Progress 08/15/08 to 08/14/11
Outputs
OUTPUTS: The economically important potyvirus family includes 30% of known plant viruses. Previously, we discovered a short open reading frame (ORF) in the potyvirus genome. This ORF, called pipo, overlaps with the P3 coding region of the potyvirus polyprotein ORF. It is translated in the +2 (or -1) reading-frame relative to the polyprotein ORF, and is required for infection of plants. To determine the expression mechanism of PIPO we performed immunoblots with antibodies against the N-terminus of the P3 protein and against PIPO. Both antibodies detected a 25 kDa protein suggesting that PIPO is expressed by ribosomal frameshifting as a fusion to the N-terminal half of the P3 protein, yielding a protein called P3N-PIPO. The predicted frameshift site, GGAAAAA and flanking bases, were inserted into a dual luciferase reporter plasmid so that the downstream luciferase could be translated only if -1 or +2 frameshifting occurred. This mRNA construct was translated in wheat germ extract and in protoplasts. This assay was used to determine whether frameshifting occurred and to identify sequences required for frameshifting. To determine the exact amino acid sequence across the frameshift site in a natural infection, which would distinguish between a -1 vs +2 frameshift as the event for expression of P3N-PIPO, attempts were made to purify the P3N-PIPO protein from Turnip mosaic virus (TuMV) -infected plants, and then to determine its exact molecular weight by mass spectrometry. To determine the function of P3N-PIPO, host proteins with which it interacts were identified by yeast two-hybrid screen with P3N-PIPO from TuMV as bait. An interacting protein identified in this assay, PCaP1, was tested for interaction in planta by transient expression in leaves followed by co-immunoprecipitation with antibodies that detect P3N-PIPO and PCaP1. The interaction was confirmed by bimolecular fluorescence complementation (BiFC) assay. Confocal microscopy was used to co-localize the P3N-PIPO-PCaP1 interaction with the plasmodesmatal protein, PCBP1. To investigate the requirement, if any, for PCaP1 by TuMV, Arabidopsis plants with T-DNA insertions that knocked out the PCaP1 gene were tested for susceptibility to infection by TuMV. To determine the need for PCaP1 for infection at the cellular level, protoplasts from the PCaP1 knockout plants were inoculated with TuMV and viral RNA accumulation levels were measured. Dissemination: Results were presented at the North Central Extension Research Activity (NCERA) Workshop on Soybean Viruses in 2010, an EMBO plant virology workshop in Italy in 2010, the AFRI awardees' meeting in 2010 and 2011, the American Society for Virology meetings in 2010 and 2011, the International Congress of Virology in 2011, and at Cambridge University (home to a collaborator), and at INRA research stations in Colmar and Avignon, France. A manuscript describing interaction of P3N-PIPO with PCaP1 is under revision. PARTICIPANTS: Individuals: John Atkins, co-PI, University of Utah. Norma Wills, University of Utah, technician. Alice Hui, Iowa State University, graduate student. Vijayapalani Paramasivan, Iowa State University, postdoc. Partner Organization: Department of Human Genetics, University of Utah. Collaborations: List new interactions made possible from project support. Dr. Dinesh-Kumar, UC-Davis, provided advice and plasmids for VIGS and BiFC experiments. Dr. Steven Whitham, Iowa State University, provided advice and the TuMV-GFP constructs. Dr. Masayoshi Maeshima, Nagoya University, Japan, provided antibodies, plasmids, and Arabidopsis knockout plants of the PCaP1 gene. Dr. Krzysztof Treder, Plant Breeding. & Acclimatization Institute in Bonin near Koszalin, Poland, assisted in attempting to purify P3N-PIPO from TuMV-infected plants. Betty Chung, University College Cork, provided some plasmids and attempted to purify P3N-PIPO from TuMV-infected plants. Dr. Andrew Maule, John Innes Institute, Norwich, United Kingdom provided a plasmid that encodes plasmodesmal callose binding protein fused to mCherry. Dr. Diane Bassham, Iowa State University, provided expertise and equipment for preparing protoplasts from Arabidopsis plants. Andrew Firth, Cambridge University, bioinformatics and research advice. Training: Alice Hui, graduate student, Interdepartmental Plant Biology Major, Iowa State University. Alice performed and is performing the work on ribosomal frameshifting. Betty Chung, biochemistry graduate student, University College Cork learned plant virus propagation, protein purification and mass spectrometry techniques. Professional development: Vijayapalani Paramasivan, postdoc, Plant Pathology Dept, Iowa State University. Performed all the research on P3N-PIPO host interactions and function. Because of her outstanding research on this project, Dr. Vijayapalani was promoted from postdoc to Research Scientist II at Iowa State University. Technical personnel: Norma Wills, Department of Human Genetics, University of Utah. Made some constructs for ribosomal frameshifting. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: As reported in a previous annual report, Specific Aim III intended to determine whether natural mutations in P3 or PIPO explained how soybean mosaic virus breaks resistance to the Rsv1 gene in soybean. However, recently another lab demonstrated that P3 and not PIPO was responsible for resistance breaking. Therefore, we changed Specific Aim III to identify host proteins that interact with P3N-PIPO and their role in viral infection. As is clear on the progress report, this change in direction proved to be highly successful.
Impacts
Changes in knowledge. The detection of a 25 kDa protein both by antibodies to the N terminus of P3 and to PIPO indicates that P3N-PIPO exists and that PIPO is translated via a -1 or +2 ribosomal frameshift. The luciferase reporter gene assays reveal that the conserved GGAAAAA motif appears to facilitate the frameshift although it is more efficient with flanking viral sequence. This reveals a new class of frameshift element, unlike the known -1 frameshift signals of other plant viruses, retroviruses and coronaviruses. Once characterized, this will reveal a new way in which ribosomes interact with mRNA during the elongation process of translation. We have been unable to purify P3N-PIPO for mass spec, in part because many host proteins are of similar size, making gel purification difficult. Transient expression of P3N-PIPO fused to GFP revealed that P3N-PIPO facilitates its own cell-to-cell movement, a property of a viral movement protein. In support of this, P3N-PIPO interacting with PCaP1 localized to the plasma membrane and plasmodesmata as revealed by co-localization with the plasmodesmal callose binding protein (PCBP1) in BiFC experiments using confocal microscopy. TuMV accumulated to slightly higher levels in PCaP1 knockout protoplasts than in those from wild type plants, yet PCaP1 knockout Arabidopsis plants allowed only limited, localized accumulation of TuMV, and they remained healthy, unlike wild type plants which were severely stunted by TuMV infection. These observations support a role for PCaP1 in virus cell-to-cell movement which is not required for infection of individual cells but is required for infection of whole plants. Combined, these observations led us to propose a model in which P3N-PIPO binds a ribonucleoprotein complex (possibly including the virion) consisting of potyviral genomic RNA, coat protein and cylindrical inclusion protein (based on previous published observations by other labs), and that by interacting with the plasma membrane protein PCaP1, P3N-PIPO localizes this complex to the plasma membrane facilitate movement of the genome complex to the plasmodesmata through which it moves to the neighboring cell. Finally, important practical new knowledge from this research is that the PCaP1 knockout may serve as a valuable recessive resistance gene, revealing a potential new strategy to obtain durable resistance to the economically important potyviruses.
Publications
- No publications reported this period
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Progress 08/15/09 to 08/14/10
Outputs
OUTPUTS: Outputs. The potyvirus genome consists of an RNA encoding a large open reading frame (ORF) which translates into a polyprotein that is cleaved into functional protein units. We discovered a small ORF, pipo, that overlaps with the P3 protein region of the polyprotein ORF. The goal of this project is to determine how the pipo ORF is expressed and the function of the PIPO protein. We found that the PIPO protein exists in cells as a fusion to the N-terminal half of the P3 protein, giving a protein called P3N-PIPO. To determine if pipo is translated by minus one (-1) ribosomal frameshifting as expected, a dual luciferase reporter vector was constructed containing the Renilla luciferase ORF fused to a 200 nt sequence from Turnip mosaic potyvirus (TuMV) flanking the predicted start of the pipo ORF (a conserved GGAAAAA motif), followed by the firefly luciferase ORF, with the pipo-derived region arranged so that a -1 change in reading frame is required for translation of the firefly ORF fused to the upstream Renilla ORF. This dual reporter mRNA was translated in wheat germ extract (wge) and the luciferase products quantitated. It was also transfected into oat protoplasts and translation products measured by luciferase assay. The wild type TuMV sequence gave 5-7% frameshifting in wge and 20% frameshifting in oat protoplasts. Point mutations in this motif (GGAGAAG) greatly reduced frameshifting. Constructs containing only 8 viral bases, including the GGAAAAAA motif gave about 4% frameshifting in wge and 8% frameshifting in vivo. In a modification to the original proposal, we sought host proteins that interact with P3N-PIPO, to gain an understanding of PIPO's function. A yeast two-hybrid screen was performed using P3N-PIPO as bait and an Arabidopsis expression library as prey. Two interacting proteins, host factors HF3 and HF6, were selected for further analysis. P3N-PIPO and HF3 were transiently co-expressed by Agroinfiltration in Nicotiana benthamiana, and the products were co-immunoprecipitated using antibodies to either hemagglutinin tagged P3N-PIPO (HA-P3N-PIPO) or to myc tagged HF3 (myc-HF3), confirming the interaction first detected in yeast. Bimolecular fluorescence complementation (BiFC) also confirmed interaction of P3N-PIPO and HF3 in N. benthamiana cells. To determine the relevance of these interactions to virus replication, Arabidopsis lines with knock-outs in HF3 and HF6 genes are being tested for ability to support TuMV replication. Finally, a plasmid designed to express a P3N-PIPO-GFP fusion protein was bombarded into onion or N. benthamiana cells, and the green fluorescence was observed to move from the initially bombarded cells into adjacent cells. GFP constructs lacking P3N-PIPO showed fluorescence only in the originally bombarded cell. Thus, P3N-PIPO mediates its own movement from cell-to-cell. Dissemination: Results were presented at the EMBO Workshop: Genomic approaches to interactions between plant viruses, their hosts, and their vectors, Fenestrelle, Italy, June 2010; American Society for Virology meeting, Bozeman, MT, July 2010; and the USDA AFRI Awardees workshop, Washington, DC, July 2010. PARTICIPANTS: PD: W. Allen Miller, Plant Pathology Dept, Iowa State University. co-PD: John Atkins, Department of Human Genetics, University of Utah. Norma Wills, Department of Human Genetics, University of Utah. Training: Alice Hui, graduate student, Plant Biology Major, Iowa State University. Professional development: Vijayapalani Paramasivan, postdoc, Plant Pathology Dept, Iowa State University. Collaborator: Betty Chung, graduate student, University College Cork, Ireland. Collaborator: Andrew Firth, lecturer, Cambridge University, United Kingdom. Collaborator: Krzysztof Treder, Plant Breeding & Acclimatization Institute, Bonin, Poland. TARGET AUDIENCES: Plant virologists, molecular biologists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We conclude that PIPO is probably translated by programmed -1 ribosomal frameshifting by an entirely novel mechanism, although a +2 or short ribosomal hop has not been ruled out. The viral sequence induces a high rate of frameshift using novel signals in the RNA. This may reveal a new way in which ribosomes move on mRNA to make protein, which is of broad relevance because protein synthesis mechanisms are essential to, and highly conserved in, all living things. We also conclude that PIPO (fused to the N-terminus of P3) likely participates in cell-to-cell movement. Our discoveries affected behavior of other labs by stimulating them to investigate PIPO. One lab localized P3N-PIPO to the plasmodesmata. This is important knowledge because potyviruses are among the most diverse and devastating viral pathogens of plants, and restriction of cell-to-cell movement is an efficient virus resistance mechanism. By understanding virus movement functions, we may be able to disrupt this process and create virus resistant plants. Also, a graduate student is being trained on this project. She passed her oral prelminary exams and advanced to candidacy for a Ph.D.
Publications
- Paramasivan, V., Miller, W.A., Hui, A.Y., Wills, N., Chung, B.W.-Y., Firth, A.E., and Atkins, J.F. 2010. Expression of the Potyviridae overlapping reading frame, pipo. Program and Abstract Book EMBO Workshop: Genomic approaches to interactions between plant viruses, their hosts, and their vectors, Fenestrelle, Italy, June 2010.
- Hui, A.Y., Paramasivan, V., Wills, N., Chung, B.W.-Y., Firth, A.E,. Atkins, J.F., and Miller, W.A. 2010. Potyviridae overlapping gene pipo is translated via a non-canonical frameshift signal. Program and Abstract Book 29th Annual Meeting of the American Society for Virology, Bozeman, MT, July 2010.
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Progress 08/15/08 to 08/14/09
Outputs
OUTPUTS: Activities. Potyviruses are the largest and most economically important family of plant viruses. Their genome consists of an RNA encoding a large open reading frame (ORF) which translates into a polyprotein that is cleaved into functional protein units. We discovered a small ORF, pipo, that overlaps with the P3 protein region of the polyprotein ORF. The goal of this project is to determine how the pipo ORF is expressed and the function of the pipo protein. Using antibodies targeted against the pipo protein, we previously identified a 25 kDa protein that was predicted to comprise the N-terminus of P3 fused to pipo. Here, we verified this by performing a western blot on total protein from infected cells using antibodies against the N-terminus of P3. As predicted we detected the 25 kDa protein, and also the 40 kDa P3 protein that is generated by normal translation. An unexpected 17 kDa protein also appeared. To determine if pipo is translated by minus one (-1) ribosomal frameshifting as predicted by the above protein sizes, a dual luciferase reporter vector was constructed containing the Renilla (jellyfish) luciferase ORF fused to the sequence from Turnip mosaic potyvirus (TuMV) around the pipo ORF, followed by the firefly luciferase ORF, with the pipo-derived region arranged so that a -1 change in reading frame is required for translation of the firefly ORF as a fusion with renilla ORF. This dual reporter mRNA was translated in wheat germ extract followed by gel electrophoresis and phosphorimagery, or by measuring luciferase activities. As expected for -1 frameshifting, a large amount of renilla luciferase and a small amounts of firefly luciferase was obtained. A second aim was to determine whether pipo is involved in suppressing the virus induced gene silencing (VIGS) resistance response in plants. Previously we showed premature stop codons in the pipo ORF rendered TuMV unable to infect Nicotiana benthamiana plants. Here we tested whether Arabidopsis plants with knockouts of genes required for VIGS could serve as hosts for the pipo-lacking TuMV mutants. We observed that the mutant-pipo TuMV-GFP genome (containing a GFP reporter) accumulated neither in wild type Arabidopsis plants, nor in Arabidopsis plants containing either dcl2/3/4 or rdr1/2/6 triple knockouts in genes essential for VIGS. In one experiment, long after inoculation, two rdr1/2/6 plants showed a small area of green fluorescence after inoculation with the pipo-knockout virus. However, sequencing revealed that the virus had reverted to wild type. Wild type TuMV-GFP infected all Arabidopsis genotypes as indicated by large fluorescent green areas. PARTICIPANTS: PI: W. Allen Miller, Plant Pathology Dept, Iowa State University. co-PI: John Atkins, Department of Human Genetics, University of Utah. Norma Wills, Department of Human Genetics, University of Utah. Training: Alice Hui, graduate student, Plant Pathology Dept, Iowa State University. Professional development: Vijayapalani Paramasivan, postdoc, Plant Pathology Dept, Iowa State University. Collaborator: Betty Chung, graduate student, University College Cork, Ireland. Collaborator: Andrew Firth, postdoc, University College Cork, Ireland TARGET AUDIENCES: plant virologists and molecular biologists PROJECT MODIFICATIONS: A new approach to determine the function of the pipo protein will be to identify the host proteins with which it interacts. To identify host proteins that interact with pipo we are performing yeast 2-hybrid screens probing Arabidopsis whole genome libraries with pipo and the P3-pipo fusion protein. This can lead to discovery of new functions not considered in the original proposal.
Impacts
We conclude that pipo is probably translated by programmed ribosomal frameshifting by an entirely novel mechanism. This is important because there is no sequence resembling the known -1 frameshift consensus signal which is present in many important plant and human viruses. This may reveal a new way in which ribosomes move on mRNA to make protein, which is of broad relevance because protein synthesis mechanisms are essential to, and highly conserved in, all living things. We also conclude that pipo is unlikely to participate in suppressing the host antiviral silencing machinery. Thus, we will test other functions for pipo. These results indicate that research in labs focusing on potyvirus suppression of host silencing should remain focused on the other viral protein(s) that, unlike pipo, are known to suppress silencing. This is important knowledge because potyviruses are among the most diverse and devastating viral pathogens of plants.
Publications
- No publications reported this period
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