Here are the questions which is about the paper of “A Virus in a Fungus in a Plant:Three-Way Symbiosis Required for Thermal Tolerance”. I attached the file and I wish I can get the correct answer for

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Here are the questions which is about the paper of “A Virus in a Fungus in a Plant:Three-Way Symbiosis Required for Thermal Tolerance”. I attached the file and I wish I can get the correct answer for that. Thanks.

1) What does it mean for a virus to ‘attenuate’ the ‘virulence’ of a fungus on a plant? Define both terms in apostrophe and give example from paper.

2) Describe the process of 1) reverse transcription, 2) amplification, and 3) cloning in your own words.

3) What does it mean for a gene sequence to be described as ‘putative’?

4) What does it mean for a virus to be transmitted ‘vertically’? What would be the opposite of ‘vertical transmission’?

5) Why did authors use simple sequence repeat analysis and sequence the rDNA region of the fungal isolate with low titer of the virus? What was the purpose of these tests?

6) Why did authors reintroduce the virus to the virus-free fungal isolate? If the fungus with reintroduced virus did not confer heat-tolerance, what would the authors have concluded?

Here are the questions which is about the paper of “A Virus in a Fungus in a Plant:Three-Way Symbiosis Required for Thermal Tolerance”. I attached the file and I wish I can get the correct answer for
to the Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Disease Research (MRCE) and by NIH grant AI53298. The DDRCC is supported by NIH grant DK52574. W.W.L. was supported by the Clinical/Translational Fellowship Program of the MRCE, the W.M. Keck Foundation, and the NIH National Research Service Award (NRSA) F32 AI069688-01. P.A.P.was supported by the NIH Institutional NRSA T32 GM07067 to the Washington University School of Medicine. Supporting Online Materials and MethodsSOM Text Figs. S1 to S4 Tables S1 and S2 References 6 November 2006; accepted 14 December 2006 10.1126/science.1137195 A Virus in a Fungus in a Plant: Three-Way Symbiosis Required for Thermal Tolerance Luis M. Márquez, 1Regina S. Redman, 2,3 Russell J. Rodriguez, 2,4 Marilyn J. Roossinck 1* A mutualistic association between a fungal endophyte and a tropical panic grass allows both organisms to grow at high soil temperatures. We characterized a virus from this fungus that is involved in the mutualistic interaction. Fungal isolates cured of the virus are unable to confer heat tolerance, but heat tolerance is restored after the virus is reintroduced. The virus-infected fungus confers heat tolerance not only to its native monocot host but also to a eudicot host, which suggests that the underlying mechanism involves pathways conserved between these two groups of plants. E ndophytic fungi commonly grow within plant tissues and can be mutualistic in some cases, as they allow plant adaptation to extreme environments ( 1). A plant-fungal symbiosis between a tropical panic grass from geothermal soils, Dichanthelium lanuginosum , and the fungus Curvularia protuberata allows both organisms to grow at high soil temperatures in Yellowstone National Park (YNP) (2 ). Field and laboratory experiments have shown that when root zones are heated up to 65°C, non- symbiotic plants either become shriveled and chlorotic or simply die, whereas symbiotic plants tolerate and survive the heat regime. When grown separately, neither the fungus nor the plant alone is able to grow at temperatures above 38°C, but symbiotically, they are able to tolerate ele- vated temperatures. In the absence of heat stress, symbiotic plants have enhanced growth rate compared with nonsymbiotic plants and also show significant drought tolerance ( 3). Fungal viruses or mycoviruses can modulate plant-fungal symbioses. The best known exam- ple of this is the hypovirus that attenuates the virulence (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica (4 ). Virus regu- lation of hypovirulence has been demonstrated experimentally in several other pathogenic fungi ( 5 –8). However, the effect of mycoviruses on mutualistic fungal endophytes is unknown. There is only one report of a mycovirus from the well- known mutualistic endophyte, Epichloë festucae, but no phenotype has been associated with this virus (9 ). Fungal virus genomes are commonly com- posed of double-stranded RNA (dsRNA) ( 10). Large molecules of dsRNA do not normally occur in fungal cells and, therefore, their presence is a sign of a viral infection (9 ). Using a protocol for nucleic acid extraction with enrichment for dsRNA ( 11), we detected the presence of a virus in C. protuberata . The dsRNA banding pattern consists of two segments of about 2.2 and 1.8 kb. Asmallersegment,lessthan1kbinlength,was variable in presence and size in the isolates analyzed and, later, was confirmed to be a sub- genomic element, most likely a defective RNA (fig.S1andFig.1,AandB).Usingtagged random hexamer primers, we transcribed the virus with reverse transcriptase (RT), followed by amplification and cloning. Sequence analysis revealed that each of the two RNA segments contains two open reading frames (ORFs) (fig. S2). The 2.2-kb fragment (RNA 1) is involved in virus replication, as both of its ORFs are similar to viral replicases. The first, ORF1a, has 29% amino acid sequence identity with a putative RNA-dependent RNA polymerase (RdRp) from the rabbit hemorrhagic disease virus. The amino acid sequence of the second, ORF1b, has 33% identity with the RdRp of a virus of the fungal pathogen Discula destructiva.ThesetwoORFs overlap and could be expressed as a single protein by frameshifting, a common expression strategy of viral replicases. The two ORFs of RNA 2 have no similarity to any protein with known function. As in most dsRNA mycovi- ruses, the 5 ′ends (21 bp) of both RNAs are conserved. Virus particles purified from C. protuberata are similar to those of other fungal viruses: spherical and ~27 nm in diameter (fig. S3). This virus is transmitted vertically in the conidiospores. We propose naming this virus Curvularia thermal tolerance virus (CThTV) to reflect its host of origin and its phenotype. The ability of the fungus to confer heat tolerance to its host plant is related to the presence of CThTV. Wild-type isolates of C. protuberata contained the virus in high titers, as evidenced by their high concentration of dsRNA (~2 mg/g of lyophilized mycelium). However, an isolate obtained from sectoring (change in morphology) of a wild-type colony contained a very low titer of the virus, as indicated by a low concentration of dsRNA (~0.02 mg/g of lyophi- lized mycelium). These two isolates were iden- tical by simple sequence repeat (SSR) analysis with two single-primer polymerase chain reac- tion (PCR) reactions and by sequence analysis of the rDNA ITS1-5.8 S-ITS2 region (figs. S4 and S5). Desiccation and freezing-thawing cycles are known to disrupt virus particles ( 12); thus, my- celium of the isolate obtained by sectoring was 1Plant Biology Division, Samuel Roberts Noble Foundation, Post Office Box 2180, Ardmore, OK 73402, USA. 2Depart- ment of Botany, University of Washington, Seattle, WA 98195, USA. 3Department of Microbiology, Montana State University, Bozeman, MT 59717, USA. 4U.S. Geological Sur- vey, Seattle, WA 98115, USA. *To whom correspondence should be addressed. E-mail: [email protected] Fig. 1. Presence or absence of CThTV in different strains of C. protuberata , detected by ethid- ium bromide staining ( A), Northern blot using RNA 1 ( B) and RNA 2 (C )transcriptsof the virus as probes, and RT- PCR using primers specific for a section of the RNA 2 ( D). The isolate of the fungus obtained by sectoring was made virus- free (VF) by freezing-thawing. The virus was reintroduced into the virus-free isolate through hyphal anastomosis ( An) with the wild type (Wt). The wild-type isolate of the fungus sometimes contains a subgenomic fragmen t of the virus that hybridizes to the RNA 1 probe (arrow). SCIENCEVOL 315 26 JANUARY 2007 513 REPORTS on September 25, 2019 Downloaded from lyophilized, frozen at–80°C, and subcultured to cure it completely of the virus. The complete absence of CThTV in this isolate was confirmed by dsRNA extraction, Northern blotting, RT-PCR (Fig. 1), and electron microscopy (no particles were observed in four grids). We assessed ex-perimentally the ability of the wild-type and virus-free isolates to confer heat tolerance by using thermal soil simulators ( 2,11 ). Plants inoc- ulated with the virus-inf ected wild-type isolate of the fungus tolerated intermittent soil temperatures as high as 65°C for 2 weeks (10 hours of heat per day), whereas both nonsymbiotic plants and plants inoculated with the virus-free isolate of the fungus became shriveled and chlorotic and died (Fig. 2). To confirm that CThTV was involved in heat tolerance in the plant-fungal symbiosis, we rein- troduced the virus into the virus-free fungal isolate and tested its ability to confer heat toler- ance. To provide a selectable marker, the virus- free isolate was transformed with a pCT74 vector containing a hygromycin-resistance gene (13) by restriction enzyme –mediated integration (REMI) transformation ( 14). Virus-containing wild-type hygromycin-sensitive (Wt) and virus- free hygromycin-resistant (VF) isolates of C. protuberata were cultured on single Petri dishes and allowed to undergo hyphal fusion or anastomosis (Fig. 3A). The mycelium from the area of anastomosis was subcultured twice with single conidiospores grown on hygromycin- containing plates. Thirty-five hygromycin-resistant isolates obtained in this way were screened for their dsRNA profiles, but only one was found to have acquired the virus (Figs. 1 and 3B). This fungal isolate, newly infected by hyphal anasto- mosis with CThTV (An), was tested for its ability to confer heat tolerance by the same experimental approach indicated above. The heat-stress exper- iment confirmed that the isolate newly infected with CThTV confers the same level of heat tol- erance as that conferred by the wild-type isolate (Fig. 2). Previously, we found that some beneficial endophytes isolated from monocots could be transferred to eudicots and still function as mutualists ( 3). Thus, we tested the ability of the C. protuberata isolates to confer heat tolerance to tomato ( Solanum lycopersicon). Using a slightly modified protocol for the heat-stress experiment ( 11 ), we obtained similar results to those obtained with D. lanuginosum (Fig. 4). However, it was not possible to attain 100% fungal colonization of tomato plants ( 11), and this may explain the higher proportion of dead plants colonized with the Wt or An fungus, compared with the experiment using D. lanuginosum. Given thatC. protuberata , when infected with CThTV, provides similar mutualistic benefits to both a monocot and a eudicot, it is possible that the underlying mechanism is conserved between these two groups of plants. Plants inoculated with C. protuberatain- fected with CThTV do not activate their stress- response system in the usual way. For example, the osmolyte concentration in these plants does not increase as a response to heat stress, although the levels are constitutively higher than in plants colonized with the virus-free isolate or the non- symbiotic plants (fig. S6). It has been hypoth- Fig. 2. (Top )Representative D. lanuginosum plants after the heat-stress experiment with thermal soil simulators. Rhizo- sphere temperature was main- tained at 65°C for 10 hours and 37°C for 14 hours/day for 14 days under greenhouse condi- tions. Plants were nonsymbiotic (NS) and symbiotic with the wild-type virus-infected isolate of C. protuberata (Wt), the hygromycin-resistant isolate new- ly infected with the virus through hyphal anastomosis (An), or the virus-free hygromycin-resistant isolate (VF). ( Bottom) The histo- gram presents the number of plants chlorotic, dead, and alive at the end of the experiment. The small letters on top of the bars indicate statistical differ- ences or similarities (chi-square test, P<0.01). Fig. 3. (A ) Anastomosis of the wild-type virus- infected isolate of C. pro- tuberata (Wt) and the virus-free hygromycin- r es ist ant isolate (VF) to produce a virus-infected hygromycin-resistant iso- late (An). (B ) After single- spore isolation to produce pure cultures, the isolate newly infected with the virus (An) retained the hygromycin-resistance and the morphology of the VF isolate. Fig. 4. (Top )Representativeto- mato ( Solanum lycopersicon ,var. Rutgers) plants after the heat-stress experiment. Plants were non- symbiotic (NS) and symbiotic with the wild-type virus-infected isolate of C. protuberata (Wt), the hygromycin- resistant isolate newly infected with the virus through hyphal anastomo- sis (An) or the virus-free hygromycin- resistant isolate (VF). Rhizosphere temperature was maintained at 65°C for 10 hours and ambient tem- perature (26°C) for 14 hours/day for 14 days under greenhouse condi- tions. ( Bottom ) The histogram presents the number of plants dead (white) and alive (black) at the end of the experiment. The small letters on top of the bars indicate statisti- cal differences or similarities (Fisher ’s exact test, P<0.05). 26 JANUARY 2007 VOL 315 514 REPORTS on September 25, 2019 Downloaded from esized that endophytes may protect their host plants by scavenging the damaging reactive oxygen species (ROS) generated by the plant defense mechanisms in response to environ- mental stress (15). The leaves of nonsymbiotic plants generated detectable ROS when stressed with heat, whereas those of symbiotically colonized plants did not (table S1). However, there was no difference in the ROS response to heat between plants inoculated with the virus-free and the CThTV-infected isolates of C. protuberata . Complex tripartite symbioses have been found among arthropods, bacteria, and mutualistic bac- teriophages (16 ,17). This study reports a three- way mutualistic symbiosis involving a virus, a fungal endophyte, and either a monocot or eudicot plant. References and Notes1. R. J. Rodriguez, R. S. Redman, J. M. Henson, Mitig. Adapt. Strategies Global Change 9, 261 (2004). 2. R. S. Redman, K. B. Sheehan, R. G. Stout, R. J. Rodriguez, J. M. Henson, Science298, 1581 (2002). 3. R. J. Rodriguez, R. S. Redman, J. M. Henson, in The Fungal Community: Its Organization and Role in the Ecosystem , J. Dighton, J. F. White Jr., P. Oudemans, Eds. (CRC Press, Boca Raton, FL, 2004), pp. 683 –695. 4. A. L. Dawe, D. L. Nuss, Annu. Rev. Genet.35, 1 (2001). 5. S. L. Anagnostakis, P. R. Day, Phytopathology69, 1226 (1979). 6. T. Zhou, G. J. Boland, Phytopathology87, 147 (1997). 7. I. P. Ahn, Y. H. Lee, Mol. Plant Microbe Interact. 14, 496 (2001). 8. Y. M. Chu et al.,Appl. Environ. Microbiol. 68, 2529 (2002). 9. I. Zabalgogeazcoa, E. P. Benito, A. G. Ciudad, B. G. Criado, A. P. Eslava, Mycol. Res.102, 914 (1998). 10. S. A. Ghabrial, Adv. Virus Res.43, 303 (1994). 11. Materials and methods are available as supporting material on ScienceOnline. 12. Y. G. Kuznetsov, A. McPherson, Virology352, 329 (2006). 13. J. M. Lorang et al.,Appl. Environ. Microbiol. 67, 1987 (2001). 14. R. S. Redman, J. C. Ranson, R. J. Rodriguez, Mol. Plant- Microbe Interact. 12, 969 (1999). 15. R. Rodriguez, R. Redman, Proc. Natl. Acad. Sci. U.S.A. 102 , 3175 (2005). 16. S. R. Bordenstein, J. J. Wernegreen, Mol. Biol. Evol.21, 1981 (2004). 17. N. A. Moran, P. H. Degnan, S. R. Santos, H. E. Dunbar, H. Ochman, Proc. Natl. Acad. Sci. U.S.A. 102, 16919 (2005). 18. We thank F. Coker, M. Hoy, Y. Chen, and R. Pescador for technical assistance and P. Xu, J. Pita, K. Craven, T. Feldman, A. Ali, G. Shen, R. Uppalapati, and C. Bastidas for providing comments and suggestions that improved the manuscript. This project was made possible by the permission, assistance, and guidelines of YNP. This work was supported by The Samuel Roberts Noble Foundation, the MSU Thermal Biology Institute, and grants from the U.S. Geological Survey, the NSF (9977922 and 0414463), and the U.S. Army Research Office (DAAHO4-96-1-01194). Sequences were deposited in GeneBank under accession numbers EF120984 (RNA 1) and EF120985 (RNA 2). The CThTV description was deposited in the ICTVdB —The Universal Virus Database, version 4 (, with the virus accession number: 0000040020. Supporting Online Materials and Methods Figs. S1 to S5 Table S1 References 12 October 2006; accepted 12 December 2006 10.1126/science.1136237 The Neural Basis of Loss Aversion in Decision-Making Under Risk Sabrina M. Tom, 1Craig R. Fox, 1,2 Christopher Trepel, 2Russell A. Poldrack 1,3,4 * People typically exhibit greater sensitivity to losses than to equivalent gains when making decisions. We investigated neural correlates of loss aversion while individuals decided whether to accept or reject gambles that offered a 50/50 chance of gaining or losing money. A broad set of areas (including midbrain dopaminergic regions and their targets) showed increasing activity as potential gains increased. Potential losses were represented by decreasing activity in several of these same gain-sensitive areas. Finally, individual differences in behavioral loss aversion were predicted by a measure of neural loss aversion in several regions, including the ventral striatum and prefrontal cortex. M any decisions, such as whether to in- vest in the stock market or to accept a new job, involve the possibility of gaining or losing relative to the status quo. When faced with such decisions, most people are markedly risk averse. For instance, people typ- ically reject gambles that offer a 50/50 chance of gaining or losing money, unless the amount that could be gained is at least twice the amount that could be lost (e.g., a 50/50 chance to either gain $100 or lose $50) (1 ). Prospect theory, the most successful behavioral model of decision-making under risk and uncertainty ( 1, 2), explains risk aversion for “mixed ”(gain/loss) gambles using the concept of loss aversion: People are more sensitive to the possibility of losing objects or money than they are to the possibility of gaining the same objects or amounts of money ( 1, 3–5 ). Thus, people typically require a potential gain of at least $100 to make up for exposure to a potential loss of $50 because the subjective impact of losses is roughly twice that of gains. Similarly, people demand substantially more money to part with objects that they have been given than what they would have been willing to pay to acquire those objects in the first place ( 6). Loss aversion also has been used to explain a wide range of economic behaviors outside the laboratory ( 7,8). Further, loss aversion is seen in trading behavior of both children as young as age five ( 9) and capuchin monkeys ( 10), which sug- gests that it may reflect a fundamental feature of how potential outcomes are assessed by the primate brain. Previous neuroimaging studies of responses to monetary gains or losses have focused on ac- tivity associated with the anticipation of im- mediate outcomes ( “anticipated ”utility) ( 11,12 ) or the actual experience of gaining or losing money ( “experienced” utility) (11,13, 14)rather than specifically investigating which brain sys- tems represent potential losses versus gains when a decision is being made ( “decision ”utility). Be- havioral researchers have shown that anticipated, experienced, and decision utilities often diverge in dramatic ways, which raises the possibility that the corresponding brain systems involved may also differ (15). In the current study, we aimed to isolate activity associated with the evaluation of a gamble when choosing whether or not to accept it (i.e., decision utility) without the expectation that the gamble would be immediately resolved. This allowed us to test whether neural responses during the evaluation of potential outcomes are similar to patterns previously reported in studies of anticipated and experienced outcomes. One fundamental question for the study of decision-making is whether loss aversion reflects the engagement of distinct emotional processes when potential losses are considered. It has been suggested that enhanced sensitivity to losses is driven by negative emotions, such as fear or anxiety (16). This notion predicts that exposure to increasing potential losses should be associated with increased activity in brain structures thought to mediate negative emotions in decision-making [such as the amygdala or anterior insula; compare with ( 17,18)]. Alternatively, loss aversion could reflect an asymmetric response to losses versus gains within a single system that codes for the subjective value of the potential gamble, such as ventromedial prefrontal cortex (VMPFC)/ orbitofrontal cortex (OFC) and ventral striatum ( 11 ,19, 20). To examine the neural systems that process decision utility, we collected functional magnetic resonance imaging (f MRI) data while partici- 1Department of Psychology, University of California Los Angeles (UCLA), Franz Hall, Box 951563, Los Angeles, CA 90095 –1563, USA. 2Anderson School of Management, UCLA, 110 Westwood Plaza, Los Angeles, CA 90095 –1481, USA. 3Brain Research Institute, UCLA, Los Angeles, CA 90095, USA. 4Department of Psychiatry and Biobehavioral Sciences, UCLA, Los Angeles, CA 90095, USA. *To whom correspondence should be addressed. E-mail: [email protected] SCIENCEVOL 315 26 JANUARY 2007 515 REPORTS on September 25, 2019 Downloaded from ERRATUM www.sciencemag.orgSCIENCEERRATUM POST DATE 13 APRIL 2007 1 CORRECTIONS & CLARIFICATIONS Reports:“A virus in a fungus in a plant: three-way symbiosis required for thermal tolerance” by L. M. Márquez et al. (26 Jan. 2007, p. 513). On page 514, in the legend to Fig. 4, the colors of the histogram are inverted: The number of dead plants is black, and the number of alive plants is white. Post date 13 April 2007 on September 25, 2019 Downloaded from A Virus in a Fungus in a Plant: Three-Way Symbiosis Required for Thermal Tolerance Luis M. Márquez, Regina S. Redman, Russell J. Rodriguez and Marilyn J . Roossinck DOI: 10.1126/science.1136237 (5811), 513-515. 315 Science  ARTICLE TOOLS MATERIALS SUPPLEMENTARY CONTENT RELATED REFERENCES This article cites 15 articles, 5 of which you can access for free PERMISSIONS Terms of Service Use of this article is subject to the is a registered trademark of AAAS. Science Science, 1200 New York Avenue NW, Washington, DC 20005. The title (print ISSN 0036-8075; online ISSN 1095-9203) is published by the Amer ican Association for the Advancement of Science American Association for the Advancement of Science on September 25, 2019 Downloaded from

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