasizing the significance of our finding we demonstrated that the native PS RNA pool inhibits in non-specific fashion translation and that tRNA fragments (tRNA halves) contribute to this inhibitory activity. Such tRNA halves are also observed in other organisms such as yeast and human cells. In yeast a specialized RNase T2 and in mammalians an RNase A-related enzyme named angiogenin cut tRNAs at their anticodon loop and produce tRNA halves under stress conditions. In plants such angiogenin-related enzymes do not exist, hence, the particular plant tRNA endonuclease(s) producing tRNA halves remains to be identified and characterized in plants.
Using biochemical approaches combined with genomic techniques we aim to find answers to the following questions:
- What are the signaling function(s) of phloem mobile RNAs?
- What are the RNA processing enzymes forming small phloem RNA fragments?