Specific targeting of protein into cells

12 Jul 2017. NUS biologists report that a bacterial pathogen hijacks a genetic process so that it can transfer its genetic codes into host plant cells.

Agrobacterium tumefaciens is a type of soil bacterium that causes plant diseases. This bacterium is also a natural genetic engineer that can transfer selected DNA and protein molecules into plant cells effectively. NUS researchers have discovered that the bacterium uses a natural uptake process called endocytosis to perform this delivery function. Endocytosis is a natural process by which a cell absorbs molecules and particles from its surroundings. Through this process, the virulence protein VirE2 from the bacterium is delivered into the host plant cells.

Delivery of macromolecules into plant cells has the potential for a wide range of biological research and industrial applications. However, it remains a challenge to efficiently deliver external macromolecules like proteins and DNA into plant cells. A team led by Prof PAN Shen Quan from the Department of Biological Sciences, NUS has been investigating the methods by which Agrobacterium delivers its virulence proteins into plant cells in a highly efficient manner. In the current study published in Science Advances, Prof Pan and his research associate Dr LI Xiaoyang have uncovered a new strategy which the bacterium uses to facilitate specific targeting of its virulence protein VirE2 into subcellular locations in the plant cell. They discovered that clathrin-mediated endocytosis is the mechanism which is responsible for this delivery process. They have also identified the protein sequence signals that facilitate the specific targeting of the protein into the plant cells.

The bacterium can transfer both single-stranded (ss) DNA (T-DNA) molecules and bacterial virulence proteins into different recipient cells. Among the many types of proteins which can be delivered by the Agrobacterium, VirE2 is an ssDNA-binding protein that is involved in the various steps of the transformation process. However, it is not clear how the plant cells receive the T-DNA or protein molecules. Spilt Green Fluorescent Protein (GFP) is a biological imaging technique used to detect and visualise interactions between proteins in plant cells.Using this approach, the team monitored the VirE2 delivery process by the bacterium inside plant cells in real time. They observed that VirE2 was absorbed by the plant cell through clathrin-mediated endocytosis via the dual endocytic motifs at the C-terminal tail of VirE2 and clathrin-associated adaptor AP2 complex. As AP2-mediated endocytosis is well conserved, the data suggests that the pathogen hijacks conserved endocytic pathways to achieve a wide range of host plants with a high degree of transformation efficiency.

The research team has developed a molecular tracking system, known as VirE2-GFP, to observe the delivery process. VirE2 is a natural protein that can self-aggregate and contains the targeting sequences so that it can efficiently move all the way from outside of a cell to the nucleus. They adopted a split-GFP approach and generated the VirE2-GFP fusion, which retains the self-aggregating property and the targeting sequences. The fusion protein is fully functional; it can be readily visualised as fluorescent and unique filamentous aggregates. This provides flexibility to obtain accurate insights into the delivery process.

Using this approach, the research team has identified an internalisation signal sequence that could be used to deliver macromolecules into targeted cells at high efficiencies. Based on these findings, novel research reagents can be developed to facilitate the work for researchers in the fields of cell biology, molecular biology and drug development. The research team also plans to develop novel drug delivery systems to facilitate efficient internalisation and targeting. Prof Pan commented, “This provides a new model that can be used to study real-time molecular events of protein delivery into cells and to develop novel technology for drug delivery”.

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Figure shows:

(a) Agrobacterim cells (in red) deliver virulence protein VirE2 (labeled as VirE2-GFP in green) from the lateral sides.

(b) Upon delivery into host cells, Agrobacterim virulence protein VirE2 (labeled as VirE2-GFP in green) accumulated on the host membranes (in red).

(c) Subsequently, virulence protein VirE2 (labeled as VirE2-GFP in green) is internalised into host cell via endomembrane compartments (in red).

[Image credit: Li Xiaoyang]

 

 

Reference

Li X; Pan SQ*, “Agrobacterium delivers VirE2 protein into host cells via clathrin-mediated endocytosis”. SCIENCE ADVANCES Volume: 3, e1601528 DOI: 10.1126/sciadv.1601528 Published: 2017.

Link for the paper: http://advances.sciencemag.org/content/3/3/e1601528 (open access)