Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Dallas-TAMU Agr Res Cntr
Non Technical Summary
Potato is the world's third largest food crop but yields are badly damaged by potyvirus and potexvirus infections. There is an urgent need in potato to advance the knowledge of genes responding to virus disease. Our overarching, long- term goal is to improve potato resistance to potyvirus and potexvirus infection. Currently, there is a significant knowledge gap of how the cellular stress machinery recognizes and responds to virus infection. Current research has shown that the potato virus X and Y viruses encode small membrane binding proteins that modify the endoplasmic reticulum (ER) membranes and activate a key ER stress response pathway. This pathway is called the IRE1/bZIP60 pathway and is a central hub for responses to adverse environmental challenges such as virus infection, heat, chemical, osmotic, and salt stress. The physiological consequences of prolonged low-level ER stress include constrained plant development and productivity, whereas chronic stress can result in death and crop losses (4, 8). This pathway is know to control the expression of genes to increase tolerance to virus infection, heat and osmotic stress. This proposed research tests a model in which ER stress sensors and are engaged to provide cellular protection against virus infection.
Animal Health Component
10%
Research Effort Categories
Basic
60%
Applied
10%
Developmental
30%
Goals / Objectives
This project's long-term goal is to identify genetic components that regulate virus pathogenesis in potato plants and translate this information into tools for germplasm improvement to reduce the impact of viral diseases on yield and marketability. Potato is the world's fourth largest food crop but yields are badly damaged by potyvirus and potexvirus infections. There is an urgent need in potato to advance the knowledge of genes responding to virus disease. Our overarching, long- term goal is to improve potato resistance to potyvirus and potexvirus infection.Potato virus X (PVX) and potato virus Y (PVY) encode small membrane binding proteins that functions as an elicitor of endoplasmic reticulum (ER) stress, activating a specific pathway known as the IRE1/bZIP60 pathway (Gaguancela et al., 2016). This ER stress pathway also contributes to the regulation of plant reproductive fitness and programmed cell death (PCD) regulation (Che et al., 2010; Duwi Fanata et al., 2013). The goal of this work was to examine how PVX and PVY modulate PCD through the ER stress machinery, and to use this information to engineer the IRE1/bZIP60 pathway in potato to regulate virus induced necrosis or improve antiviral defense. PVX and PVY are economically damaging viruses of potato, affecting both yields and marketability. Cultivated potato is the worlds' fourth most important food and in Texas is one the most important crops in production. This research will provide innovative solutions to improve yields and marketability by diminishing the impact of virus pathogens on production.Currently, there is a significant knowledge gap of how the cellular ER stress machinery recognizes and responds to virus infection. Current research has shown that the potyvirus 6K2 protein and potexvirus TGB3 protein modify the ER (and chloroplast) membranes and activate the plant IRE1/bZIP60 ER stress response pathway (Lu et al., 2016; Williams et al., 2014; Ye et al., 2012; Ye et al., 2011a; Ye et al., 2011b; Ye et al., 2013; Zhang et al., 2015). It is well known that the ER is a central hub for responses to adverse environmental challenges such as virus infection, heat, chemical, osmotic, and salt stress. The physiological consequences of prolonged low-level ER stress include constrained plant development and productivity, whereas chronic stress can result in death and crop losses (Duwi Fanata et al., 2013; Williams et al., 2014). Three well known monitoring/sensing pathway use the transcription factors bZIP60, bZIP28 and/or bZIP17 to activate cellular adaptive responses. They coordinate the transcription of molecular chaperones, including the ER lumen binding protein (BiP). It is well known that Arabidopsis plants overexpressing certain BiP isoforms display greater tolerance to virus infection, heat and osmotic stress. The N. benthamiana and potato BiP isoforms that respond specifically to virus infection are not known. In this context, our central hypothesis motivating the proposed research is that bZIP60, bZIP28, and bZIP17 differentially upregulate expression of BiP isoforms that ameliorates ER stress and cell death in these solanaceous plants. This proposed research tests a model in which ER stress sensors and BiP isoforms are engaged to provide cellular protection against virus infection.Objectives:(A) Develop novel gene silencing tools for functional analysis of ER stress associated candidate genes in potato. Most potato cultivars are not amenable to the current virus induced gene silencing system used for functional analysis. We will develop transgenic as well as transient gene silencing tools to enable discovery of genes that control virus infection.(B) Develop genetic tools to study ER related factors that interact with PVX and PVY proteins in Arabidopsis and potato. Annotate ER stress associated genes in potato and Arabidopsis that perform antiviral functions.(C) PVX and PVY both activate the IRE1/bZIP60 pathway. bZIP60 activates promoters containing cis elements, P-UPRE and ERSE, including activating its own transcription and BiP genes. We will examine gene expression of key cis elements in response to PVX and PVY inoculation
Project Methods
For optimizing virus induced gene silencing, we will deliver gene fragments to plants using TRV as a virus vector. We will identify genes in potato that are homologues for the Arabidopsis bZIP60, IRE1, BiP, bZIP17, bZIP28, and Bax inhibitor 1 and clone fragments of these genes to introduce into the viral vector. We will also develop a transient gene silencing. We will also work with Marco Molina to establish a potato transformation method to prepare plants that can transgenically silence or overexpress the same genes. This objective will take 18 months. We have preliminary success with transient gene silencing in two potato varieties. Currently 60% of plants tested in an experiment are silenced to a significant extent. If we fail to make improvements, we will use Solanum venturii which is a diploid relative of potato. This is reported in the literature that this wild species of potato is amenable to virus induced gene silencing, the genes involved in ER stress in this plant are more related to S. tuberosum than N. benthamiana (which is the most widely used model plant host) orthologues.The potato genome is not well annotated. We will use bioinformatic tools to identify the potato orthologue (s) of these genes and to study their phylogenetic relationships across Plantae. We will identify coding regions, promoters with UPRE elements, and alternate mRNA splice sites which may lead to alternate protein products with altered subcellular targeting signals. These data will be reported back to the SPUD database and NCBI. This work will also inform our experiments in part A and part C. This work should take 18 months.While it is known in Arabidopsis hat bZIP60 controls expression of BiP3, in potato there are 5 BiP orthologues and preliminary data suggests that these are somewhat distant from the Arabidopsis genes. Therefore, we cannot assume that the expression of these genes is solely driven by bZIP60 in potato. We will clone the promoter regions (~2kb) of IRE1, BIP2, and bZiP60 from potato. These promoters will be placed upstream of the GUS reporter gene in a binary plasmid and used for transient expression assays in N. benthamiana during PVX and PVY infection. Additionally, using in silico data mining of the potato genome sequence, we have identified 43 promoter sequences in potato that contain UPRE-like elements. The presence of a UPRE-like element suggests that these genes could be controlled by bZIP60. Promoters of these selected genes (BiP, transcription factor, and Frigida) have been cloned and will be used for transient GUS-fusion assays to test if expression is affected by virus gene expression.