Progress 01/01/14 to 06/30/18
Outputs
Target Audience:Potato breeders and producers. Changes/Problems:We stated in the proposal experimental plan that the Halterman lab have experience with Agrobacterium-mediated transformation of potato in separate projects. PVX and PVY susceptible potato cultivar 'Katahdin' will be transgenically silenced using lab standard transformation methods. A unique 400 bp fragment of bZIP60, SGT1, and SKP1 transcripts will be amplified by RT-PCR using Gateway adapted PCR primers for cloning [70]. Ten to fifteen independent transformed plants were to be generated and analyzed for the presence or absence of silencing constructs using PCR and the copy number of the insertion events will be determined using DNA blotting techniques. Two stable transgenic lines with the most profound reduction in bZIP60, SGT1 and SKP1 were to be selected for further studies. Transgenic lines will be assessed by qRT-PCR to report transcript accumulation. Elongation factor 1α (EF1α) and 18s RNA genes will be used as internal controls for gene expression (Chen and Halterman, 2011). Dr Halterman did not prepare these transgenic plants, although he did prepare some constructs. We used the constructs for leaf infiltration to silence these genes in leaves, but we do not have the transgenic potato. The results of silencing and transient gene expression studies were carried out in the Verchot laboratory and then published in 2017. We have subcontracted with Marco Molina to complete the potato transformation. Plants in in the process of being regenerated and will be ready later this year to study. What opportunities for training and professional development has the project provided?Omar Arias Gaguancela joined this project and completed his MS degree. His first publication was from this project and produced the cover article for MPMI. He is pursuing PhD studies at another University Lisabeth Zuniga, Alex Vela-Arias, Evelyn Vasquez participated as visiting researchers from ESPE in Ecuador and their work was part of their senior thesis. Both have moved onto graduate school and were co-authors in a publication last year. Professional development includes new valuable collaborations for my program with IE Johansen (University of Copenhagen), Aiming Wang (Ontario, Canada) Y Yamaji (Univ. Tokyo) and Randy Allen (Oklahoma State University). Other professional development includes participation in conferences listed above. Gabriela Orquera-Tornakian participated in potato transformation and gene silencing as a post-doctoral researcher. How have the results been disseminated to communities of interest?Research has been included in classroom education in graduate courses including: PLP5562 Molecular Plant Microbe Interactions, and PLP5013, Plant Virology We have also reported peer review journal articles and presented at scientific conferences What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The potexvirus TGB3 and potyvirus 6K2 are recognized by IRE1a and IRE1b which splice the bZIP60 mRNA to produce a truncate transcription factor that activates expression of genes such as BI-1, SGT1 and SKP1. Knockdown of these genes in Arabidopsis and N. benthamiana increases virus accumulation in inoculated and systemic leaves. We demonstrated that knockdown of IRE1a and IRE1b interferes with bZIP60 induction but is not responsible for BI-1, SGT1, or SKP1. Knockdown of bZIP60, a transcription factor changes the pattern of BI-1, SGT1 and SKP1 expression. Thus, we discovered BI-1, a cell death inhibitor, is linked to virus accumulation in potato and its expression is linked somehow to IRE1 and bZIP60. There are two models for BI-1 function: a) it suppresses IRE1 and blocks bZIP60 activation; and b) it binds calmodulin as an adaptor for interactions with other ER lumen factors. We completed studies in N. benthamiana, which shows that knockdown of bZIP60 leads to increased accumulation of PVX-GFP and PVY-GFP in inoculated leaves. We also noted that knockdown of bZIP60 leads to increased necrosis due to PVY and chlorosis due to PVX inoculated N. benthamiana leaves. These combined data indicate that bZIP60 expression provides protection to virus infection. bZIP60 is activated by two IRE1 orthologues known as IRE1a and IRE1b. IRE1a plays a greater role in restricting the overall accumulation of potexviruses and IRE1b contributes to restricting potyvirus infection. To understand the interactions involving the 6K2 and TGB3 proteins with the IRE1a and IRE1b, we contracted with Hybrigenics to conduct a library screen of plant proteins that bind the potyvirus 6K2 and potexvirus TGB3. We are confirming these candidate interactions in vivo and testing whether these are critical for IRE1 activation. We identified orthologues of IRE1, bZIP60, BI-1 and BiP genes in potato, N. benthamiana and Arabidopsis. We determined that some alleles in potato are not directly related to Arabidopsis. The sequence phylogeny indicates that further experiments are needed to confirm that the candidate genes in potato function in ER stress similar to their counterparts in Arabidopsis. We are preparing transgenic potato overexpressing these genes to learn if they suppress virus accumulation. bZIP60 activates promoters containing cis elements, P-UPRE and ERSE, including activating its own transcription and BiP3 genes. We identified 4 genes in potato that are likely BiP orthologues. Phylogenetic studies of these genes and found that the BiP phylogeny for Solanaceae is complicated. We completed qRT-PCR analysis of virus induced gene expression and identified which BiP-like transcripts in potato were induced by virus infection and which were not. We cloned the promoters for Arabidopsis and potato BiP genes into GUS expression vectors. Constructs were transformed to Arabidopsis and we collected T2 seeds. Plants will be used in the next few months after the completion of this funding to examine gene expression of key cis elements in response to PVX and PVY inoculation. This research proposal has led to comprehensive advances toward how prolonged exposure to virus infection can constrain plant growth and productivity. This research identified the critical ER stress regulating genes that interact with PVX TGB3 and PVY 6K2 in N. benthamiana and potato. By completion of this project we devised a new routine method for transient gene silencing in S. tuberosum and significantly advanced our technology for conducting forward genetic screens in potato. We cloned StBI-1 and St bZIP60 from potato, developed silencing constructs that work in N. benthamiana and provided the genetic sequences to NCBI. We are developing a generation of ihpRNA constructs for transient gene silencing in S. tuberosum. TRV-pds silencing does not work in potato. We are developing new constructs to silence ClaI or chloroplast genes as indicators of gene silencing in plants.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Gaguancela, OA, Zuniga, LP, Arias, AV, Halterman, D, Flores, FJ, Johansen, IE, Wang, A, Yamaji Y, and Verchot J (2016). The IRE1/bZIP60 pathway and bax inhibitor 1 suppress systemic accumulation of potyviruses and potexviruses in Arabidopsis and Nicotiana benthamiana plants. Molec Plant Microbe Interact. 29: 750-766.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Verchot J (2016) Plant virus infection and the ubiquitin proteasome machinery: Arms race along the endoplasmic reticulum. Viruses, 8(11), 314; doi:10.3390/v8110314
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Verchot, J. (2016) How does the stressed out ER find relief during virus infection? Current Opinion in Virology 17: 74-79.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Zhang, L, Chen H, Brandizzi, F, Verchot J, Wang A (2015). The UPR branch IRE1-bZIP60 in plants plays an essential role in viral infection and is complementary to the only UPR pathway in yeast. PLoS Genet. 2015 11(4):e1005164. doi: 10.1371/journal.pgen.1005164.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Verchot, J. (2014) The ER quality control and ER associated degradation machineries are vital for viral pathogenesis. Frontiers in Plant Biology Vol 5 p 66-71 10.3389/fpls.2014.00066.
- Type:
Journal Articles
Status:
Other
Year Published:
2014
Citation:
Brett Williams, Jeanmarie Verchot and Marty Dickman (2014) When Supply Does Not Meet Demand-ER Stress and Plant Programmed Cell Death. Frontiers in Plant Biology Vol 5: 211-216 10.3389/fpls.2014.0021
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2017
Citation:
Omar Gaguancela (2017) Role of IRE/bZIP60 pathway in regulating potyvirus and potexvirus infection in potato.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
American Society Virology, UMD. bZIP60 and bZIP28 control plant virus infection in potato through regulation of PCD.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
American Society Plant Biology, Honolulu HI. IRE1/bZIP60 pathway controls plant virus infection in potato.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
International Society Plant Microbe Interactions, ER and unfolded protein response to potexviruses and potyviruses Portand Oregon.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
American Society Plant Biology, ER and unfolded protein response to potexviruses and potyviruses
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Korean Society Plant Pathology, ER and unfolded protein response to potexviruses and potyviruses
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
The 5th International Conference on Biotic Plant Interactions, Xiamen China. Presented research paper: IRE1/bZIP60 pathway controls plant virus infection in potato.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
The 5th International Conference on Biotic Plant Interactions, Xiamen China. IRE1/bZIP60 pathway controls plant virus infection in potato.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Viruses 2016, Basel Switzerland How viruses deal with a stressed out ER
|
Progress 01/01/17 to 12/31/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:We stated in the proposal experimental plan that the Halterman lab have experience with Agrobacterium-mediated transformation of potato in separate projects. PVX and PVY susceptible potato cultivar 'Katahdin' will be transgenically silenced using lab standard transformation methods. A unique 400 bp fragment of bZIP60, SGT1, and SKP1 transcripts will be amplified by RT-PCR using Gateway adapted PCR primers for cloning [70]. Ten to fifteen independent transformed plants were to be generated and analyzed for the presence or absence of silencing constructs using PCR and the copy number of the insertion events will be determined using DNA blotting techniques. Two stable transgenic lines with the most profound reduction in bZIP60, SGT1 and SKP1 were to be selected for further studies. Transgenic lines will be assessed by qRT-PCR to report transcript accumulation. Elongation factor 1α (EF1α) and 18s RNA genes will be used as internal controls for gene expression (Chen and Halterman, 2011). Dr Halterman did not prepare these transgenic plants, although he did prepare some constructs. We used the constructs for leaf infiltration to silence these genes in leaves, but we do not have the transgenic potato. The results of silencing and transient gene expression studies were carried out in the Verchot laboratory and then published in 2017. We have subcontracted with Marco Molina to complete the potato transformation. What opportunities for training and professional development has the project provided?Omar Arias Gaguancela joined this project and completed his MS degree. His first publication was from this project and produced the cover article for MPMI. Lisabeth Zuniga,, and Alex Vela, Arias both worked as technicians part time on the project They were visiting students from ESPE in Ecuador and their work was part of their senior thesis. Both have moved onto graduate school and were co-authors in a publication last year. Professional development includes new valuable collaborations for my program with IE Johansen (University of Copenhagen), Aming Wang (Ontario, Canada) Y Yamaji (Univ. Tokyo). Other professional development includes participation in conferences listed above. How have the results been disseminated to communities of interest?Research has been included in classroom education in graduate courses including : PLP5562 Molecular Plant Microbe Interactions, and PLP5013, Plant Virology We have also reported peer review journal articles and presented at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?Objective 3: we will examine gene expression of key cis elements in response to PVX and PVY inoculation. Objective 1 &2, We are preparing transgenic potato overexpressing and silencing bZIP60, BI-1.
Impacts
What was accomplished under these goals?
We used qRT-PCR to demonstrate that bZIP60, BI-1, SGT1 and SKP1 are induced by potexvirus TGB3 and potyvirus 6K2 proteins. Knockdown of these genes in N. benthamiana increases virus accumulation in inoculated and systemic leaves. We demonstrated that knockdown of IRE1a and IRE1b interferes with bZIP60 induction but is not responsible for BI-1, SGT1, or SKP1. Knockdown of bZIP60, a transcription factor, can result in changes to BI-1, SGT1 and SKP1 expression but is not directly responsible for changes in expression. Thus, we discovered BI-1, a cell death inhibitor, is linked to virus accumulation in potato and its expression is linked somehow to IRE1 and bZIP60. There are two models for BI-1 function: a) it suppresses IRE1 and blocks bZIP60 activation; and b) it binds calmodulin as an adaptor for interactions with other ER lumen factors. IRE1, leads three major ER stress response signaling branches: a) bZIP60 transcriptional activation, b) mRNA decay (RIDD) and c) autophagy. These branches are separately activated by IRE1a and IRE1b. We used genetic knockouts of IRE1a and IRE1b to inoculate with poty- and potexviruses and, we used GFP to monitor the spread of virus infection over time. Combining the analysis of local and systemic infection, IRE1a plays a greater role in restricting the overall accumulation of poty- and potexviruses. IRE1b contributes to restricting virus movement and there is a greater change when both genes are knocked out. Similarly, bZIP60 and BI-1 were knocked down and plants were inoculated with the viruses. Virus accumulation was significantly greater in both plants. Surprisingly, BI-1 knockdown did not result in cell death. These data indicate that BI-1, a cell death suppressing protein, plays a role in virus infection that is independent of cell death regulation. To understand the interactions involving the 6K2 and TGB3 proteins with the IRE1a and IRE1b, we contracted with Hybrigenics to conduct a library screen of plant proteins that bind the potyvirus 6K2 and potexvirus TGB3. We are planning to confirm these candidate interactions in vivo and test whether these are critical for IRE1 activation. We identified orthologues of IRE1, bZIP60, BI-1 and BiP genes in potato, N. benthamiana and Arabidopsis. We determined that the some alleles in potato are not directly related to Arabidopsis. The sequence phylogeny indicates that further experiments are needed to confirm that the candidate genes in potato function in ER stress similar to their counterparts in Arabidopsis. Our next step is to clone these potato genes and transform Arabidopsis mutant plants that are defective in these same genes to learn if these are functional orthologues. bZIP60 activates promoters containing cis elements, P-UPRE and ERSE, including activating its own transcription and BiP3 genes. We identified 4 genes in potato that are likely BiP orthologues. Phylogenetic studies of these genes and found that the BiP phylogeny for Solanaceae is complicated. We completed qRT-PCR analysis of virus induced gene expression and identified which BiP-like transcripts in potato were induced by virus infection and which were not. We are currently cloning the promoters for Arabidopsis and potato BiP genes into GUS expression vectors to examine gene expression of key cis elements in response to PVX and PVY inoculation. This is a final goal of the proposed research that is not yet met but will be completed by the final report. This research proposal has led to comprehensive advances toward how prolonged exposure to virus infection can constrain plant growth and productivity. Virus infection and other biotic assaults cause malformed proteins to accumulate in plant cells and impose stress on the endoplasmic reticulum (ER). The Potato virus X TGB3 and Potato virus Y 6K2 are small membrane binding protein that are recognized by an ER resident sensor of stress known as IRE1. Activation of IRE1 leads to splicing of bZIP60 mRNA which produces a truncated transcription factor. The bZIP60 transcription factor upregulates several genes to restore ER homeostasis and suppress programmed cell death during virus infection. While the IRE1/bZIP60 pathway is well described in Arabidopsis, their orthologues are not identified in potato or N. benthamiana. This research identified the critical ER stress regulating genes that interact with PVX TGB3 and PVY 6K2 in N. benthamiana and potato.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Gaguancela, OA, Zuniga, LP, Arias, AV, Halterman, D, Flores, FJ, Johansen, IE, Wang, A, Yamaji Y, and Verchot J (2016). The IRE1/bZIP60 pathway and bax inhibitor 1 suppress systemic accumulation of potyviruses and potexviruses in Arabidopsis and Nicotiana benthamiana plants. Molec Plant Microbe Interact. 29: 750-766.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Verchot J (2016) Plant virus infection and the ubiquitin proteasome machinery: Arms race along the endoplasmic reticulum. Viruses, 8(11), 314; doi:10.3390/v8110314
- Type:
Theses/Dissertations
Status:
Other
Year Published:
2017
Citation:
Role of IRE/bZIP60 pathway in regulating potyvirus and potexvirus infection in potato (2017) by Omar Gaguancela.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
June 26-30: American Society Plant Biology, Honolulu HI. IRE1/bZIP60 pathway controls plant virus infection in potato
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Aug 17-21, The 5th International Conference on Biotic Plant Interactions, Xiamen China. Presented research paper: IRE1/bZIP60 pathway controls plant virus infection in potato.
|
Progress 01/01/16 to 12/31/16
Outputs
Target Audience:Target audience includes growers and researchers in the potato industry. Efforts to deliver sicence based knolwedge in the classroom at Oklahoma State University where I teach Molecular Plant Microbe Interactions and Plant Virology at the graduate level. In addition this project has attracted 3 interns from ESPE Ecaudor who worked in the laboratory for 1 or 2 semesters. Their work was either funded by themselves or as hourly interns on this project. The work was toward their undergraduate thesis. This practicum experience provided outreach to a potato growing region of the world, and provides a unique experience to latino students who cannot obtain such practicum experience in their own countries. Changes/Problems:Plans for next phase is improvement to vectors for gene silencing in potato. Now that we have in place a system for comparing silencing and KO of genes in Arabidopsis and potato and how those genes affect virus infection we plan to develop improved vectors for gene silencing in potato that will enable us to expand comparative studies between these two hosts. Production of transgenic potato is ongoing that overexpresses desired proteins. Delays in hiring at U. Wisconsin has slowed this step down. What opportunities for training and professional development has the project provided?There is one graduate student and one post doc working on this project. The post doc is now moving to Monsanto. The graduate student is on track to graduate. Three interns from ESPE in Ecuador joined the project in seperate semesters. One is now in graduate school and one is applying. They each cite this experience as a reason to move forward in their careers. Presenting this work at ASPB, we began talks with CASIS which enabled us to begin discussion of a project. In January 2016 I led 3 undergraduates to obtain a NASA Travel Grant to learn about Agriculture at NASA and the role of agriculture science in Space Biology. The NASA Travel Grant was $3000, Dec 2015. This project established new collaborations: Marty Dickman (Texas A&M University) to produce invited review articles on ER stress,Elisabeth Johansen (Denmark) who provided the infectious clone of PVY, Aiming Wang (Ontario) to study TuMV-GFP ER stress, and Shigetou Namba (University of Tokyo) to study PlAMV-GFP. How have the results been disseminated to communities of interest? This research has gained the attention of the Center for Advancement of Science on the International Science in Space (CASIS) when I presented it at the ASPB conference in 2015. CASIS is interested to fund impactful research to study challenging environments affect food production. With regard to growing plants on the ISS, the concerns for limited water supply and growth media are linked to causes of dehydration/osmotic stress. This research on virus-ER stress interactions also involves pathways that overlap with drought stress and we can examine both responses in parallel. CASIS is also interested in potato production with the goal of producing potato for the next mission to Mars. We are now discussing a project that involves viruses and drought stressors that will provide new knowledge concerning the constraints on plant growth and productivity in response to subtle or profoundly adverse conditions in space as on earth, where preserving local and healthful food production must be carefully managed and encouraged. Such conditions as virus infection, limiting water or nutrients are known to contribute to the activation of ER stress adaptive pathways which include the UPR, in plants which can affect crop yields, flowering time, etc. Furthermore, it is not known whether prolonged exposure to microgravity contributes to: a) the accumulation of malformed proteins, b) diseases of protein conformation, or c) enhanced ER stress responsiveness, or d) exacerbates the impacts of dehydration or virus infection on cell survival. We are working together to establish an experimental system that we can launch on the ISS that will provide new insights into this pivotal topic of stress biology, will dissect the biological mechanisms using Arabidopsis or potato as a genetic system and, will enable us to model for understanding the role that gravity plays in cellular and genetic mechanisms regulating ER stress responses. I have been invited to prepare an unsolicited research proposal and have submitted a NASA research initiation grant. Thus the value of this research goes far beyond food production on earth. What do you plan to do during the next reporting period to accomplish the goals?I have begun discussions with Dr Steven Howell at Iowa State about the overlap between drought/heat stress tolerance and virus infection. We are sharing germplasm to look at which ERstress related transcription factors, including bZIP60, bZIP17 and bZIP28 contribute to both suppression of virus infection and drought/heat stress tolerance. We also began a project to look for factors downstream of bZIP60 that contribute to virus infection. This is vital for renewed funding. We are looking for the partner that interacts with the potyvirus 6K2 and potexvirus TGB3 which activates these UPR pathways. We are also focusing on the third objective of promoter studies.
Impacts
What was accomplished under these goals?
Demonstrated that IRE1/bZIP60 interacts with multiple potyviruses and potexviruses in Arabidopsis, N. benthamiana, and potato. We began this work proposing to use N. benthamiana as a model for potato. The drawback of using N. benthamiana is that we cannot use knockout mutations as we can with Arabidopsis to study how host genes affect virus disease in whole plants. We were not able to work well in potato because it was not as efficient for silencing as N. benthamiana. In addition, we lacked an infectious clone of PVY or a potexvirus that infected Arabidopsis. We now have in place infectious clones of two potexviruses and two potyviruses that infect Arabidopsis, N. benthamiana and potato and demonstrated that the same genes induce the same pathways in all three hosts. The work achieved exceeded the scope of the first two objectives by using more viruses and two model plants. We published data showing bZIP60 and BI-1 contribute to suppression of virus systemic infection across host species. We identified IRE1a and IRE1b contribute seperately to potyvirus and potexvirus infection, although both monitor bZIP60 mRNA splicing. This study demonstrated that potexviruses and potyviruses interact with the IRE1/bZIP60 pathway in a manner that downregulates virus infection. The data also suggests that the TGB3 protein has a preferential relationship with IRE1a while 6K2 interacts with both proteins. Since the interaction is likely not a direct interaction there are likely alternative intermediate factors recognized by each potexvirus or potyvirus protein that mediates IRE1 recognition. Our current experiments show that bZIP60 and bZIP28 or bZIP60 and bZIP17 regualte virus movement into flowers. It seems that bZIP60 coordinates with these other two transcription factors. We are trying to understand if they dimerize on the promoters.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Gaguancela, OA, Zuniga, LP, Arias, AV, Halterman, D, Flores, FJ, Johansen, IE, Wang, A, Yamaji Y, and Verchot J (2016). The IRE1/bZIP60 pathwya and bax inhibitor 1 suppress systemic accumulation of potyviruses and potexviruses in Arabidopsis and Nicotiana benthamiana plants. Molec Plant Microbe Interact. 29: 750-766.
|
Progress 01/01/15 to 12/31/15
Outputs
Target Audience:Current target audience is academic scientists working on crop improvement, potato specialists, growers Changes/Problems:There was a hold up for releasing funds through the USDA to Dennis Halterman. This delayed work in his lab by at least 6 months. What opportunities for training and professional development has the project provided?Omar Arias is a current MSc student working to characterize the pattern of virus infection in plants (0.5 FTE graduate student). Alexis Velas was a visiting researcher from Ecaudor who came voluntarily to work on this project and established qRT-PCR (0.5 FTE). This was a component of his undergraduate thesis and he is hoping to return to pursue graduate school. Elisabeth Pena was a visiting researcher who continued the qRT-PCR analysis and is currently applying for PhD in Animal science (0.5 FTE). This training has helped her gain skills and English proficiency for further education. Abdulbasset was a visiting scientist from Iran who helped make constructs for this project (0.2 FTE). Sanchita Vaghchhipawala (1.0 FTE post doc) has been hired to carry out promoter cloning, GUS-fusion assays, and potato transformation in the Halterman lab. After taking several years off from laboratory work in order to raise a family, she is using this opportunity to refamiliarize herself with laboratory protocols in order to pursue a permanent career at the end of the project. How have the results been disseminated to communities of interest?Two publications were prepared. One is published and one submitted How a stressed out ER manages virus infection.(2015) Jeanmarie Verchot, Current Opinion Virology, submitted The UPR Branch IRE1-bZIP60 in Plants Plays an Essential Role in Viral Infection and Is Complementary to the Only UPR Pathway in Yeast. (2015). Lingrui Zhang, Hui Chen, Federica Brandizzi, Jeanmarie Verchot, Aiming Wang, PLOS Genetics 10.1371/journal.pgen.1005164 Talks given Korean Society Plant Pathology How a stressed out ER manages virus infection American Society Plant Biology How a stressed out ER manages virus infection University of Tokyo department of Plant Pathology Seminar Series.How a stressed out ER manages virus infection University of Helsinki Department of Plant Pathology Seminar. What do you plan to do during the next reporting period to accomplish the goals?Need to complete research to document gene silencing of bZIP60 and SKP1 in N. benthamiana and potato. We need to characterize the impact of these genes on virus infection in these hosts. This will enable us to write a larger research article to submit to Plant Cell. Using in silico data mining of the potato genome sequence, Dennis Halterman identified 43 promoter sequences in potato that contain UPRE-like elements. This list includes promoters of two luminal-binding protein genes (BiPs), a transcription factor, and a gene with similarity to the flowering time gene FRIGIDA. 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. We are also following up on these exciting results by determining if changes in flowering, which has been observed in virus infected IRE1 mutant plants, relate to changes in Frigida expression. These data could demonstrate a genetic explanation of how viruses can impact other physiological host responses, including tuberization, flowering, or yield.
Impacts
What was accomplished under these goals?
Toward objective 1: research was conducted using PVX, PVY and parallel work with TuMV and PlaMV, which infect Arabidopsis. We were concerned that silencing genes in potato and N. benthamiana may reduce but not knock out expression and that minimal expression of bZIP60 might be sufficient to allow virus infection. Therefore, we used three hosts and four viruses. We demonstrated that the potyvirus 6K2, and potexvirus TGB3 proteins activate bZIP60 and SKP1 expression, however SKP1 expression does not appear to depend upon bZIP60 but is an early induced gene linked to ERAD. Toward objective 2: we demonstrated that 6K2 and TGB3 trigger IRE1-dependent splicing of bZIP60 transcripts. Arabidopsis plants that are defective for IRE1 show early spread of TuMV and PlAMV infection which was opposite of the phenotype we were expecting. These data suggest that IRE1 regulates long distance movement of the virus and that in the absence of UPR, virus titre is increased. We also determined that flowering is altered in virus infected IRE1 mutant plants suggesting there is a link to flowering, virus infection via the UPR. In comparative study we show that PVX TGB3, PlAMV TGB3, PVY6K2, TuMV 6K2 trigger bZIP 60 expression in an IRE1 dependent manner. Experiments were carried out using qRT-PCR to monitor gene expression. Protein blotting was used to confirm the patterns of virus infection. GFP fluorescence was quantified to track changes in virus accumulation relative to control plants. Toward objective 3: we have successfully cloned the promoter regions (~2kb) of IRE1, BIP2, and bZip60 from potato. These promoters have been placed upstream of the GUS reporter gene in a binary plasmid and are being used for transient expression assays in N. benthamiana during PVY infection and 6K2 and TGB3 expression.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
The UPR branch IRE1-bZIP60 in plants plays an essential role in viral infection and is complementary to the only UPR pathway in yeast.
Zhang L, Chen H, Brandizzi F, Verchot J, Wang A.
PLoS Genet. 2015 Apr 15;11(4):e1005164. doi: 10.1371/journal.pgen.1005164. eCollection 2015 Apr.
PMID: 25875739
|
Progress 01/01/14 to 12/31/14
Outputs
Target Audience: The goal is to solve the devastation caused by plant viruses to the potato industry. So the audience includes biotechnology companies and potato breeders. Changes/Problems: The USDA has held up Dennis Halterman's ability to hire on a technician to do the work in potato. This has been a problem. We are moving forward however with N. benthamiana and looking at PVY, PVX and we added 3 other viruses that are agronomically important for comparative outcomes. The data set we are getting is robust but we have had to adjust our work due to the lack of technical support. What opportunities for training and professional development has the project provided? This project provided training for a graduate student and two visiting scientists from Ecuador. Alexis Valegis is completing his thesis work at ESPE and participated in this project for 6 months. Then Liz Pena has joined this work for 6 months from Ecuador to obtain job training post graduation from ESPE. How have the results been disseminated to communities of interest? Two peer reviewed publications have resulted from this work. This work has been presented at the American Society Virology conference in 2015. Additional collaborations are being discussed with Aiming Wang at University of Guelph and Simon Gilroy at University of Wisconsin. What do you plan to do during the next reporting period to accomplish the goals? We have mostly completed objective 1, to examine protein expression changes in response to viral inputs in N. benthamiana. Since there has been a hold up for Dennis Halterman's work we have expanded to studies involving the infectious clone of Bean common mosaic virus and included Arabidopsis mutants to try to get at the mechanism of IRE1 and bZIP60 regulation of virus infection. This is already providing comparative data that will benefit the research in potato. We are now expanding micropropagation of potato plants and plan to run gene silencing experiments to define the roles of these genes in virus infection.
Impacts
What was accomplished under these goals?
ER stress resulting from virus infection can chronically impact cell survival and is linked to the transcriptional activation of the UPR and components of the ER associated degradation (ERAD) machineries. Virus infection triggers ER stress to coordinate the action of a common set of molecular chaperones to cope with the demand for increasing protein folding capacity and to mitigate the toxic accumulation of malformed proteins. RNA viruses create a huge biosynthetic burden on the ER and transiently enhance UPR to manipulate the production of protein chaperones and foldases to meet their needs. The Verchot/Halterman collaboration is to report on the transcriptional activation of bZIP60, BIP, and SKP1 and their importance for ER stress responses to virus infection in potato and tobacco. This research will determine the impact of overexpressing or knocking-out/down critical ER stress factors in virus susceptibility and cold tolerance with the goal of uncovering critical new information that can be exploited in a next generation of crops with engineered abiotic stress tolerance and virus resistance. This work will provide a global view of the similar and different outcomes that result when unrelated triggers activate the UPR to overcome a condition that is singly termed ER stress.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Verchot, J. (2014) Invited Review for Frontiers in Plant Biology special issue in Plant Membrane Biology. The ER quality control and ER associated degradation machineries are vital for viral pathogenesis. 10.3389/fpls.2014.00066. Vol 5 p 66.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Brett Williams, Jeanmarie Verchot and Marty Dickman (2014) Invited Review for Frontiers in Plant Biology special issue in Plant Membrane Biology. When Supply Does Not Meet Demand-ER Stress and Plant Programmed Cell Death. 10.3389/fpls.2014.0021. Vol 5: 211.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
The ER quality control and ERAD machineries contribute to plant viral pathogenesis (oral presentation). Verchot J
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