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Interaction between viral RNA silencing suppressors and host factors in plant immunity

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Interaction between viral RNA silencing suppressors and host factors in plant immunityKenji S Nakahara and Chikara MasutaTo elucidate events in the molecular arms race between the host and pathogen in evaluating plant immunity,a zigzag model is useful for uncovering aspects common to different host–pathogen interactions.By analogy of the steps in virus–host interactions with the steps in the standard zigzag model outlined in recent papers,we may regard RNA silencing as pattern-triggered immunity(PTI)against viruses,RNA silencing suppressors(RSSs)as effectors to overcome host RNA silencing and resistance gene(R-gene)-mediated defense as effector-triggered immunity(ETI)recognizing RSSs as avirulence proteins.However,because the standard zigzag model does not fully apply to some unique aspects in the interactions between a plant host and virus,we here defined a model especially designed for viruses.Although we simplified the phenomena involved in the virus–host interactions in the model,certain specific interactive steps can be explained by integrating additional host factors into the model.These host factors are thought to play an important role in maintaining the efficacy of the various steps in the main pathway of defense against viruses in this model for virus–plant interactions.For example,we propose candidates that may interact with viral RSSs to induce the resistance response.AddressesPlant Breeding Science,Research Faculty of Agriculture,Hokkaido University,Sapporo,060-8589,JapanCorresponding authors:Nakahara,Kenji S(knakahar@res.agr.hokudai.ac.jp)andMasuta,Chikara(masuta@res.agr.hokudai.ac.jp)Current Opinion in Plant Biology2014,20:88–95This review comes from a themed issue on Biotic interactions2014Edited by Makoto Hayashi and Martin Parniske/10.1016/j.pbi.2014.05.0041369-5266/#2014Elsevier Ltd.All rights reserved. IntroductionPlants use two major strategies to defend against patho-gens;the resistance(R)-protein-mediated strategy works effectively against diverse pathogens,including fungi, bacteria and viruses,while the RNA silencing strategy is a major antiviral mechanism[1–3].Most viruses encode RNA silencing suppressors(RSSs)to interfere with RNA silencing[4,5].As a consequence of the particular strategy used in the battle between virus and host,infected plants develop various symptoms[6].According to the zigzag model(Figure1a)to explain the two-branched immune system of plants in response to a plant pathogen[7 ,8],R-protein-mediated resistance developed to control a pathogen that had overcome basal resistance or innate immunity,thefirst line of preformed,inducible defenses against the major groups of pathogens.Basal resistance starts with the detection of pathogen-associated molecular patterns(PAMPs),such as bacterialflagellin and fungal chitin,by the host’s pattern-recognition receptors(PRRs).In the zigzag model,it is defined as pattern-triggered immunity(PTI).PRRs for bacteria and fungi have been identified,and these are mostly receptor-like kinases,which were once classified in the R-protein family.For example,the host transmem-brane FLS2protein recognizes theflg22peptide from Pseudomonasflagellin[9].To circumvent basal defense (PTI),pathogens produce effector proteins.When pathogen effectors overcame PTI,plants next evolution-ally developed R-proteins to activate effector-triggered immunity(ETI),by which host proteins recognize the effectors as avirulence(Avr)factors,which then induces an amplified version of resistance comparable to PTI.R-protein-mediated resistance is often accom-panied by a hypersensitive response(HR),which is observed as local necrotic lesions.Therefore,we regard the HR-associated resistance response as a consequence of R-protein-mediated resistance,unless the HR pathway is independent of this resistance pathway.There are not many comparative studies between anti-viral and antibacterial/antifungal immune responses. Mandadi and Scholthof[10]have once reviewed analo-gous viral and nonviral immune concepts,but it was found not to be so simple to define viral PTI,ETI and ETS finding concrete examples;they did not actually integrate RNA silencing into their model.On the other hand, because RNA silencing against viruses is reminiscent of basal resistance against fungi and bacteria,by regarding RNA silencing as a type of PTI,viruses can be also integrated in a modified zigzag model;here,viral double-stranded RNA(dsRNA)corresponds to a PAMP [11,12].However,there are certainly differences between viruses and other pathogens in their molecular interactions with plant hosts.In devising a model for viruses,we here integrate additional host factors to explain certain virus–host interactions and highlight aspects of the anti-viral defense that differ from theAvailable online at ScienceDirectstandard zigzag model for fungi and bacteria.In addition, we focus on the molecular cross-talk between RNA silencing and R-protein-mediated resistance.Figure1c shows our entire scheme to explain the host–virus inter-actions in our model described here.Comparison of the viral version of PTI and ETI with those in the standard zigzag model proposed for bacterial or fungal pathogens To encompass all the phenomena involved in the com-plicated arms race between a particular host and pathogen,the zigzag model is quite useful.The concept of the model may be applied also to host–virus inter-actions,paying attention to some analogous phenomena in PTI and ETI.For example,one review totallyfit a model for host defense against viruses to the standard zigzag model,regarding viral dsRNAs as PAMPs,host RNA silencing as PTI and counterattack by viral RSSs as ETS and so on[12].Consistent with this review,based on extreme resistance observed for tobacco plants expressing the P19protein,an RSS of tombusvirus,Sansregret et al.[13 ]showed that the general scheme of host induction and viral suppression of RNA silencing could be adapted to the classical frame of PTI and ETI.However,in another review,although some degree of analogy of PTI and ETI between viruses and other pathogens was drawn,the author indicated a clear difference and the uniqueness in virus–host interactions[11,14 ].Fungal and bacterial pathogens have various Avr proteins in their arsenal when ETI is activated;one can be replaced by other redundant effectors.However,viruses have a lim-ited number of proteins that are all important for their survival.When one of the viral proteins is recognized by a host R protein,viruses cannot easily replace it with another;rather they modify it by changing the amino acid sequence while retaining the protein structure necessary for the function.Whether the host R protein still recognizes the modified version depends on the LRR domain in the R protein with a varying degree of affinity. Alternatively,according to the bait and switch model,a host co-factor that binds to an R protein may affect the specificity of the host recognition for the viral Avr protein [10].Therefore,for virus–host interactions,we cannot draw an actual zigzag model in which multiple rounds of ETS followed by ETI are repeated with different com-binations of host R protein and viral Avr.As such,the molecular virus–host interaction must be explained by a limited number of players.Although the idea that RNA silencing and its suppression by viral RSSs can be rationalized within the PTI–ETI framework is attractive,we need more experimental evidence because there are actually viral proteins that are not RSSs but are recognized by R-proteins.Instead of expanding on the standard zigzag model tofit virus–host interactions,we can create a model that allows a quick overview of the molecular phenomena in the virus–host arms race,the strategies unique to viruses and the steps that are analogous to the standard zigzag model.As we will discuss,we consider that the host response branches from the general course of antiviral response instead of repeating the ETI;the strategies at these branches vary depending on the specific host and virus and the particu-lar point of the interaction.RNA silencing and viral RNA silencing suppressorsRNA silencing functions as an antiviral mechanism in plants[2,4](Figure1b–d).As a counterdefense,viruses developed RNA silencing suppressor(RSS)proteins, which function to inhibit RNA silencing through diverse modes of action.The main mechanism for the RSSs appears to be binding with long dsRNA or siRNA duplexes,subsequently inhibiting siRNA biogenesis or RISC formation[15].Another mechanism is binding to the components in the silencing pathway such as AGO1. Several RSSs(TCV CP,CMV2b,TBSV P19,PVX P25, Polerovirus P0and P1of Sweet potato mild mottle virus) have been reported to repress or interfere with the func-tion of AGO1[16–21].Diverse RSSs appear to reduce AGO1in infected plant tissues[22].However,although viral RSSs interfere with host RNA silencing and are mostly effective,hosts have some mechanisms to activate another or secondary defense.For example,Arabidopsis thaliana encodes10AGO proteins.AGO1performs not only antiviral RNA silencing,but also silences endogen-ous genes by cleaving viral RNA and endogenous target mRNA.Recent screening of other AGO proteins in antiviral defense using knockout mutants revealed that AGO2is also induced and functions in the defense against TCV and CMV when the viral RSSs targeted AGO1[23].This study suggested that AGO2is involved in antiviral RNA silencing,which is induced via infection by viruses that encode RSSs targeting AGO1or via miRNA-mediated RNA silencing because AGO2expres-sion is repressed by AGO1via miR403.In addition, miRNA-mediated RNA silencing appears to control other RNA silencing components,including DCLs,DRB4, RDR6,and AGOs[24],implying their involvement in secondary antiviral RNA silencing.Interestingly, AGO2is also involved in the induction and secretion of antimicrobial pathogenesis-related protein1,in addition to antiviral RNA silencing[25].Since DRB4is involved not only in RNA silencing but also in R-gene-mediated resistance,if many R-genes are controlled by miRNAs,seemingly,when a virus suppresses RNA silencing,diverse secondary defense systems could be activated.Viral RNA silencing suppressors as Avr determinantsDirect and indirect interactions occur between R-gene-mediated resistance and RSSs.For example,a link between ETI-like phase and RNA silencing has beenZigzag model of the arms race between plants and viruses Nakahara and Masuta8990Biotic interactions2014Figure1The interactions between viral RSS(SIS)and host factors involved in plant immunity.(a)Model of the arms race between pathogens and plants using the standard zigzag model.(b)Model of molecular virus–host interactions involving RNA silencing and R-protein(NB-LRR)-mediated resistance.Unlike the innate immunity against other pathogens,the first layer of the immunity against viruses is RNA silencing.RNA silencing is induced by intramolecularly and intermolecularly formed double-stranded RNAs(dsRNAs)of the viral genome or its transcripts.Then,dsRNA is processed into siRNAs by the DCL4–DRB4complex and DCL2in Arabidopsis.AGO1binds siRNA and cleaves viral RNA guided by the incorporated siRNA.Most viruses counteract this by expressing RNA silencing suppressors(RSSs).Plants coevolved an immune system that is associated with the HR in response to the RSS.In this figure,the HR that is not associated with SA-mediated resistance is defined as programmed cell death(PCD).Recent studies have suggested that host cofactors such as DRB4,a tobacco calmodulin-like protein,rgs-CaM,and the Arabidopsis NAC transcription factorsuggested for Cucumber mosaic virus(CMV),Tobacco etch virus(TEV),and Potato virus Y(PVY).The RSS of CMV, the2b protein(CMV2b),inhibits the salicylic acid(SA)-mediated defense response[26].Some examples of molecular interactions have been reported between a viral RSS and an R-protein[27–29].Well-established examples of host recognition of an RSS are the coat protein(CP)of Turnip crinkle virus(TCV)and the replicase of Tobacco mosaic virus(TMV).The TCV CP serves as an RSS,but also as the TCV Avr protein that induces R-gene(the HRT gene)-mediated resistance in Arabidopsis ecotype Di-17[30,31].TMV replicase has RSS activity[32],and the p50helicase domain in the replicase can induce an HR,serving as the Avr determi-nant in tobacco carrying the N-gene,which is the well-known R-gene working for ETI against TMV. Using an agroinfiltration assay to study the ability of viral RSSs to elicit HR-like necrosis,Angel and coworkers[33]also found that the P19protein(P19)of Tomato bushy stunt virus(TBSV)was recognized by a putative R-protein in Nicotiana species,which then induced an HR-like ing a similar agroinfil-tration assay for Capsicum annuum,Ronde and colleagues [34]recently showed that the RSS of Tomato spotted wilt virus(TSWV),the NSs protein,function as the Avr determinant in C.annuum carrying the R-gene(Tsw) against TSWV.They discussed their pathosystem in light of the putative interplay between RNA silencing and the R-gene-mediated resistance.Consistent with the reports by Angel and colleagues [33,35],P19was recently demonstrated to function as the Avr protein that induced extreme resistance(ER), characterized by strong SA-dependent resistance without visible HR lesions,in Nicotiana tabacum [13 ].In addition,the binding of P19to small RNA (sRNA)was necessary to induce ER,suggesting that RNA silencing and an ETI-like phase are linked to each other.Similarly,the2b protein(TAV2b)of Tomato aspermy virus(TAV),an RSS of TAV,was found to induce HR on the leaves of N.tabacum and Nicotiana benthamiana infected with the TMV vector expressing TAV2b[36].In this case,Lys21and Arg28,both located within the N-terminal region of TAV2b,were critical for the HR induction.These positively charged residues were later shown to be involved in sRNA binding and thus the RSS activity of TAV2b[37]. These studies thus suggest the existence of an R-protein that recognizes TAV2b with RSS activity as an Avr protein in Nicotiana species.However, when its expression was driven by its own parent virus or when2b of CMV,which is closely related to TAV, was expressed by the TMV vector,no necrotic lesions were observed[36].These contradictory observations imply additional involvement of other viral or host factors in the HR-associated resistance responses in specific combinations of RSS and host.In further support of2b serving as an Avr protein,we recently demonstrated that CMV2b induced weak necrosis and SA and hydrogen peroxide accumulation in Arabidopsis thaliana Col-0ecotype(hereafter, Arabidopsis),suggesting that the plant has an R-protein that recognizes CMV2b as an Avr protein.In fact,CMV Y strain(CMV-Y)causes mosaics withfine necrotic spots in the upper leaves,but not typical HR-like necrosis, although we observed slightly stronger necrosis on Col-0 infected with CMV-HL(a lily strain).From the results of an in situ molecular interaction study,the necrosis on Arabidopsis seemed to have been driven by a specific interaction between CMV2b and the Arabidopsis catalase-3(CAT3)[38 ,39],a key enzyme in cellular scavenging of hydrogen peroxide and induction of HR.If this type of HR-like induction is indeed part of the host ETI-like phase,then an Arabidopsis R-protein may recognize CMV2b as a complex with a host factor(s)that includes CAT3.The affinity between CMV2b and CAT3seems to be important for determining the degree of necrosis because the observed necrosis depends on the CMV strain and the Arabidopsis ecotype.miRNA-mediated regulation of the R-genes against virusesRecent studies demonstrated that plant microRNAs (miRNAs)target and negatively regulate R-gene expression via RNA silencing[29–33].This miRNA-mediated R-gene regulation was actually inhibited upon viral infection,suggesting that RNA silencing is linked to R-protein-mediated resistance.Downregu-lation of R-gene expression by RNA silencing is perhaps because plants prevent unwanted autoimmu-nity by overexpressing the R-gene in the absence of viruses.Although RNA silencing primarily targets viruses in the model,we also consider that RNA silencing can also affect the subsequent host defense governed by R-proteins as discussed here.Zigzag model of the arms race between plants and viruses Nakahara and Masuta91 (Figure1legend continued)(TIP)help putative NB-LRRs to recognize RSSs[46 ].Salicylic-acid(SA)-mediated defense responses were found to be suppressed by RSSs such as CMV2b and TCV CP,suggesting viral evasion of induced HR,which is associated with the SA-mediated immunity to prevent viral infection.Here,an SA-mediated immunity suppressor is designated SIS.(c)Entire scheme to explain the host–virus interactions,integrating steps unique to viruses compared with the standard zigzag model.(d)Branches from the main path of the model,where viral factors(RSS and SIS)participate, represent other virus–host interactions that are mediated by the same viral factors.For example,the SA-mediated basal immunity involving TIP[58]and the rgs-CaM-directed degradation of RSS via autophagy[51 ]are also thought to contribute to antiviral immunity,although TIP and rgs-CaM seem to be independent of the general course of host defense.TCV CP counteractively suppresses the basal immunity by binding to TIP[14 ].Recent evidence has indicated that plant small RNAs (sRNAs)(siRNAs and miRNAs)are involved in the basal resistance against pathogens.For bacteria,the bacterial peptideflag22actually induces miRNA393,which targets auxin receptors,which in turn mediate the sig-naling that activates the SA resistance pathway[40]. In fact,an RNA-induced silencing complex(RISC) containing Argonaute2(AGO2)programmed with miR393plays a critical role in ETI against Pseudomonas syringae[25].The suppression of auxin signaling by miR393ultimately activates the SA-mediated defense response,which is also one of the main mechanisms in the antiviral PTI-like phase;interference with the miR393-mediated regulation of auxin receptors by viral suppressors must impair the PTI-like and ETI-like phases against viruses.Some sRNA can target R-gene transcripts directly.With the recent discovery of many new miRNAs through deep-sequencing studies(e.g.RNASeq)bioinformatics analyses of the sRNA libraries obtained have identified novel miRNAs and putative functions that potentially target host R-genes.For example,He and colleagues[41] found an miRNA in Brassica rapa(named bra-miR1885) that was induced by Turnip mosaic virus(TuMV)infec-tion and potentially targeted the mRNAs of R-genes encoding TIR-NB-LRR class proteins;unfortunately, whether the R-gene targets are involved in the host resistance to TuMV was not determined.As another example,after searching tobacco sRNA libraries for N-gene-related sRNAs,Li and coworkers[42 ]found that two newly discovered plant miRNAs(nta-miR6019 and nta-miR6020)could guide cleavage of the N-gene transcript in tobacco,conferring resistance to TMV.In a search for phased,trans-acting siRNAs(phasiRNAs) isolated from Medicago after deep sequencing,Zhai and colleagues[43]revealed that the majority of phasiRNAs were produced from R-genes,suggesting a close association between RNA silencing and the R-gene-mediated resistance response.Although the phasiRNAs targets have not been identified,we should consider generation of phasiRNAs when trying to understand R-gene-mediated immunity.Host factor(s)that regulate the interactions between RNA silencing and R-gene-mediated resistance in a model for virusesIn our model for viruses(Figure1b–d),virusesfirst produce dsRNAs in infected plants.In turn,plants acti-vate RNA silencing as a PTI-like phase to target the viral RNAs.Then,the viruses produce RSSs as viral effectors to suppress RNA silencing.In the subsequent ETI-like phase,to generate an effective defense,the plants should activate an R-protein that specifically recognizes the viral RSSs as the Avr protein,thus leading to the HR and SA-dependent resistance.Although we have simplified each phase to give a general overview,depending on the particular host–virus combination, additional host factors should be integrated into the model for understanding certain specific stages in host–virus interactions.For example,the RNA silencing component DRB4is potentially one such mediator of PTI-like phase.DRB4is a dsRNA-binding protein that associates with a dicer-like protein4(DCL4)to produce virus-specific siRNA[44,45].A recent study revealed that the Arabidopsis R-protein requires DRB4for the sub-sequent HRT(an R protein to TCV)-mediated HR against the RSS or CP of TCV[46 ].Notably,DRB4 interacts with both HRT and the TCV CP and stabilizes HRT,but inhibits the interaction between HRT and the TCV CP.Although we do not yet know how DRB4 contributes to the HR,we do know that DRB4is also involved in R-gene-mediated resistance against bacteria, implying that it is involved in ETI.Another candidate mediator is the plant calmodulin-like protein,rgs-CaM, which we describe in detail next.Possible branches in the model between plants and virusesRSSs that can suppress SA-related defense responses include CMV2b,CaMV P6,and TCV CP[14 ,26, 47–49].Since viral RSSs participate in an arms race between viruses and plants,and since RNA silencing is a PTI-like phase against diverse viruses,RSSs reduce host defense,shifting the phase to effector-triggered susceptibility(ETS)-like in the model.On the other hand,because some RSSs also behave as an SA-mediated immunity suppressor(SIS),those RSSs can be considered to create another ETS-like phase,implying additional branches in the model.All of these RSSs also function as avirulence proteins,which can elicit the HR in plants that possess the corresponding R-gene.In these cases,the HR is closely associated with SA-related defense responses. Integrating all these data,we propose a model in which viral SIS has been developed to repress or evade the HR-mediated resistance against viruses(Figure1b and d).If a virus has RSS or other viral proteins with SIS ability,SIS might be able to mask the R-protein-mediated defense responses,resulting in a phenotype similar to that seen in a susceptible plant.Therefore,we believe that many potential resistant interactions be-tween viruses and plants are still hidden.For example, the exacerbation of the HR and symptoms that accom-panies necrosis in plants infected with virus vectors that express heterologous viral RSSs or other proteins[36,50] might be explained by the induction of defense responses to the expressed viral proteins,which are not induced by the parental viruses because of the viral SISs.In addition to R-gene-mediated resistance,recent studies have suggested that plants have additional counter-coun-terdefense systems against viral RSSs.We discovered an antiviral counter-counterdefense that involves rgs-CaM in tobacco[51 ,52].When rgs-CaM was initially found,92Biotic interactions2014rgs-CaM was reported to interact with the TEV RSS, HC-Pro and act as an endogenous RSS[53,54].Later, we found another function for rgs-CaM in antiviral defense.Our previous study suggested that rgs-CaM binds not only HC-Pro,but also other RSSs,including CMV and TAV2b,via its affinity to the negatively charged dsRNA-binding domains of RSSs.Then, rgs-CaM presumably reinforces antiviral RNA silencing by directing the degradation of its associated RSSs via autophagy(Figure1c and d).Calmodulin-like proteins are one of the three protein families of EF-hand Ca2+ sensors in plants and are thought to coordinate the functions of several endogenous proteins by binding to the targets as a hub protein in response to the Ca2+ stimulus[55].Since they are known to function in countering abiotic and biotic stresses,we suspect that rgs-CaM functions in antiviral defense[41].Recent studies on the interaction of the TCV CP with the Arabidopsis NAC transcription factor(TIP)imply that an alternative branch should be included in the model between viruses and plants.TCV CP is a viral RSS [56,57]and the avirulence protein recognized by the R-gene,HRT in Arabidopsis Di-17[31],suggesting that TIP is involved in the RSS activity and HR induction by the TCV CP.However,TIP is not required for either the RSS activity or HR induction.Instead,TIP was recently shown to be involved in SA-mediated basal immunity in Arabidopsis[58].TCV CP suppresses the SA-mediated basal immunity via its binding to TIP[14 ].These studies indicate that TCV CP suppresses both the RNA silencing and SA-mediated basal immunity to facilitate the initial infection of Arabidopsis with TCV (Figure1d).In the canonical zigzag model for the inter-action between other microorganisms and plants,PTI should be an induced defense,which partly shares defense responses with ETI after the perception of PAMPs by receptor-like kinases.Therefore,the TIP-associated PTI seems to be integrated in the viral model as another interaction between virus and host.Here,we draw those new interactions as branches in the viral model.More interestingly,the basal resistance involving TIP also affects CMV accumulation,indicating that the basal resistance is not specific to TCV[58].For many other viruses,similar sets of defense-related genes have been reported to be induced during viral infection of a susceptible plant[59,60],suggesting thatthe host resist-ance response is somehow suppressed in those plants, although it is partly activated.As such interactions are uncovered,we can better organize the branches from the main course of defense involving RNA silencing in the model for host–virus interactions. AcknowledgementsThis work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education,Culture,Sports,Science and Technology of Japan and the Sumitomo Foundation.References and recommended readingPapers of particular interest,published within the period of review, have been highlighted as:of special interestof outstanding interest1.Ding SW:RNA-based antiviral immunity.Nat Rev Immunol2010,10:632-644.2.Shimura H,Pantaleo V:Viral induction and suppression of RNAsilencing in plants.Biochim Biophys Acta2011,1809:601-612.3.Ding SW,Lu R:Virus-derived siRNAs and piRNAs in immunityand pathogenesis.Curr Opin Virol2011,1:533-544.4.Burgyan J,Havelda Z:Viral suppressors of RNA silencing.Trends Plant Sci2011,16:265-272.5.Giner A,Lopez-Moya JJ,Lakatos L:RNA silencing in plants andthe role of viral suppressor.In RNA interference and viruses.Edited by Martinez MA.Norfolk,UK:Caister Academic Press;2010:25-46.6.Wang MB,Masuta C,Smith NA,Shimura H:RNA silencingand plant viral diseases.Mol Plant Microbe Interact2012,25:1275-1285.7.Jones JD,Dangl JL:The plant immune system.Nature2006,444:323-329.This is thefirst article on a zigzag model that explains PTI and ETI together.8.Monaghan J,Zipfel C:Plant pattern recognition receptorcomplexes at the plasma membrane.Curr Opin Plant Biol2012, 15:349-357.9.Chinchilla D,Bauer Z,Regenass M,Boller T,Felix G:TheArabidopsis receptor kinase FLS2bindsflg22anddetermines the specificity offlagellin perception.Plant Cell 2006,18:465-476.10.Mandadi KK,Scholthof KB:Plant immune responses againstviruses:how does a virus cause disease?Plant Cell2013,25:1489-1505.11.Moffett P:Mechanisms of recognition in dominant R genemediated resistance.Adv Virus Res2009,75:1-33.12.Zvereva AS,Pooggin MM:Silencing and innate immunity inplant defense against viral and non-viral pathogens.Viruses2012,4:2578-2597.13.Sansregret R,Dufour V,Langlois M,Daayf F,Dunoyer P,Voinnet O,Bouarab K:Extreme resistance as a hostcounter-counter defense against viral suppression of RNAsilencing.PLoS Pathog2013,9:e1003435.This article is thefirst to demonstrate a direct link between RSS activity and the Avr determinant for the ETI(R-protein-mediated resistance)in the zigzag model.14.Donze T,Qu F,Twigg P,Morris TJ:Turnip crinkle virus coatprotein inhibits the basal immune response to virus invasion in Arabidopsis by binding to the NAC transcription factor TIP.Virology2014,449:207-214.The Arabidopsis NAC transcription factor TIP is known to interact with the TCV CP,which behaves as a viral RSS and avirulence protein in Arabi-dopsis Di-17with the R-gene,HRT.Therefore,TIP was suspected of being involved in the RSS activity of TCV CP or its recognition by the R-gene,HRT.However,this study showed that in reality,TIP is involved in SA-mediated basal immunity and that TCV CP suppresses the basal immunity by binding to TIP.katos L,Csorba T,Pantaleo V,Chapman EJ,Carrington JC,Liu YP,Dolja VV,Calvino LF,Lopez-Moya JJ,Burgyan J:SmallRNA binding is a common strategy to suppress RNA silencing by several viral suppressors.EMBO J2006,25:2768-2780. 16.Derrien B,Baumberger N,Schepetilnikov M,Viotti C,De Cillia J,Ziegler-Graff V,Isono E,Schumacher K,Genschik P:Degradation of the antiviral component ARGONAUTE1by the autophagypathway.Proc Natl Acad Sci U S A2012,109:15942-15946. 17.Azevedo J,Garcia D,Pontier D,Ohnesorge S,Yu A,Garcia S,Braun L,Bergdoll M,Hakimi MA,Lagrange T et al.:ArgonauteZigzag model of the arms race between plants and viruses Nakahara and Masuta93。

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蛋白质-RNA相互作用界面预测与设计

蛋白质-RNA相互作用界面预测与设计


Biological function RNA splicing regulation RNA metabolic regulation
mRNA degradation gene regulation RNA degradation gene regulation
mRNA degradaton and translation post-transcriptional gene regulation
(Biomolecular Physics and Modeling Group, School of Physics, Huazhong University of Science and Technology, Wuhan 430074, P. R. China)
Abstract: RNA-protein interactions play key roles in many biological processes. The three dimensional (3D) structure of protein-RNA complexes can be determined experimentally by structural biologists. The recognition between protein and RNA can be understood from the 3D atomic structure. However, the structure determination of protein-RNA complexes by experimental methods is often difficult and costly, and limited to the binding strength. Thus, the prediction and design of protein-RNA complex structures is important in biological medical research. In this review, we will discuss the recent progress in protein-RNA interface prediction and design, which includes the following aspects: (1) protein-RNA docking and the conformational change on binding; (2) the recognition mechanism of protein-RNA binding; (3) the molecular design based on the protein-RNA interface. Improvement of the protein-RNA docking algorithm will help us annotate a large number of proteins and RNA with unknown function, and molecular design based on macromolecular interactions will be useful in drug design.
细胞器之间相互作用在非酒精性脂肪性肝病发生发展中的作用

细胞器之间相互作用在非酒精性脂肪性肝病发生发展中的作用

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细胞器之间相互作用的异常与疾病的发生发展密切相关。

近年来，细胞器之间相互作用在非酒精性脂肪性肝病（ＮＡＦＬＤ）发生发展中的作用受到关注，特别是线粒体、脂滴与其他细胞器之间的相互作用。

关键词：非酒精性脂肪性肝病；细胞器；线粒体；脂肪滴基金项目：国家自然科学基金面上项目（８２０７０６１８）ＲｏｌｅｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｉｎｔｈｅｄｅｖｅｌｏｐｍｅｎｔａｎｄｐｒｏｇｒｅｓｓｉｏｎｏｆｎｏｎａｌｃｏｈｏｌｉｃｆａｔｔｙｌｉｖｅｒｄｉｓｅａｓｅＬＩＵＴｉａｎｈｕｉ．（ＬｉｖｅｒＲｅｓｅａｒｃｈＣｅｎｔｅｒ，ＢｅｉｊｉｎｇＦｒｉｅｎｄｓｈｉｐＨｏｓｐｉｔａｌ，ＣａｐｉｔａｌＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｂｅｉｊｉｎｇ１０００５０，Ｃｈｉｎａ）Ｃｏｒｒｅｓｐｏｎｄｉｎｇａｕｔｈｏｒ：ＬＩＵＴｉａｎｈｕｉ，ｌｉｕ＿ｔｉａｎｈｕｉ＠１６３．ｃｏｍ（ＯＲＣＩＤ：００００－０００１－６７８９－３０１６）Ａｂｓｔｒａｃｔ：Ｉｎａｄｄｉｔｉｏｎｔｏｉｔｓｏｗｎｓｐｅｃｉｆｉｃｆｕｎｃｔｉｏｎｓ，ａｎｏｒｇａｎｅｌｌｅｃａｎａｌｓｏｉｎｔｅｒａｃｔｗｉｔｈｏｔｈｅｒｏｒｇａｎｅｌｌｅｓｔｏｃｏｍｐｌｅｔｅｉｍｐｏｒｔａｎｔｐｈｙｓｉｏｌｏｇｉｃａｌｆｕｎｃｔｉｏｎｓ．Ｔｈｅｄｉｓｏｒｄｅｒｓｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｓａｒｅｃｌｏｓｅｌｙａｓｓｏｃｉａｔｅｄｔｈｅｄｅｖｅｌｏｐｍｅｎｔａｎｄｐｒｏｇｒｅｓｓｉｏｎｏｆｖａｒｉｏｕｓｄｉｓｅａｓｅｓ．Ｉｎｒｅｃｅｎｔｙｅａｒｓ，ｔｈｅｒｏｌｅｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｓｈａｓａｔｔｒａｃｔｅｄｍｏｒｅａｔｔｅｎｔｉｏｎｉｎｔｈｅｐｒｏｇｒｅｓｓｉｏｎｏｆｎｏｎａｌｃｏｈｏｌｉｃｆａｔｔｙｌｉｖｅｒｄｉｓｅａｓｅ，ｅｓｐｅｃｉａｌｌｙｔｈｅｉｎｔｅｒａｃｔｉｏｎｓｂｅｔｗｅｅｎｍｉｔｏｃｈｏｎｄｒｉａ，ｌｉｐｉｄｄｒｏｐｌｅｔｓ，ａｎｄｏｔｈｅｒｏｒｇａｎｅｌｌｅｓ．Ｋｅｙｗｏｒｄｓ：Ｎｏｎ－ａｌｃｏｈｏｌｉｃＦａｔｔｙＬｉｖｅｒＤｉｓｅａｓｅ；Ｏｒｇａｎｅｌｌｅｓ；Ｍｉｔｏｃｈｏｎｄｒｉａ；ＬｉｐｉｄＤｒｏｐｌｅｔｓＲｅｓｅａｒｃｈｆｕｎｄｉｎｇ：ＮａｔｉｏｎａｌＮａｔｕｒａｌＳｃｉｅｎｃｅＦｏｕｎｄａｔｉｏｎｏｆＣｈｉｎａ（８２０７０６１８） 细胞器可以通过膜接触位点与其他细胞器相互作用，完成物质与信息的交换，形成互作网络［１］。

2014SCI CMVCP- Coat protein mutations in an attenuated Cucumber mosaic virus

2014SCI CMVCP- Coat protein mutations in an attenuated Cucumber mosaic virus

Coat protein mutations in an attenuated Cucumber mosaic virusencoding mutant2b protein that lacks RNA silencing suppressoractivity induces chlorosis with photosynthesis gene repression andchloroplast abnormalities in infected tobacco plantsTomofumi Mochizuki n,Ryota Yamazaki,Tomoya Wada,Satoshi T.OhkiGraduate School of Life and Environmental Sciences,Osaka Prefecture University,1-1,Gakuen-cho,Naka-ku,Sakai,Osaka599-8531,Japana r t i c l e i n f oArticle history:Received28February2014Returned to author for revisions23March2014Accepted6April2014Available online24April2014Keywords:Coat proteinCucumber mosaic virusChloroplastChlorosisTobaccoViral suppressor2b Proteina b s t r a c tIn tobacco plants,the Cucumber mosaic virus(CMV)pepo strain induces mosaic symptoms,includingpale green chlorosis and malformed tissues.Here,we characterized the involvement of2b protein andcoat protein(CP)in the development of mosaic symptoms.A2b mutant(R46C)that lacks viralsuppressor of RNA silencing(VSR)activity showed an asymptomatic phenotype with low levels of virusaccumulation.Tomato spotted wilt virus NSs protein did not complement the virulence of the R46C,although it did restore high-level virus accumulation.However,R46C mutants expressing mutated CP inwhich the amino acid P129was mutated to A,E,C,Q,or S induced chlorosis that was associated withreduced expression of chloroplast and photosynthesis related genes(CPRGs)and abnormal chloroplastswith fewer thylakoid membranes.These results suggest that the CP of the CMV pepo strain acquiresvirulence by amino acid mutations,which causes CPRG repression and chloroplast abnormalities.&2014Elsevier Inc.All rights reserved.IntroductionIn plant pathology studies,the molecular mechanisms thatdrive the development of symptoms during viral infection havereceived much attention.Thus,the identification and character-ization of viral virulence factors in certain host species is impor-tant.Cucumber mosaic virus(CMV)is a well-studied plant virusthat belongs to family Bromoviridae,genus Cucumovirus.CMVinfects over1200species in over100families of both monocotsand dicots and causes variable disease symptoms,includingmosaicism,stunting,chlorosis,necrosis,dwarfism,and leaf mal-formation.The range of disease symptoms develop in a hostspecies-and CMV strain-dependent manner.CMV encodes at leastfive proteins(reviewed in Palukaitis and García-Arenal(2003)):the1a and2a viral RNA replication proteins,the2b viral suppres-sor of RNA silencing(VSR;Brigneti et al.,1998),the3a movementprotein,and a coat protein(CP).Although all CMV proteins act asvirulence factors in certain host species,2b and CP are the majorvirulence factors in most host plants(reviewed in Mochizuki andOhki(2012)).The CMV pepo strain(subgroup IA)causes mosaic disease ininfected tobacco plants.Affected symptomatic tissues in mosaicleaves show pale green chlorosis with malformed tissues.A CMVpepo strain that expressed mutant2b protein showed attenuatedvirulence with low virus accumulation in tobacco plants(Kobori etal.,2004,2005;Ryang et al.,2004).In contrast with wild-type CMVpepo,theΔ2b mutant strain did not efficiently infect developingtobacco tissues(Sunpapao et al.,2009).Importantly,loss-of-functionmutations in RNA silencing components(e.g.,DCL2,DCL4,RDR6,AGO1,and AGO2)in Arabidopsis resulted in the restoration ofsystemic viral infection and virulence in theΔ2b mutant(Diaz-Pendon et al.,2007;Wang et al.,2010,2011).Thus,the2b proteinindirectly contributes to CMV virulence by facilitating increasedsystemic virus accumulation in leaves by suppressing anti-viralRNA silencing.Indeed,the2b protein was not required for CMV toinduce pathology in RNA silencing-defective Arabidopsis strains.Furthermore,constitutive transgenic expression of2b in Arabidopsiscaused disease-like phenotypes by perturbing the expression ofmiRNA-mediated plant genes(Lewsey et al.,2007;Zhang et al.,2006).The2b proteins of CMV-HL(subgroup I)and CMV-Q and-M(subgroup II)also induced necrosis in Arabidopsis by inhibitingcatalase activity by direct protein–protein interactions(Inaba et al.,2011;Masuta et al.,2012).Consequently,the2b protein can directlyelicit the development of symptoms.Because complementationexperiments using RNA silencing-deficient tobacco have not beenContents lists available at ScienceDirectjournal homepage:/locate/yviroVirology/10.1016/j.virol.2014.04.0100042-6822/&2014Elsevier Inc.All rights reserved.n Corresponding author.Tel.:þ81722548459;fax:þ81722549410.E-mail address:tomochi@plant.osakafu-u.ac.jp(T.Mochizuki).Virology456-457(2014)292–299reported,the role of 2b protein VSR activity in the development of mosaic symptoms in infected tobacco plants remains unclear.CP is another major CMV virulence factor in tobacco plants (Shintaku et al.,1992;Suzuki et al.,1995).We previously reported a potential link between the properties of the amino acid at position 129in CP and the types of symptoms caused in tobacco plants,e.g.,polar uncharged mutants (129C,129Q,and 129S)caused severe white mosaicism,whereas acidic group mutants (129D and 129E)induced pale green mosaicism (Mochizuki and Ohki,2011).These CP mutations governed the degree to which the expression of chlor-oplast and photosynthesis related genes (CPRGs)was reduced,and the magnitude of the reduction in chloroplast thylakoid membrane number.Both phenotypes were correlated with chlorosis severity in the chlorotic tissues infected with each CP mutant (Mochizuki and Ohki,2011;Mochizuki et al.,2014).However,it is not clear whether the CPRG repression and chloroplast abnormalities induced by CP mutants are dependent on 2b protein VSR activity or whether mutant CP alone can induce CPRG repression,chloroplast abnorm-alities,and chlorosis in mosaic leaves.The aim of this study was to characterize the roles of 2b protein and CP from the CMV pepo strain in the development of mosaic symptoms in tobacco plants.We showed that the VSR activity of 2b protein was required for high-level systemic viral accumulation in infected tobacco plant leaves.However,in RNA silencing-de ficient transgenic tobacco plants designed to prevent anti-viral RNA silencing and promote high-levels of viral accumulation,the 2b protein VSR activity was not suf ficient to drive the development of mosaic symptoms.In a VSR-activity-de ficient 2b mutant CMV strain,we found that CP point mutations in which residue P129was mutated to A,E,C,Q,or S induced chlorosis but not leaf malformation.Interestingly,these virulent CP mutants without 2b protein VSR-activity induced CPRG repression and caused a reduction in thylakoid membrane numbers.The roles of 2b protein and CP in the expression of mosaic symptoms in tobacco plants are also discussed.ResultsSymptoms,VSR activity,and the amounts of pepo and R46C 2b-mutant viruses in tobacco plantsWe first con firmed the effect of 2b protein mutation on virulence,VSR activity,and viral abundance.Because residue 46is involved in the siRNA-binding activity of 2b protein and affects VSR activity and CMV virulence (Goto et al.,2007),we used an attenuated virulence mutant of pepo CMV,R46C (Kobori et al.,2004,2005).Upon inoculation on GFP-silenced transgenic tobacco (Sm2;Kubota et al.,2003),pepo induced pale green mosaic symptoms in uninoculated systemic leaves,whereas R46C caused no symptoms (Fig.1A,upper panels).GFP fluorescence was recovered in the newly developed pepo-infected mosaic leaves (Fig.1A,lower panels).Notably,GFP fluorescence was observed only in symptomatic chlorotic tissues and not in the dark green islands in pepo-infected mosaic leaves.By contrast,GFP fluorescence was not detected in the R46C-and mock-inoculated Sm2tobaccos.Abundant accumulation of GFP protein and mRNA were observed in the symptomatic tissues of pepo-infected leaves;expression was similar to that of a constitutive GFP-expression line (Sm1,Kubota et al.,2003)(Fig.1B and C).By contrast,no GFP protein and lower amounts of GFP mRNA were detected in R46C-and mock-infected Sm2plants.The accumulation of GFP-derived siRNA in pepo-infected tissues was also lower than in R46C-and mock-infected tissues (Fig.1C).The amount of pepo RNA and CP was higher than the amount of R46C RNA and CP (Fig.1B and C).Furthermore,the amount of CMV RNA-derived siRNA was higher in pepo-infected tissues than in R46C-infected tissues (Fig.1C),indicating that 2b protein did not suppress the accumulation of CMV siRNA.These results con firmed that the R46C mutation disrupts virulence by impairingVSR.Fig.1.(A)Tissues with symptoms (upper panels)and recovery of GFP fluorescence (lower panels)in GFP-silenced tobacco plants (G3Sm2)inoculated with CMV-pepo or 2b protein R46C mutants.The photographs were taken at 10days post-inoculation.GFP fluorescence signals were observed using a blue LED lamp (470nm)and a wide path filter ($540nm).Note that GFP fluorescence was only recovered in the symptomatic tissues of mosaic leaves infected with pepo.Bars ¼ca.1cm.(B)The accumulation of CMV RNAs,GFP mRNA,CMV siRNA,and GFP siRNA in GFP-silenced tobacco plants (G3Sm2)inoculated with CMV-pepo or R46C.Virus accumulation was analyzed using samples from pepo-infected symptomatic tissues of mosaic leaves or from R46C-infected asymptomatic tissues.Mock-inoculated G3Sm1,which constitutively expresses erG3GFP (Kubota et al.,2003),was used as a positive control for GFP detection.GFP mRNA,GFP siRNA,CMV RNAs,and CMV siRNA were detected by Northern blot analysis with speci fic probes.(C)The accumulation of CMV CP and GFP in G3Sm2inoculated with CMV-pepo and R46C.CMP CP and GFP were detected by Western blot analysis using speci fic antibodies.T.Mochizuki et al./Virology 456-457(2014)292–299293Coat protein mutations in the R46C 2b mutant cause chlorosis induction by CMVWe previously reported that substituting CP amino acid P129with A,D,or E did not alter the pale green chlorosis caused by pepo CMV,whereas substitution with C,Q,or S induced severe white chlorosis (Fig.2A)(Mochizuki and Ohki,2011).To further elucidate whether the mutant CPs compensate for the virulence of R46C,the symptoms of tobacco plants inoculated with double mutants containing substitutions in both 2b protein and CP (R46C/CP mutant)were examined (Fig.2B).Substitution of CP residue 129in the R46C strain with D (R46C-D)resulted in an asympto-matic phenotype similar to that of R46C.By contrast,R46C-A and R46C-E (R46C containing CP with 129A or 129E)caused pale green chlorosis and R46C-C,R46C-Q,and R46C-S (R46C containing CP with 129C,129Q,or 129S)induced severe white chlorosis in uninoculated systemic leaves.Thus,R46C-A,-E,-C,-Q,and -S mutations induced chlorosis that was similar in severity to the corresponding parental CP mutants (Fig.2A vs.B).Interestingly,unlike the parental CP mutants,these R46C/CP mutants did not induce obvious leaf malformations (Fig.2A vs.B),and showed a recovery phenotype in which the chlorotic tissues gradually disappeared on newly developing leaves as the plant grew.The amounts of viral RNA and CP in symptomatic chlorotic tissues (R46C-A,-E,-C,-Q,and -S)or asymptomatic tissues (R46C and R46C-D)of plants infected with the R46C/CP mutants were compared those infected with the parental CP mutants by North-ern and Western blot analyses (Fig.3).The amounts of viralRNAFig.2.(A)The symptoms in tobacco plants infected with the CMV pepo strain and CP mutants.Photographs were taken at 7–14days post-inoculation.The pale green mosaic symptoms that developed upon pepo infection were not altered by the substitution of amino acid 129P in CP with A,D,or E,but the symptoms were altered to white mosaic after substitution with C,Q,or S.(B)The alteration of the asymptomatic phenotype caused by R46C infection through substitutions at amino acid position 129in CP.Amino acid 129P in the CP of R46C virus was substituted with A,D,E,C,Q,or S (termed R46C/CP mutants).The photographs were taken at 7–14days post-inoculation.More than five individual tobacco plants were inoculated with each R46C/CP mutant and experiments were conducted three times,with all individuals showing similar symptoms.Bars ¼ca.1cm.T.Mochizuki et al./Virology 456-457(2014)292–299294and CP for all R46C/CP mutants were less than those of the parental CP mutants.These results indicated that the substitution of amino acid P129with A,E,C,Q,or S in CP altered the chlorotic virulence of R46C,which was dose-independent.Constitutive expression of VSR in tobacco does not complement the virulence of R46CIt remained possible that the loss of virulence in R46C and R46C-D could have resulted from reduced virus accumulation because of the loss of VSR activity in R46C 2b protein.To test this possibility,representative R46C/CP mutants (R46C,R46C-D,R46C-A,and R46C-Q)were inoculated into NSs protein-expressing transgenic tobacco (line 15,Hirai et al.,2008)and symptoms and viral abundance wereexamined.The NSs protein is a strong VSR encoded by Tomato spotted wilt virus (TSWV)(Takeda et al.,2002).R46C and R46C-D did not produce any symptoms in NSs-transgenic tobacco plants (Fig.4A).The amounts of viral RNA and CP in the R46C/CP mutant-infected NSs-transgenic tobacco plants were higher than those in infected wild-type tobacco plants (Fig.4B),con firming that anti-viral RNA silencing of the R46C/CP mutants could be inhibited by NSs expression.R46C-A and -Q induced chlorotic symptoms without causing obvious leaf malforma-tions in NSs-transgenic tobacco plants,similar to infected wild-type tobacco (Fig.4A).Four independent experiments were performed and that yielded similar results.These results showed that the virulence of R46C and R46C-D in tobacco plants was not complemented,although viral abundance recovered as a result of the suppression of anti-viral RNA silencing.Expression of chloroplast and photosynthesis related genes (CPRGs)reduced in chlorotic tissues infected with R46C/CP mutantsWe previously reported that the CP mutants caused a reduction of CPRG expression and that the degree of CPRG repression was closely associated with the severity of chlorosis (Mochizuki et al.,2014).Thus,the mRNA levels of representative CPRGs from chlorotic tissues infected with R46C/CP mutants were examined (Fig.5).The mRNA levels of the antenna genes (LHCB1and LHCB6),the chlorophyll synthesis enzyme gene (CHLH ),and the rubisco small subunit gene (RBC S )were analyzed by Northern blotting.The mRNA levels of all CPRGs in the asymptomatic tissues infected with R46C and R46C-D were similar to those of mock-inoculated tissues.By contrast,the mRNA amounts of LHCB6and CHLH both in the pale green and white chlorotic tissues infected with R46C-A,-E,-C,-Q,and -S were reduced and the amounts of LHCB1and RBC S mRNAs were lower in the white chlorotic tissues infected with R46C-C,-Q,and -S.These results indicated that the virulent R46C/CP mutants induced CPRG repression independent of 2b protein VSR activity.Morphological analysis of tissues and chloroplasts in tobacco infected with R46C/CP mutantsBecause the R46C/CP mutants did not induce obvious leaf malformations (Fig.2B),morphological changes in the chlorotic tissues were compared between the R46C/CP mutants and the corresponding parental CP mutants.Wild-type tobacco plants were inoculated with representative CMV strains (pepo,R46C,129D,R46C-D,129A,R46C-A,129Q,or R46C-Q)and then at least six tissue samples per experimental group were embedded in Spurr resin and semi-thin sections (3m m)were analyzed using light microscopy.The experiment was repeated twice.AmarkedFig.3.The accumulation of CMV RNAs and CP of the R46C/CP mutants and the corresponding parental CP mutants (wt 2b protein)in the systemic leaves of tobacco plants.CMV RNAs and CP were detected using Northern and Western blot analyses,respectively.Fig.4.(A)Symptoms of Tomato spotted wilt virus NSs protein-expressing transgenic tobacco (NSs)inoculated with the representative R46C/CP mutants (R46C,R46C-D,R46C-A,and R46C-Q).The photographs were taken at 13days post-inoculation.Bars ¼ca.1cm.(B)Accumulation of CMV RNAs and CP of R46C/CP mutants in NSs or wild-type tobacco plants and the corresponding parental CP mutants (wt 2b protein)in wild-type tobacco plants.The accumulation was analyzed using samples from pepo-,129D-,129A-,129Q-,R46C-A-,or R46C-Q-infected symptomatic tissues of mosaic leaves and from R46C-or R46C-D-infected asymptomatic tissues.CMV RNAs and CP were detected using Northern and Western blot analyses,respectively.Fig.5.Northern blot analysis of chloroplast-and photosynthesis-related genes in total RNA extracted from white chlorotic (R46C-C,-Q,and -S),pale green chlorotic (R46C-A and -E),and asymptomatic (mock infected,R46C,and R46C-D)tissues of tobacco leaves infected with R46C/CP mutants.The genes that encode the antenna proteins (LHCB1and LHCB6),the chlorophyll synthesis enzyme (CHLH ),and the small subunit of rubisco (RBC S )were analyzed by Northern blot using DIG-labeled gene-speci fic RNA probes.Three samples from four individual plants per R46C/CP mutant were investigated.T.Mochizuki et al./Virology 456-457(2014)292–299295difference was found in the palisade tissues;abnormal palisade tissues were observed on the adaxial side of the chlorotic tissues infected with pepo,129D,129A,and 129Q (Fig.6A,upper panels).By contrast,palisade tissues with normal structures,which were similar to the palisade tissues of R46C-,R46C-D-,and mock-inoculated asymptomatic leaf tissues,were observed on the adaxial side of chlorotic tissues from R46C-A and R46C-Q-infected tobacco plants (Fig.6A,lower panels).These findings indicated that the abnormal development of palisade tissues was associated with leaf malformation in CP mutant-infected tobacco plants.Additionally,wild-type 2b protein was required to induce the abnormal development of palisade tissues.Abnormal chloroplasts with fewer thylakoid membranes and granal stacks were observed in the chlorotic tissues infected with the CP mutants (Mochizuki and Ohki,2011).To verify whether abnormal chloroplasts also existed in the chlorotic tissues infected with R46C-A and R46C-Q,the ultra-structures of the chloroplasts in cells from R46C-,R46C-D-,R46C-A-,and R46C-Q-infected tobacco plants were observed.Ultra-thin sections (silver to gold)were prepared from at least three Spurr resin-embedded samples that were used first for light microscopy,and then for electron microscopy analyses.Representative images of chloroplasts are shown in Fig.6B.Predictably,many abnormal chloroplasts with fewer thylakoid mem-branes could be observed in the cells of chlorotic tissues infected with R46C-A and R46C-Q,although a few normal chloroplasts could also be observed in those tissues.By contrast,only normal chlor-oplasts were found in the cells of asymptomatic tissues from R46C-,R46C-D-,and mock-inoculated tobacco plants.DiscussionWe investigated the contribution of the 2b protein and CP CMV virulence factors to the development of mosaic symptoms in tobacco plants using 2b protein and CP double-mutant viruses.We showed that substituting the amino acid P129in CP with A,E,C,Q,or S altered the virulence of an attenuated VSR-de ficient 2bmutant (R46C).Virulent R46C/CP mutants induced CPRG repres-sion and abnormal chloroplasts with fewer thylakoid membranes,but did not induce leaf malformations or palisade abnormalities.These results indicated that the virulent mutant CPs caused chlorosis via CPRG repression,but that wild-type 2b protein was essential for inducing leaf malformation.For Arabidopsis mutants such as dcl2,dcl4,rdr6,ago1,and ago2,the virulence and viral titer of Δ2b-mutant CMV can be restored by the disruption of anti-viral RNA silencing pathways (Diaz-Pendon et al.,2007;Wang et al.,2010,2011).In addition,the 2b protein directly contributes to CMV virulence (Du et al.,2014;Lewsey et al.,2007,2009;González et al.,2010;Zhang et al.,2006).The binding and sequestration of siRNA of the 2b protein is critical to its VSR function (Duan et al.,2012;González et al.,2012).However,Lewsey et al.(2009)showed that the mutation of two putative phosphorylation sites,residues 40and 42in the 2b protein,resulted in mild symptoms in infected tobacco plants.These mutations did not affect the siRNA binding ability of the 2b protein,indicating that 2b protein VSR function alone is insuf ficient to induce symptoms.Similarly,in this present study,we found that the constitutive expression of NSs,a strong VSR encoded by TSWV (Takeda et al.,2002),could not complement the virulence of a VSR-activity-de ficient R46C 2b protein mutant in transgenic tobacco plants,even though the amounts of R46C and R46C-D in the asymptomatic tissues of NSs tobacco plants were restored (Fig.4).This finding conclusively indicated that the VSR activity of 2b protein could promote viral accumulation by suppressing anti-viral RNA silencing in tobacco plants,but this activity alone was insuf ficient to induce mosaic symptoms.Thus,the results of our complementation experiments using RNA silencing-de ficient tobacco supports the direct contribu-tion of the 2b protein to the development of mosaic symptoms in CMV-infected tobacco plants.We showed that the 2b protein induced developmental defects and palisade malformation in CMV infected-tobacco leaves (Fig.6A).It has been suggested that the 2b protein perturbs the small RNA-mediated host gene expression system,leading to palisade malformation and leaf developmental defects intobaccoFig.6.(A)Morphological observations of leaf tissues from tobacco plants infected with R46C/CP mutants (R46C,R46C-D,R46C-A,and R46C-Q)and the corresponding parental CP mutants [wt pepo (129P),129D,129A,and 129Q].Spurr resin-embedded transverse sections were obtained from wt pepo (129P)-,129D-,129A-,129Q-,R46C-A-,or R46C-Q-infected symptomatic tissues of mosaic leaves and from R46C-,R46C-D-,or mock-infected asymptomatic tissues at 14days post-inoculation.Note that distinct palisade tissues could be observed in the samples from R46C/CP mutants,but not in the samples from parental CP mutants.Bars ¼100m m.(B)Representative electron micrographs of chloroplasts in tobacco cells infected with R46C/CP mutants (R46C,R46C-D,R46C-A,and R46C-Q).Note that fewer thylakoid membranes could only be observed in the cells of chlorotic tissues infected with R46C-A and R46C-Q.Scale bars ¼5m m.T.Mochizuki et al./Virology 456-457(2014)292–299296plants.The2b protein perturbs the miRNA and trans-acting small interfering RNA(tasiRNA)pathways by directly binding and thereby inhibiting AGO1(Feng et al.,2013;Zhang et al.,2006). The2b protein also affects host gene expression through the inhibition of RNA-directed DNA methylation by directly binding to AGO4and AGO4-related siRNA(Hamera et al.,2012).Lewsey et al.(2007)reported that the expression of the2b protein alone, cloned from the severe Fny strain in Arabidopsis,induced abnormal development and altered the mRNA stability of the miRNA-regulated auxin-responsive transcription factors ARF10,ARF17, and NAC1,but not ARF8,SCL6-IV,and SPL3.Du et al.(2014)also demonstrated that the Fny-2b protein prevented the function of miR159,which regulated the expression of MYB33and MYB65,and thereby contributed to the developmental defects in Arabidopsis infected with Fny-CMV.Therefore,it will also be necessary to investigate the contributions of miRNA-regulated transcriptional factors(MYB,ARF,and others)to the development of mosaic symptoms in tobacco plants infected with CMV.Some of the double-mutants(R46C-A,R46C-E,R46C-C,R46C-Q, and R46C-S)that expressed R46C2b protein and mutated CP could induce chlorosis in uninoculated systemic leaves(Fig.2).These results indicated that CPs were virulent for chlorosis induction if they encoded an amino acid at position129of A,E,C,Q,or S,but not P or D.We previously showed that the chlorosis-inducing CP mutants repressed CPRG expression(Mochizuki et al.,2014)and decreased thylakoid membrane numbers(Mochizuki and Ohki,2011).Such CPRG repression was observed in chlorotic tissues infected with virulent R46C/CP mutants,and fewer thylakoid membranes were also observed in chlorotic tissues infected with R46C-A and-Q.By contrast,CPRG repression and fewer thylakoid membranes were not observed in R46C and R46C-D infected asymptomatic tissues (Figs.5and6).These results suggested that the mutated virulent CPs induced chlorosis by repressing CPRGs and reducing thylakoid membrane numbers independently of the2b protein VSR activity. The amounts of viral RNA and CP from R46C-A,-E,-C,-Q,and-S were lower than those from the parental CP mutants(Fig.3),indicating that the repression of CPRG and the reduction in thylakoid mem-brane numbers by the virulent R46C/CP mutants were not dose-dependent.We speculate that small amounts of virulent R46C/CP mutants could induce stress responses that alter CPRG expression,by a molecular mechanism that is affected by the properties of amino acid residue129.One possibility is that the mutant CP molecules indirectly repressed CPRGs expression by affecting chloroplast-to-nucleus retrograde signals.When chloroplasts are exposed to oxida-tive stress,such as ROS,the expression of nuclear-encoded CPRGs, including the antenna protein genes(LHCB1and LHCB6)and chlor-ophyll synthesis enzyme gene(CHLH),are reduced as a result of chloroplast-to-nucleus retrograde signals to avoid excess ROS gen-eration(Barajas-Lopez et al.,2013).Indeed,abundant H2O2accumu-lated in chloroplasts in cells of CMV-infected tomatoes and cucumbers(Song et al.,2009).Thus,the expression level of nuclear-encoded CPRGs might be suppressed by retrograde signaling between the chloroplast and nucleus to avoid the oxidative damage induced by the mutated CP molecule.It remains unclear where the mutated CP molecules induce stress response.CMV virions and CP molecules are thought to exist in the cytoplasm,although the presence of CMV CP in chloroplasts has also been described(Liang et al.,1998).Additionally,the possibility remains that the CP amino acid mutations alter the subcellular localization of CP.Thus,it is not clear whether mutated CPs directly damages the chloroplasts.Future studies that observe the subcellular localization of mutant CPs will be necessary.Importantly,reduced levels of CPRG expression and thylakoid membrane numbers were also observed in the pale green chlorotic tissues infected with either wild-type pepo or the129D CP mutant (Mochizuki and Ohki,2011;Mochizuki et al.,2014),but not in tissues infected with the R46C or R46C-D mutants(this study,Figs.5and6). Thesefindings suggested that in addition to its role in the induction of morphological changes,wild-type2b protein was also involved in CPRG repression and the reduction in thylakoid membrane number in pepo-and129D-infected tissues.Although it has not been investigated,with regard to the effect of2b protein on chlorosis, there are at least two possible mechanisms for2b protein-mediated induction of chlorosis.One possibility is that the chlorosis induction is a side effect of the morphological changes in leaf tissues.The2b protein most likely induced palisade malformation and leaf devel-opmental defects(Fig.6A)by perturbing small RNA-mediated host gene expression,leading to stress-induced CPRG repression,thyla-koid membrane loss,and pale green mosaicism.Another possibility is that CPRG expression is regulated by small RNAs(miRNAs or tasiRNAs)and that the2b protein VSR activity perturbed these regulatory mechanisms.Furthermore,the possibility remains that wild-type2b protein and other CMV proteins synergistically induce CPRG repression and reduce the number of thylakoid membranes during CMV infection.In this study,we suggest that CMV CP alone can induce the repression of CPRGs,leading to chlorosis in CMV-infected tobacco plants.However,it has not been demonstrated whether the expression of CMV CP alone is sufficient to produce symptoms that resemble chlorotic phenotypes in transgenic plants.Recently, Wang et al.(2013)showed that the expression of CMV CP molecules in Cyanobacteria affected photosynthesis metabolism and resulted in a chlorosis-like phenotype.A detailed molecular analysis of CP-expressing transgenic tobacco or transient CP-expression systems will be needed to better understand the chlorosis-inducing mechanisms of CMV CP in tobacco plants. Materials and methodsVirus,plants,and inoculationThe pepo strain of CMV(subgroup IA){GenBank accession nos. AB124834(RNA1),AB124835(RNA2),and AF103991(RNA3)} was used in this study.R46C is a modified pepo strain that contains an R-to-C substitution at residue46in the pepo2b protein(Kobori et al.,2004,2005).Coat protein(CP)mutants contain a modified CP in which the P at residue129is substituted with A,D,E,C,Q,or S(Mochizuki and Ohki,2011).Wild-type(Nicotiana tabacum cv.Samsun),erG3GFP-silenced (Sm2,Kubota et al.,2003),and Tomato spotted wilt virus NSs protein-expressing tobacco lines(line15,Hirai et al.,2008)were used as experimental plants.The largest leaf offive-leaf-stage tobacco plants was mechanically inoculated with purified virus (100μg/ml),and the inoculated plants were grown in a growth chamber(CLE-303,TOMY Seiko,Tokyo,Japan)under a16h light(251C)/8h dark(221C)cycle.Transcription and inoculation of infectious viral RNA from clones Infectious viral RNAs were synthesized using the T7RiboMAX large-scale RNA production system(Promega,Madison,WI),and tobacco plants were mechanically inoculated with the transcribed RNAs.For the R46C/CP mutants,RNA1and R46C-RNA2transcripts were mixed with each mutated RNA3transcript,containing a substitution at amino acid129in CP,and inoculated.Purified viruses were obtained from the inoculated leaves.GFPfluorescence observationFor the detection of GFPfluorescence in inoculated Sm2 tobacco leaves,plants were irradiated with a blue LED lampT.Mochizuki et al./Virology456-457(2014)292–299297。

RNA互作网络结构和功能解析

RNA互作网络结构和功能解析

RNA互作网络结构和功能解析随着生物学研究的不断深入，我们对于非编码RNA（Non-coding RNA，ncRNA）的重要性和功能逐渐加深认识。

作为其中的一种重要类型，RNA互作网络在细胞内起着关键的调控作用。

RNA互作网络是指RNA与其他分子（如蛋白质、DNA等）之间通过相互作用形成的复杂网络结构。

这些互作事件可以在转录后调控、转录调控、转录双靶等生物学过程中发挥重要的功能。

RNA互作网络的结构可以分为直接和间接相互作用两种形式。

直接相互作用是指RNA分子之间的物理接触，如RNA双链结构的形成、RNA与RNA之间的碱基对配对等。

间接相互作用则是指RNA与其他分子之间通过蛋白质介导的作用而实现的互作。

这两种形式的相互作用是RNA互作网络的基础。

在RNA互作网络中，RNA分子之间的相互作用可以通过多种机制实现。

其中包括互补配对、蛋白质介导的相互作用、RNA结构调控等。

互补配对是指两条RNA链之间的碱基互补配对，形成一个稳定的RNA双链结构。

这种互补配对产生的相互作用可以通过稳定RNA结构、促进RNA折叠等方式影响RNA的功能。

蛋白质介导的RNA相互作用是RNA互作网络中另一个重要的机制。

在细胞中，有很多蛋白质与RNA之间发生相互作用，调控RNA的稳定性、定位、转运等。

一些蛋白质可以结合到RNA的特定序列上，形成蛋白-核酸复合物，进而调控RNA的功能。

这种蛋白质介导的相互作用在维持细胞的稳态和正常功能方面起着重要的作用。

除了直接和间接的相互作用外，RNA互作网络中的功能也是非常复杂和多样的。

RNA互作网络可以通过调控转录后修饰、RNA的定位、转运、翻译调控等多种方式影响细胞的发育、分化和功能。

例如，一些具有miRNA活性的非编码RNA可以通过与mRNA结合形成RNA-蛋白质复合物，进而调控靶基因的翻译。

另外，一些长链非编码RNA还可以作为“桥梁”分子，将其他分子组装在一起，形成复杂的功能单元。

通过这些功能调控，RNA互作网络可以调节细胞的基因表达、细胞周期、细胞凋亡等多种生物学过程。