Research Projects
We are currently conducting research on the following topics.
（Click on the numbers to go to each item.）
（１）Studies on the mechanism of cell-to-cell movement of plant viruses
（２）Research on the regulatory mechanism of viral resistance gene expression
（３）Functional analysis of Dof-type transcription factors involved in disease resistance
（４）Research on the mechanism of cell death-independent virus resistance
（５）Studies on the formation mechanism of Makomotake (stem gall in Z. latifolia)
Plants have plasmodesmata, which are cytoplasmic channel structures connecting neighboring cells. At plasmodesmata, cell membranes between adjacent cells are connected and play an important role in the transport of micro-molecules and macro-molecules between cells. Plant viruses use plasmodesmta to move from infected cells to surrounding cells, and this process of viral infection is called "cell-to-cell movement". Virally encoded movement proteins (MPs) are known to interact with various viral and plant factors. The molecular mechanisms of how plant viruses support cell-to-cell transfer through contacts are not fully understood.
Recent studies have indicated a link between plasmodesmata and membrane rafts (lipid rafts). Membrane rafts are microdomains on the plasma membrane defined as "heterogeneous, highly dynamic sterol- and sphingolipid-rich membrane domains 10-200 nm in diameter. Specific proteins accumulate on these membrane rafts and are thought to play important roles in the response to abiotic and biotic stresses. One plant-specific membrane raft protein is known as "remorin," which has been shown to be localized in plasmodesmata. Potato remorin has been reported to inhibit the cell-to-cell transfer of potato virus X (PVX) and to interact with the MP of PVX, and the role of remorin and membrane rafts in the cell-to-cell transfer of plant viruses has attracted much attention.
We cloned the cDNA of tobacco remorin (hereafter referred to as NtREM), which is most similar to potato remorin, and have been analyzing its function. Our experiments have shown that infection with tomato mosaic virus (ToMV) alters the subcellular localization of NtREM and that NtREM and ToMV MP interact. Interestingly, unlike the case of potato remorin, NtREM does not inhibit ToMV intercellular transfer, suggesting that it may have a slightly facilitatory effect (Sasaki et al., 2018). It is possible that different types of remorin play different roles in viral infection. We are currently attempting to identify membrane raft-associated proteins involved in viral infection by screening for NtREM interactors, while analyzing the functional domains involved in NtREM's plasma membrane localization and interaction with MPs.
One of the plant responses to pathogens is hypersensitive reaction/response (HR), in which plant resistance factors recognize avirulence factors (elicitors) of pathogens, resulting in the infected tissue inducing a defense response with cell death and localized pathogen invasion. Tobacco mosaic virus (TMV) is a pathogenic virus that infects tobacco and causes mosaic symptoms in susceptible tobacco plants. Tobacco varieties carrying the N gene can induce HR against TMV infection and locally contain the virus infection
The N gene is characterized by the fact that its expression is normally repressed, but is specifically induced by TMV infection. Plants have adapted to effectively and efficiently express the required amount of resistance genes in response to pathogen infection, as constant expression of large amounts of resistance genes in the absence of pathogens is just a waste of energy. However, little is known about how N gene expression is regulated in response to elicitor recognition, and to what extent such N gene expression regulation actually contributes to the induction of virus resistance.
We established a model experimental system to analyze the induction of viral infection-specific gene expression of the N gene using a transient gene expression system based on the Agrobacterium-mediated infiltration method. Our experiments revealed that the regulation of N gene expression in response to elicitor recognition involves four introns in its own gene. It was also suggested that N sequences with the introns induce viral resistance more efficiently than those without the introns (Taku et al., 2018). A recent study found that intron 1 and intron 2 function cooperatively to increase transcript levels (Ikeda et al., 2021). Currently, functional analysis is underway with the goal of elucidating the role of each of the four introns in regulating N gene expression and inducing resistance.
Dof (DNA-binding with one finger) proteins are known as plant-specific transcription factors. Dof proteins have a highly conserved Dof domain in many plants, which acts as a DNA-binding domain and is thought to specifically recognize and bind AAAG motif sequences on genomic DNA. Dof proteins have been reported to regulate the expression of genes involved in metabolism, differentiation and development, and abiotic stress responses such as carbon fixation and nitrogen assimilation, secondary metabolism, lipid metabolism in seeds, germination, vascular formation, plant hormone signaling, photoperiodic flowering and flower drop. On the other hand, the role of Dof proteins in biotic stress responses such as induction of resistance to pathogens has not been fully investigated.
In the course of our research on the regulatory mechanism of N gene expression in response to elicitor recognition (see above), we have obtained data suggesting that tobacco Dof proteins (BBF1, BBF2, and BBF3) may regulate the expression of a group of genes involved in disease resistance induction (Haque et al, 2009; Takano et al. 2013; Sasaki et al. 2015). Since these data were obtained from transient gene expression experiments using the Agrobacterium-mediated infiltration method, we are currently analyzing transgenic tobacco overexpressing BBF2 or BBF3 under the control of the constitutive CaMV 35S promoter and genome-edited tobacco using the CRISPR/Cas9 genome editing system to investigate the disease resistance of BBF proteins. Based on some reports that viruses and bacteria have an antagonistic relationship in disease resistance responses in plants, Ralstonia solanacearum as well as TMV are used in inoculation experiments.
Infection of tobacco plants carrying the N gene with TMV induces HR, and viral infection is localized to inoculated leaves. HR appears to prevent the spread of viral infection by cell death, but in fact, cell-to-cell movement of the virus is stopped before cell death occurs. Since cell death always occurs during HR induction, it is difficult to determine what changes in cell-to-cell movement are responsible for viral resistance. Therefore, the molecular mechanism by which the N gene (the N protein) suppresses the cell-to-cell movement of TMV is largely unknown.
Therefore, we constructed an experimental system to induce N-gene-mediated resistance in Nicotiana benthamiana, a related species of tobacco, in a mimic way by the Agrobacterium-mediated infiltration method. We found that in Nicotiana benthamiana with induced N-gene resistance, cell death was not induced and cell-to-cell transfer of ToMV is significantly suppressed (Sasaki et al. , 2013: 2021). In addition, we observed that MPs were unable to localize to plasmodesmata in cells in which viral cell-to-cell transfer was suppressed (Sasaki et al. , 2021). Using this cell death-free system for inducing viral resistance in Nicotiana benthamiana, we would like to explore the molecular mechanism by which viral cell-to-cell movement is restricted before cell death is induced.
Bamboo grass (Zizania latifolia) is a perennial plant in the family Poaceae. It grows wild mainly in the waters of East Asia, and its fruits are called wild rice. However, when infected with a pathogenic fungus (Ustilago esculenta), the plant does not produce ears, but instead produces a thickened stem that produces edible stem gall (Makomotake in Japanese). Although the University grows and sells Makomotake, it is difficult to stabilize yields because the timing of the maturation of stem gall differs depending on individuals and varieties, and some strains do not produce stem gall. In order to solve this problem, it is necessary to elucidate the formation mechanism of the stem gall.
We are investigating the relationship between changes in the level of U. esculenta in plants and the formation efficiency of the stem gall during the growing season. We are also trying to isolate U. esculenta and conduct characterization and genome analysis to determine the genetic type of U. esculenta that is effective for cultivation of Makomotake.