RESEARCH
The eukaryotic cell has evolved highly elaborate subcellular organelles/structures that specialize in distinct biochemical/biophysical processes. The biogenesis of organelles/structures, progression of localized biochemical reactions and cellular integration of these processes involve extensive subcellular traffic and localization of a wide variety of molecules. Within an organelle, the spatially clustered biochemical reactions also require traffic and precise localization of all the molecules involved. Thus, organized traffic and localization of molecules are hallmarks of a living cell. Elucidating the underlying pathways and regulatory mechanisms has vast importance to understanding the basic principles of life.
In plants, increasing evidence indicates that a variety of proteins and RNAs traffic from cell to cell through plasmodesmata and from organ to organ through the phloem.
Such macromolecular traffic appears to be an important means of global
coordination of gene expression. These findings change our traditional view that proteins and RNAs function only in the cells in which they are
produced. Thus, elucidating the mechanisms of protein and RNA traffic will help understand how biological processes at the individual cell level are integrated at the whole plant level, which is the basis of how a plant grows, develops, and deals with biotic and abiotic stresses.
Our research addresses the structure of function of plasmodesmata and the phloem
in relation to traffic of proteins, nucleic acids, viruses, and viroids. We also
study RNA traffic within the nucleus, which is of great importance to the
function of a cell and is yet little understood in plants. Finally, we also
study how a viroid RNA replicates in a cell and interacts with cellular factors
to cause diseases, gaining insights about the mechanisms of RNA regulation of
gene expression in plants.
We use a wide range of research methods in our research. These include gene cloning, transgenic plants, mutagenesis, light microscopy, electron microscopy, fluorescence microscopy, confocal microscopy, in situ hybridization, immunocytochemistry, genetic screening, biophysics, biochemistry, and RNA structural analysis.
Intercellular Protein Trafficking
Viruses make specific movement proteins to assist cell-to-cell spread of their infectious genomes. We
use viral proteins as tools to study the mechanisms that regulate protein
traffic. Furthermore, we used green fluorescent protein (GFP) as a reporter by
fusing to a viral movement protein, allowing tracking of the movement of the
fusion protein by fluorescence and confocal microscopy. We have developed
methods to produce a fusion protein transiently in single cells or stably with a
tissue-specific promoter in transgenic plants and then follow its movement by
fluorescence and confocal microscopy.
Recent work indicates that 1) plasmodesmata change structure and protein trafficking functions during plant
development, 2) plasmodesmata between different cell/tissue types have different protein trafficking
functions, and 3) plasmodesmata between symplasmically isolated cells, which
were thought to be closed for transport of small molecules, could mediate
traffic of selective proteins. Based on these progresses, we are
using the fusion protein to investigate the cellular and molecular parameters
that control protein traffic between specific cells.
Systemic RNA Trafficking
Once considered as merely the bridge for genetic information flow from DNA to protein, RNA is rapidly emerging as a key player in regulating a wide variety of cellular processes. Various functions require precise subcellular localizations of the RNAs, the mechanism of which is being intensively investigated by many labs. Elucidating the mechanisms of subcellular RNA localization also is essential for the development of RNA-based gene technologies such as gene therapy the success of which depends ultimately on the delivery of a designed RNA to the subcellular compartment where the target is localized. In plants, RNA traffic goes beyond subcellular level. Selected RNAs can traffic from cell to cell and from organ to organ to regulate gene expression and developmental processes.
Pathogens that partially or wholly depend on cellular factors to replicate themselves must be localized to the proper subcellular compartments to access these factors. Furthermore, systemic infection involves intercellular movement. Therefore, studying the infection patterns of pathogens can contribute greatly to our understanding of the basic mechanisms of traffic. We use Potato spindle tuber viroid (PSTVd) infection as a model system to study RNA traffic in plants. Viroids are small RNA molecules, 250-600 nucleotides long, that infect many economically important crops. These RNAs do not encode any proteins, and yet they replicate and traffic systemically throughout a host plant to establish systemic infection. PSTVd replicates in the nucleus. Therefore, systemic infection of PSTVd includes nuclear import, replication, intranuclear traffic of replication intermediates, nuclear export, plasmodesma-based cell-cell movement, and phloem-based long-distance movement. What is most remarkable is that the small viroid RNA genome itself possesses all of the necessary structural motifs that interact with cellular factors to accomplish each of these functions. This is truly a marvel of evolution. We are investigating all aspects of RNA traffic by using PSTVd as a model. Here we briefly describe some of our recent studies to illustrate how studying PSTVd traffic has provided novel insights about the basic cellular mechanisms that regulate intranuclear and phloem traffic of an RNA.
Intranuclear RNA trafficking The nucleus is the most conspicuous organelle in a eukaryotic cell. Despite the vital importance of the nucleus to the life of a cell, its structure is among the least understood. A theme emerging from recent studies is that the nuclear structure is highly organized and yet flexible. This feature has probably evolved to improve efficiency of local biochemical reactions while establishing coordination and regulation of such reactions at the whole nuclear level in response to growth and environmental conditions. In particular, the nuclear architecture may play a role in transcription control by targeting genes and regulatory factors to specific sites within the nucleus. When proteins and RNAs involved in a particular function or process associate with one another, they increase local concentration to form visible structures. In addition to facilitating traffic and organizing metabolism, the nuclear structure also plays an important role in retaining macromolecules within the nucleus. Elucidating the dynamics of nuclear organization including the principles that govern the various traffic and localization processes is a major goal for cell biology.
Phloem-mediated RNA trafficking The phloem tissue in plants transports not only photoassimilates to supply nutrients to the young growing parts of a plant, but also a wide range of molecules that regulate developmental and physiological processes as well as plant responses to biotic and abiotic stresses. Emerging evidence indicates that phloem-mediated systemic traffic of RNAs plays key roles in gene silencing and development. Thus, many basic cellular processes underlying growth and development may be coordinated at the organism level in part by the systemic traffic of selective RNAs as informational and regulatory molecules. Despite the fundamental importance of systemic RNA traffic to plant function, how an RNA enters, moves within and exits the phloem during systemic traffic is unknown. We use PSTVd traffic as a highly tractable model system to address these issues. Available evidence suggests that a viroid RNA does not diffuse passively from cell to cell; rather, it interacts with cellular factors for traffic. PSTVd appears to enter and exit the phloem via different mechanisms. We are investigating the viroid RNA structures and the cognitive cellular factors that interact to mediate the traffic. The findings will have direct relevance to understanding the mechanisms that control endogenous RNA traffic.
Viroid Replication
To achieve successful infection, a pathogen must have the capacity to recruit host machinery to assist its replication and movement as well as suppress host defense. A good understanding of the interactions between a pathogen and host factors for replication should provide basic insights about the normal functions of the host factors in growth and development and form a basis to engineer crops for pathogen resistance. A viroid RNA is small in size and has no protein-coding capacity. Thus, viroid infection showcases remarkably how evolution has produced a versatile RNA that more than any other pathogens utilizes host machinery to replicate inside a cell and then moves systemically.
We recently developed efficient protocols to characterize PSTVd replication in single cells of tobacco, Nicotiana benthamiana and tomato. We have isolated PSTVd mutants that show differing replication capacities at the cellular level. Remarkably, a single nucleotide difference in the viroid RNA can determine replication levels and host range. Continuing studies will allow us to gain insights about how a pathogenic RNA evolves structural motifs to interact with cellular factors for replication. From a broad perspective, this experimental system offers unique opportunities to study the principles of RNA-protein interactions that control many cellular functions.
Viroid Regulation of Cellular Gene Expression and
Disease Mechanisms
Development of disease symptoms in a host plant when infected by a particular pathogen is the result of complex pathogen-host interactions. The pathogen usually has pathogenicity determinants that elicit the types and degrees of severity of host symptoms. On the host side, altered cellular functions lead to changes in physiology and/or development that exhibit as disease symptoms. Thus, understanding the cellular and molecular mechanisms of disease formation will not only provide the basis for developing rational approaches to combat pathogen infection, but will also provide insights about the basic cellular processes underlying normal plant growth and development.
Viroid-infected plants can be symptomless or develop symptoms that range from mild to lethal ones, depending on viroid-host combinations. We recently found that a single nucleotide change in a PSTVd strain that usually causes relatively mild symptoms converted it into a lethal strain that essentially kills an infected plant. Underlying this lethal symptom is inhibited cell growth and shoot development, marked by the repressed expression of a tomato expansin gene implicated in cell growth. The feasibility of correlating viroid RNA sequence/structure to altered expression of specific host genes, cellular processes, and developmental patterns makes viroid infection a valuable system to investigate host factors for symptom expression and also to characterize the mechanisms of RNA regulation of gene expression in plants.
