the subcellular localization and functional analysis of ... · short sequences: onesequence formsan...

Research ArticleThe Subcellular Localization and Functional Analysis ofFibrillarin2 a Nucleolar Protein in Nicotiana benthamiana

Luping Zheng12 Jinai Yao3 Fangluan Gao12 Lin Chen2 Chao Zhang2

Lingli Lian2 Liyan Xie2 Zujian Wu2 and Lianhui Xie2

1Key Laboratory of Biopesticide and Chemical Biology Ministry of Education Fujian Agriculture and Forestry UniversityFuzhou 350002 China2Key Laboratory of Plant Virology of Fujian Province Fujian Agriculture and Forestry University Fuzhou 350002 China3Institute of Plant Protection Fujian Provincial Academy of Agricultural Sciences Fuzhou 350002 China

Correspondence should be addressed to Lianhui Xie xielhfafucom

Received 14 October 2015 Accepted 1 December 2015

Academic Editor Jialiang Yang

Copyright copy 2016 Luping Zheng et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Nucleolar proteins play important roles in plant cytology growth and development Fibrillarin2 is a nucleolar protein ofNicotianabenthamiana (N benthamiana) Its cDNA was amplified by RT-PCR and inserted into expression vector pEarley101 labeled withyellow fluorescent protein (YFP) The fusion protein was localized in the nucleolus and Cajal body of leaf epidermal cells ofN benthamiana The N benthamiana fibrillarin2 (NbFib2) protein has three functional domains (ie glycine and arginine richdomain RNA-binding domain and 120572-helical domain) and a nuclear localization signal (NLS) in C-terminal The protein 3Dstructure analysis predicted that NbFib2 is an 120572120573 protein In addition the virus induced gene silencing (VIGS) approach wasused to determine the function of NbFib2 Our results showed that symptoms including growth retardation organ deformationchlorosis and necrosis appeared in NbFib2-silenced N benthamiana

1 Introduction

Fibrillarin is a major nucleolar protein playing multifunc-tional roles in RNA biogenesis It localizes in nucleolus andCajal bodies (CBs) subnuclear dynamic particles involvedin RNA transcription and editing [1] Fibrillarin is an evolu-tionarily conserved protein Human fibrillarin shares 942sequence identity with mouse fibrillarin and 829 sequenceidentity with amphibian fibrillarin [2] Homologs of humanfibrillarin are also reported in advanced plants [3]

Fibrillarin usually consists of three domains including aglycine and arginine rich domain (GAR domain) an RNA-binding domain and an 120572-helical domain The GAR domainis critical for the function of fibrillarin This domain islocalized in the N-terminal of the protein and its arginineresidues are methylated [4] In human and plants theGAR domain is involved in the translocation of fibrillarininto nucleoli The RNA-binding domain which interactswith RNA [5] and 120572-helical domain which interacts with

nucleolar protein 56 (Nop56) [6] is localized in the mid-dle and C-terminal of fibrillarin respectively The RNA-binding domain and 120572-helical domain together form anAdo-Met-dependent methyltransferase- (MTase-) like regionTheMTase-like region which is evolutionarily conserved con-tains S-adenosylmethionine (SAM the methionine groupdonor) binding motifs and encodes a MTase required for themethylation of 21015840-O-ribose [5]

In addition to understanding its functions in pre-rRNA processing modification and ribosomal assembly [7]researches in fibrillarin are focused on its interactions withvirus-encoded-proteins and the roles of these interactionsin viral movement and infection For example Melen et alfound that fibrillarin interacted with nonstructural protein1 (NS1) in Influenza A H3N2 subtype virus via C-terminalnuclear localization signal 2 (NLS2) [8] Kim et al foundthat fibrillarin is involved in the long distance movementand infection of Groundnut rosette virus (GRV) [9 10]Specifically ORF3 (movement protein) in GRV migrates

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 2831287 9 pageshttpdxdoiorg10115520162831287

2 BioMed Research International

into nucleolus via CBs binds with fibrillarin in nucleolusrelocates into cytoplasm and finally assembles with virusribonucleoprotein (vRNP) particles in cytoplasm for long-distance movement and systemic infection It was alsoreported that fibrillarin interacts with viral genome-linkedprotein (VPg) in Potato virus A (PVA) and 2b silencingsuppressor protein in Cucumber mosaic virus (CMV) [11 12]

It is known that genes in some plants can be cosuppressedif the plants are transformed with homologous transgenesThis mechanism will result in new types of intercellularcommunication and viral defensemechanisms [13]The eventin which virus vectors carrying the host-derived sequencesilence the corresponding host genes in the infected plantsis defined as virus-induced gene silencing (VIGS) [14] VIGSis themanifestation of an RNA-mediated defensemechanismand is believed to be a fast and powerfulmethod to determinegene function In 2001 a novel VIGS vector TRV wasmodified from the RNA virusTobacco rattle virusThis vectorsuccessfully silenced endogenous genes such as phytoenedesaturase (PDS) andNicotiana FLOLEY (NFL) inNicotianabenthamiana (N benthamiana) [14] Since then TRV hasbecome themost commonly used vector inVIGS studiesThevector used in this study is a modified TRV [15] containingduplicated CaM V 35S promoters (2 times 35S) and nopalinesynthase terminator (NOSt) in the C-terminal which canensure the accumulation of viral RNAs to a higher level Thenew TRV has two genomes designated as pTRV1 and pTRV2(pYL156)The latter genome acts as a VIGS vector containingmultiple cloning sites

Fibrillarin2 is a protein in Fibrillarin family In our previ-ous study we identified the importance of fibrillarin2 fromN benthamiana (NbFib2) during the process of Rice stripevirus infection [16] However the subcellular localizationthe 3D structure and functions of NbFib2 were not fullyresolved The objectives of this study include (1) determiningthe subcellular localization of fibrillarin2 in N benthami-ana (NbFib2) (2) predicting the functional domains and 3dimensional (3D) structure ofNbFib2 and (3) identifying theroles of NbFib2 in plant growth and development

2 Results

21 The Functional Domains of NbFib2 NbFib2 is highlyhomologous to AtFib2 (GenBank accession AAG10153) andHsaFib (GenBank accession AAH19260) They share morethan 74 amino acid sequence identity (Figure 1(a)) Theprotein has 314 amino acid (aa) residues and consists of threefunctional regions including a GAR region an RNA bindingregion and an 120572-helical region (Figure 1(b))TheGAR regionhas 61 aas (aa8ndash68) and is located in the N-terminal of theproteinTheRNAbinding region is seated in themiddle of theprotein (aa131ndash221) in which the most possible interactionsites with RNA are from aa176 to aa183 The 120572-helical regionis located near to the C-terminal (aa231ndash279) followed by anuclear localization signal (NLS aa307ndash313) motif It is worthmentioning that proline encoded by aa131 is predicted tobe involved in most protein activities of NbFib2 The RNAbinding region 120572-helical region and NLS together form aMTase domain implying that NbFib2 will localize in nucleus

In addition the web server I-TASSER [17] was adoptedto predict the 3D structure of NbFib2 using PDB ID 1g8sA(Pyrococcus horikoshii fibrillarin) as template (Figure 1(c))The analysis predicted that NbFib2 belongs to 120572120573 proteins aclass of structural domains in which the secondary structureis composed of alternating 120572-helices and 120573-sheets along thebackbone The 120573-sheet is an external structure while mostpart of 120572-helices is internal The GAR region mostly consistsof hairpin-120573 motif The RNA binding region forms a 120573-120572-120573motif and is randomly coiled (Figure 1(c)) The C-terminalregion is 120572-helical and the subcellular localization signalpeptide is exposed outside

Furthermore the quality and reliability of protein struc-ture predictionwere evaluated by several assessmentmethodsincluding the C-score TM-score and root-mean-squaredeviation (RMSD) A high C-score indicates a high confi-dence in prediction and vice versa and a protein structureprediction is considered as reliable if its C-score is in the rangebetween minus5 and 2 Similarly the TM-score and RMSD areoften used to measure the accuracy of a structure modelingif a reference structure is known For NbFib2 the C-scoreof the predicted structure is minus234 and the estimated TM-score and RMSD are 044plusmn014 and 118plusmn45 A respectivelyThese results suggest that the predicted structure of NbFib2 isreliable and closely matched to the topology of the referenceprotein (eg PDB ID 1g8sA)

We then used the COFACTOR server [18] to predict thefunctions of NbFib2 including the Enzyme Classification(EC) number Gene Ontology (GO) and protein-ligandbinding sites using the protein structure (PDB ID 2ipxA)as template The analysis found that NbFib2 and humanfibrillarin share high similarity in ligand-binding sites with anEC value of 0437 which is in the range of reliable scores forthe EC prediction Thus the NbFib2 protein might also havea similar active site at residue 131 like human fibrillarin Thefunction prediction also revealed that NbFib2 has a NLS andseveral regions targeting nucleolus andCajal body suggestingthat the protein may localize in the two organelles Thesepredictions were confirmed by DAPI staining

22 Subcellular Localization of NbFib2 and Western BlotAnalysis NbFib2 was found in the nucleolus and Cajal bodyof N benthamiana epidermis cells by DAPI fluorescencestaining (Figure 2)Western blot analysis was used to confirmthe protein expression In the leaves inoculated withAgrobac-terium carrying 35S-GFP a protein with a molecular weightidentical to GFP (30 kD) was detected while in the leavesinoculated with Agrobacterium carrying pEarley101-NbFib2a protein with a molecular weight identical to NbFib2-YFPfusion protein (75 kD) was detected The two proteins werenot detected in the negative control leaves (Figure 3)

23 Verifying the Function of NbFib2 Using VIGS Tendays after infiltrating plants with different genes silencedstarted to develop different phenotypes At about three weekspostinoculation (dpi) the obvious and typical symptomsappeared The leaves in the NbPDS-silenced plants (positive

BioMed Research International 3

TT TTT TTT TT

P GGGF GGRG G GGR G GGF GGRG GG RGGG G GG G G GRG G A R K MV TRGRG R DG G G R G G SAMK R GGGR G RG GRG G F G P GGGF GGRG G GGR G GGF GGRG GG RGGG G GG G G GRG G R S K M PLTGS S RG Y D S G R FSDR R RGRGPPRG ARG PAGRG M G

111 P GGGF GGRG G GGR G GGF GGRG GG RGGG G GG G G GRG G K R Q M GFSPR GF D G R G R GFRGRG G GGGG G RGGGGFHS GNR RGGKR N S

NbFib2AtFib2HsaFibConsensus gt 50


PGGGFGGRGGGGRGGGFGGRGGGRGGGGGGGGGRGG

TT TT TT

G V VEPHRH GVFI GKEDAL TKNLVPGE VY EKR S D K EYR WNPFRSKLAAA LGGVD I IKPGAKVLY K A I VQN V V V NK V G A C N E GT D WG V VEPHRH GVFI GKEDAL TKNLVPGE VY EKR S D K EYR WNPFRSKLAAA LGGVD I IKPGAKVLY K A I VQN T V I SK I A A V N E GT N WG V VEPHRH GVFI GKEDAL TKNLVPGE VY EKR S D K EYR WNPFRSKLAAA LGGVD I IKPGAKVLY R S V ISE I A I KN M E C V G G D Q HGVVEPHRHGVFIGKEDALTKNLVPGEVYEKRSDKEYRWNPFRSKLAAALGGVDIIKPGAKVLY

157 LGAASGTTVSHVSD VGP G VYAVEFSHRSGRDL AKKRT IP I DARHP KY ML MVDVIF DVAQPDQ RI ALN L V M V I V S A L G V T D G A G GLGAASGTTVSHVSD VGP G VYAVEFSHRSGRDL AKKRT IP I DARHP KY ML MVDVIF DVAQPDQ RI ALN L V M V I V S A L E C N N E A R G

166 LGAASGTTVSHVSD VGP G VYAVEFSHRSGRDL AKKRT IP I DARHP KY ML MVDVIF DVAQPDQ RI ALN I I L I V I A T V D L N N E H R ALGAASGTTVSHVSDVGPGVYAVEFSHRSGRDL$AKKRTIPIDARHPKYMLMVDVIFDVAQPDQRIALN

TT TT

241 A FL GGHFV SIKANCIDST AEAVF EVKK Q E KP EQ TLEP ERDHA VVG YR P K K KA V AQ L A Q V F A SY M P F M C A A K Q AAA FL GGHFV SIKANCIDST AEAVF EVKK Q E KP EQ TLEP ERDHA VVG YR P K K KS V QT L Q Q V F A SY I P F A C G M K P ATAA

250 A FL GGHFV SIKANCIDST AEAVF EVKK Q E KP EQ TLEP ERDHA VVG YR P K K RN A AS M Q N L Y RN A AS M Q N L Y N HT I S M Q V V P HT I S M Q V V P P V AFLGGHFVSIKANCIDSTAEAVFEVKK$QEKPEQTLEPERDHAVVGYRPKK

1205721

1205731 1205732 1205733 1205734 1205722 1205735

1205723 1205736 1205724 1205781 1205737 1205725 1205738 1205726

1205739 1205727 1205728 12057310 12057311



TTT

737783

(a)

GAR

GAR-region ldquoR regionrdquo ldquo120572-rich regionrdquo

131Phe RBS NLS

[176ndash183]GVVYAVEF

N-terminal domain MTase domain

120572

(b) (c)

Figure 1 The structural and functional characteristics of NbFib2 (a) Amino acid sequence alignment among NbFib2 AtFib2 and HsaFib(b) a sketch of functional domains in NbFib2 (c) 3Dmodel of NbFib2 in which the 120572-helical is in red the 120573-sheet region is in yellow randomcoil region is in green and NLS is in cyan

control Figure 4(e)) became bleach whereas the NbFib2-silenced plants developed curved interior leaves (Figures 4(a)4(b) and 4(d)) chlorotic full leaves (Figures 4(b) 4(c) and4(d)) and short stems and internodes and became stunted(Figure 4(d)) However the plants infiltrated by empty TRVvector (negative control Figure 4(e)) did not develop anysymptom

24 Semiquantitative RT-PCR Analysis of NbFib2 mRNAAccumulation The accumulation of NbFib2 transcripts inthe silenced plants is significantly lower than that in the plantsinoculated by empty TRV vector A negative correlationwas shown between transcript level of target genes and theseverity of disease symptom in other words silenced plantswith lower transcript in target genes usually showed moresevere symptoms (Figure 5)

3 Discussion

NbFib2 shares high homology (more than 74 aa sequence)with AtFib2 and HsaFib they also contain three functional

domains a GAR domain an RNA binding domain andan 120572-helix domain (Figure 1(a)) This result confirmed thatNbFib2 belong to fibrillarin protein family a highly con-served protein family Previous literatures suggested thatthe GAR domain always contains a nucleolar localizationsignal and a targeting site of fibrillarin in cells [19 20] Inaddition hordeiviral movement protein encoded by the firstgene of the triple-gene block (TGBp1) in Poa semilatent virus(PLSV) interacts with AtFib which occurs between the GARdomain of AtFib and the N-terminal of TGBp1 [21] ThusGAR region might be a potential domain for NbFib2 tobind to virus-encoded proteins The RNA-binding domainis essential for the presence of fibrillarin in nucleoli [5] TheC-terminal region of Fib protein family always contains twoshort sequences one sequence forms an 120572-helix structurewhich targets fibrillarin to CBs [1] and interacts directly withNop56 [6] the other is NLS The feature of those domainsreveals that NbFib2 should localize in cell nucleus whichis consistent with the results of subcellular localization ofNbFib2 The RNA-binding region and C-terminal regioncan constitute a conserved methyltransferase- (MTase-) like


(a) (b)

(c) (d)

(e) (f)

Figure 2 Subcellular localization of pEarley101-NbFib2 (a) pEarley101-NbFib2 (b) bright field (c) overlay of (a) and (b) (d) DAPIfluorescence staining (e) pEarley101-NbFib2 (f) overlay of (d) and (e)


72

65

34

26

(kD)

1 2 3M

Figure 3 Western blot analysis of proteins expression in Nicotianabenthamiana leavesM proteinMarker 1 pEarley101-NbFib2 2 CK(healthy plant) 3 35S-GFP

domain responsible for the methyltransferase activity offibrillarin [22]

3D structure is very important in determining the func-tions and localizations of proteins and their interaction withother molecules Homology modeling is one of the mostpopular approaches to predict the 3D structure of proteinsHomology modeling requires the identification of a templatesequence that matches best the query sequenceThe templatecould be identified using homology search programs suchas PSI BLAST against a PDB database using consecutive orspaced seed techniques [23 24]

The COFACTOR server [18] provides a variety of anno-tations for functional prediction of proteins One of the mostimportant advantages of this algorithm is the combinationof the global and local structural comparisons In additionsince COFACTOR takes into account for the global structuresimilarity it is more robust than those methods relying ononly local pocket comparisons

VIGS has emerged as a powerful method to study genefunctions In this study we used VIGS to identify thefunction of NbFib2 in the growth and development of Nbenthamiana We found that NbFib2-silenced N benthami-ana plants developed growth retardation organ deformationand necrosis The silenced plants can be divided into twogroups based on the reduction of NbFib2 expression Theplants in the first group developed curly chlorotic anddeformed interior and upper leaves while those in the secondgroup developed deformed upper leaves developed shortenedstems and internodes and became stunted No symptomswere developed on the negative control (TRV-empty) Webelieve that the symptoms of NbFib2 infiltrated plants arecaused by the inserted sequences carried by TRV vectorrather than TRV vector itself The RT-PCR result confirmsthat the difference in symptom development is consistentwith the level of NbFib2 silenced suggesting that NbFib2contributes to the growth and development of plants in Nbenthamiana Fibrillarin is a catalytic component of box CD

small nucleolar ribonucleoproteins (snoRNPs) [25] whichis an essential type of proteins for plant growth [26 27]fibrillarin also plays important roles on biogenesis of differentRNAs and ribosomal subunits those show that NbFib2 isinvolved in plant growth and development

An important feature of plant viral proteins is to interactwith fibrillarin and this feature is not restricted to one ortwo taxonomic groups [28] Plant viruses are able to recruitfibrillarin to facilitate their infections in various stages of hostdevelopment which suppresses host defense responses SinceN benthamiana is a model plant and the host of many plantviruses the study of the functions genetics and 3D structureof NbFib2 in N benthamiana is important to understand theinteraction between hosts and viruses

4 Materials and Methods

41 Plant Growth Conditions The N benthamiana plantswere grown and maintained in a greenhouse at 25∘C

42 Plasmid Construction Total RNA was extracted fromN benthamiana leaves using EasyPure Plant RNA Kitmanufactured by Beijing Transgen Biotech Co Ltd (Bei-jing China) Reverse transcription was carried out usingFastQuant RT Kit with gDNase (Tiangen biotech Co LtdBeijing China) cDNA encoding NbFib2 were amplified byPCR using primers (Table 1) designed from N benthamianasequences (GenBank accession AM269909) downloadedfrom GenBank pEarley101-NbFib2 construct was generatedby first inserting NbFib2 cDNA into entry vector pDONR221and then inserting it into destination vector pEarley101 usingGateway recombination system [29] The constructs wereconfirmed by capillary sequencing conducted by TakaraBiotechnology Co Ltd (Dalian China)The PCR product ofNbFib2 was digested with EcoRI and BamHI and ligated intoTRV vector by pYL156 digested with the same enzymes

43 Functional Domain and 3D Structure Prediction ofNbFib2 Fibrillarin sequences from three species werealigned using the MAFFT program (httpmafftcbrcjpalignmentsoftware) Subcellular localization of NbFib2was predicted by web-based program WolfPsort (httpwolfpsortorg) Homology identification was performed bysubmitting NbFib2 sequences into I-TASSR server [17] andthe 3D structure of NbFib2 was constructed using Pyrococcushorikoshii fibrillarin as reference on the same server TheNbFib2 function was predicted using the COFACTOERserver [18]

44 Agrobacterium-Mediated Transient Expression Agrobac-terium tumefaciens (A tumefaciens) strain EHA105 carry-ing pEarley101-NbFib2 and strain GV3101 carrying pTRV1pYL156-NbPDS and pYL156-NbFib2 were grown separatelyto OD

600= 08 at 28∘C on Luria-Bertani liquid medium

supplemented with 50120583g120583L of rifampicin and 50 120583g120583Lof kanamycin They were then transferred to inductionmedia (10mM MES pH 56 10mM MgCl

2 and 150120583M


(a) (b)

(c) (d)

(e) (f)

Figure 4 Symptoms of NbFib2 silenced Nicotiana benthamiana (a)ndash(d) NbFib2 silenced (e) NbPDS (positive control) (f) empty pYL156(negative control)


Table 1 The sequences homologous recombination and restriction sites of PCR primers

Primer and purpose Sequence (51015840 rarr 31015840)a ModificationConstruction for entry vector pDONR221

NbFib2-GF ggggacaagtttgtacaaaaaagcaggcttcATGGTTGCACCAACTAGAGG Homologous recombinationNbFib2-GR ggggaccactttgtacaagaaagctgggtcGGCAGCAGCCTTCTGCTTCT Homologous recombination

Construction for VIGS vector pYL156NbFib2-VF CGgaattcCGATGGTTGCACCAACTAGAGGTCGCG EcoRINbFib2-VR CGggatccCGTTAAATTTTCTAGGCAGCAGCCTTC BamHI

Semiquantitative RT-PCR analysis of NbFib2mRNA accumulationNbFib2-F ATGGTTGCACCAACTAGAGGTCGCGNbFib2-R GGCAGCAGCCTTCTGCTTCTTCCGGCActin-F ACTGATGAAGATACTCACAGAActin-R TGGAATTGTATGTGGTTTCATaThe lowercased letters indicate homologous recombination sequence or a restriction enzyme site

1 2 3

(a)

(b)

(c)

(d)

(e)

(f)

Figure 5 RT-PCR analysis for the accumulation of NbFib2 tran-script inNicotiana benthamiana plants 1ndash3 PCR amplifications after30 35 and 40 cycles (a)ndash(d) amplifications of NbFib2 from corre-sponding plants shown in Figures 4(a)ndash4(d) (e)-(f) amplificationsof NbFib2 and Nbactin from plant in Figure 4(f)

Acetosyringone) The induction media with the bacterialcultures were incubated at room temperature for 3 hours

Subcellular localization of NbFib2 was determined byinfiltrating culture of A tumefaciens harboring pEarley101-NbFib2 onto the fully grown upper leaves ofN benthamianaIn the VIGS assay A tumefaciens harboring pTRV1 wasmixed in an equal proportion of A tumefaciens harboringpYL156-NbFib2 pYL156-NbPDS and empty pYL156 respec-tively (VV) and infiltrated onto fully grown upper leavesSix-week-old N benthamiana was used for the experiment

45 Confocal Imaging Analysis Subcellular locations of pro-teins were monitored at 48 hours after infiltration under aconfocal microscope (Microsystems CMS GmbH Leica TCSSP5) The fluorophores in YFP were excited using 514 nmlight and images were taken using BA535ndash565-nm emissionfilters To locate the fluorescent proteins in nuclei the Nbenthamiana leaves were infiltrated with PBS containing4101584061015840-diamidino-2-phenylindole (DAPI)

46Western Blot Analysis Leaves infiltratedwith pEarley101-NbFib2 (500mg) were collected at 72 hours after inocula-tions and homogenized in 2mL extraction buffer containing50mM phosphate (pH 80) 10mM Tris (pH 80) 500mMNaCl 01 Tween20 01 NP-40 01 120573-mercaptoethanol1mM PMSF and 14 of Roche Protease inhibitor cocktailMINI tablet The crude extracts were centrifuged at 12000 gfor 10 minutes Supernatant was transferred into a new cen-trifuge tube and centrifuged at 12000 g for another 15 min-utes 10 120583L supernatant was mixed with 2120583L 5 times SDS-PAGEloading buffer Proteins in the extracts were separated byelectrophoresis in 12 SDS-PAGE at 80V for 1 hour and thenat 120V for another 40 minutes Proteins in gels were trans-ferred onto polyvinylidene difluoride PVDF membranes byElectrophoresis Cell at 60V for 1 hour (Beijing WoDeLifeSciences Instrument Company Beijing China) and probedwith a rabbit-anti-GFP polyclonal antibody (GenScript CoLtd Nanjing China) The polyclonal antibody was a goat-anti-rabbit IgG conjugated with alkaline phosphatase (SigmaSt Louis MO USA) and used at 1 10000 (VV) dilutionProteins on the membrane were visualized by NBT-BCIPsolution (Promega)


47 Analysis of Fibrillarin-Silenced Plants When silencedplants developed symptoms which usually occur at tendays after infiltration the upper leaves of silenced plantswere collected and maintained individually Accumulationof NbFib2 mRNA was analyzed by RT-PCR using specificprimers (Table 1) designed from NbFib2 sequence NbActinmRNA (280 bp) was used as a control for the RT-PCRanalysis

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Luping Zheng and Jinai Yao contributed equally to this work

Acknowledgments

This work was supported by National Science Foundationof China (31401715 and 31171821) and Fujian Science andTechnology Agency of China (2014R1024-3) The authorswould like to thank Dr Xinzhong Cai at Zhengjiang Univer-sity China for providing vectors TRV and plasmid pYL156-NbPDS and Dr Aiming Wang at AAFC-Southern CropProtection and Food Research Centre Canada for providingvectors pDNOR221 and pEarleyGate101 The authors alsothank Dr Jiasui Zhan at Fujian Agriculture and ForestryUniversity China

References

[1] S Snaar K Wiesmeijer A G Jochemsen H J Tanke and RW Dirks ldquoMutational analysis of fibrillarin and its mobility inliving human cellsrdquo The Journal of Cell Biology vol 151 no 3pp 653ndash662 2000

[2] J P Aris and G Blobel ldquocDNA cloning and sequencing ofhuman fibrillarin a conserved nucleolar protein recognized byautoimmune antiserardquo Proceedings of the National Academy ofSciences of the United States of America vol 88 no 3 pp 931ndash935 1991

[3] D L Pearson R D Reimonenq and KM Pollard ldquoExpressionand purification of recombinant mouse fibrillarinrdquo ProteinExpression and Purification vol 17 no 1 pp 49ndash56 1999

[4] Q Liu and G Dreyfuss ldquoIn vivo and in vitro arginine methyla-tion of RNA-binding proteinsrdquoMolecular and Cellular Biologyvol 15 no 5 pp 2800ndash2808 1995

[5] V V Barygina V P Veiko and O V Zatsepin ldquoAnalysis ofnucleolar protein fibrillarin mobility and functional state inlivingHeLa cellsrdquoBiochemistry vol 75 no 8 pp 979ndash988 2010

[6] T Lechertier A Grob D Hernandez-Verdun and P RousselldquoFibrillarin and Nop56 interact before being co-assembled inbox CD snoRNPsrdquo Experimental Cell Research vol 315 no 6pp 928ndash942 2009

[7] D Tollervey H Lehtonen M Carmo-Fonseca and E C HurtldquoThe small nucleolar RNP protein NOP1 (fibrillarin) is requiredfor pre-rRNA processing in yeastrdquo The EMBO Journal vol 10no 3 pp 573ndash583 1991

[8] K Melen J Tynell R Fagerlund P Roussel D Hernandez-Verdun and I Julkunen ldquoInfluenza A H3N2 subtype virus NS1

protein targets into the nucleus and binds primarily via itsC-terminal NLS2NoLS to nucleolin and fibrillarinrdquo VirologyJournal vol 9 no 1 article 167 13 pages 2012

[9] S H Kim E V Ryabov N O Kalinina et al ldquoCajal bodies andthe nucleolus are required for a plant virus systemic infectionrdquoThe EMBO Journal vol 26 no 8 pp 2169ndash2179 2007

[10] H K Sang S MacFarlane N O Kalinina et al ldquoInteraction ofa plant virus-encoded protein with the major nucleolar proteinfibrillarin is required for systemic virus infectionrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 104 no 26 pp 11115ndash11120 2007

[11] I Gonzalez LMartınez D V Rakitina et al ldquoCucumbermosaicvirus 2b protein subcellular targets and interactions theirsignificance to RNA silencing suppressor activityrdquo MolecularPlant-Microbe Interactions vol 23 no 3 pp 294ndash303 2010

[12] M-L Rajamaki and J P T Valkonen ldquoControl of nuclear andnucleolar localization of nuclear inclusion protein a of picorna-like Potato virus a in Nicotiana speciesrdquo Plant Cell vol 21 no 8pp 2485ndash2502 2009

[13] D Robertson ldquoVIGS vectors for gene silencing many targetsmany toolsrdquo Annual Review of Plant Biology vol 55 pp 495ndash519 2004

[14] F Ratcliff A M Martin-Hernandez and D C BaulcombeldquoTechnical advance tobacco rattle virus as a vector for analysisof gene function by silencingrdquo Plant Journal vol 25 no 2 pp237ndash245 2001

[15] Y Liu M Schiff and S P Dinesh-Kumar ldquoVirus-induced genesilencing in tomatordquo Plant Journal vol 31 no 6 pp 777ndash7862002

[16] L Zheng Z Du C Lin et al ldquoRice stripe tenuivirus p2 mayrecruit or manipulate nucleolar functions through an inter-action with fibrillarin to promote virus systemic movementrdquoMolecular Plant Pathology vol 16 no 9 pp 921ndash930 2015

[17] A Roy A Kucukural and Y Zhang ldquoI-TASSER a unified plat-form for automated protein structure and function predictionrdquoNature Protocols vol 5 no 4 pp 725ndash738 2010

[18] A Roy J Yang and Y Zhang ldquoCOFACTOR an accuratecomparative algorithm for structure-based protein functionannotationrdquo Nucleic Acids Research vol 40 no 1 pp W471ndashW477 2012

[19] K T Pih M J Yi Y S Liang et al ldquoMolecular cloningand targeting of a fibrillarin homolog from Arabidopsisrdquo PlantPhysiology vol 123 no 1 pp 51ndash58 2000

[20] S A Levitskiy K S Mukharyamova V P Veiko and OV Zatsepina ldquoIdentification of signal sequences determiningthe specific nucleolar localization of fibrillarin in HeLa cellsrdquoMolecular Biology vol 38 no 3 pp 405ndash413 2004

[21] M A Semashko I Gonzalez J Shaw et al ldquoThe extremeN-terminal domain of a hordeivirus TGB1 movement proteinmediates its localization to the nucleolus and interaction withfibrillarinrdquo Biochimie vol 94 no 5 pp 1180ndash1188 2012

[22] HWang D Boisvert K K Kim R Kim and S-H Kim ldquoCrys-tal structure of a fibrillarin homologue from Methanococcusjannaschii a hyperthermophile at 16 A resolutionrdquoThe EMBOJournal vol 19 no 3 pp 317ndash323 2000

[23] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST amp PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[24] J Yang and L Zhang ldquoRun probabilities of seed-like patternsand identifying good transition seedsrdquo Journal of Computa-tional Biology vol 15 no 10 pp 1295ndash1313 2008


[25] J Venema and D Tollervey ldquoRibosome synthesis in Saccha-romyces cerevisiaerdquo Annual Review of Genetics vol 33 no 1 pp261ndash311 1999

[26] D L J Lafontaine and D Tollervey ldquoNop58p is a commoncomponent of the box C+D snoRNPs that is required forsnoRNA stabilityrdquo RNA vol 5 no 3 pp 455ndash467 1999

[27] A Narayanan W Speckmann R Terns and M P Terns ldquoRoleof the box CDmotif in localization of small nucleolar RNAs tocoiled bodies and nucleolirdquoMolecular Biology of the Cell vol 10no 7 pp 2131ndash2147 1999

[28] M E Taliansky JW S BrownM L Rajamaki J P T Valkonenand N O Kalinina ldquoInvolvement of the plant nucleolus invirus and viroid infections Parallels with animal pathosystemsrdquoAdvances in Virus Research vol 77 pp 119ndash158 2010

[29] M Karimi D Inze and A Depicker ldquoGATEWAY vectors forAgrobacterium-mediated plant transformationrdquo Trends in PlantScience vol 7 no 5 pp 193ndash195 2002


into nucleolus via CBs binds with fibrillarin in nucleolusrelocates into cytoplasm and finally assembles with virusribonucleoprotein (vRNP) particles in cytoplasm for long-distance movement and systemic infection It was alsoreported that fibrillarin interacts with viral genome-linkedprotein (VPg) in Potato virus A (PVA) and 2b silencingsuppressor protein in Cucumber mosaic virus (CMV) [11 12]

It is known that genes in some plants can be cosuppressedif the plants are transformed with homologous transgenesThis mechanism will result in new types of intercellularcommunication and viral defensemechanisms [13]The eventin which virus vectors carrying the host-derived sequencesilence the corresponding host genes in the infected plantsis defined as virus-induced gene silencing (VIGS) [14] VIGSis themanifestation of an RNA-mediated defensemechanismand is believed to be a fast and powerfulmethod to determinegene function In 2001 a novel VIGS vector TRV wasmodified from the RNA virusTobacco rattle virusThis vectorsuccessfully silenced endogenous genes such as phytoenedesaturase (PDS) andNicotiana FLOLEY (NFL) inNicotianabenthamiana (N benthamiana) [14] Since then TRV hasbecome themost commonly used vector inVIGS studiesThevector used in this study is a modified TRV [15] containingduplicated CaM V 35S promoters (2 times 35S) and nopalinesynthase terminator (NOSt) in the C-terminal which canensure the accumulation of viral RNAs to a higher level Thenew TRV has two genomes designated as pTRV1 and pTRV2(pYL156)The latter genome acts as a VIGS vector containingmultiple cloning sites

Fibrillarin2 is a protein in Fibrillarin family In our previ-ous study we identified the importance of fibrillarin2 fromN benthamiana (NbFib2) during the process of Rice stripevirus infection [16] However the subcellular localizationthe 3D structure and functions of NbFib2 were not fullyresolved The objectives of this study include (1) determiningthe subcellular localization of fibrillarin2 in N benthami-ana (NbFib2) (2) predicting the functional domains and 3dimensional (3D) structure ofNbFib2 and (3) identifying theroles of NbFib2 in plant growth and development

2 Results

21 The Functional Domains of NbFib2 NbFib2 is highlyhomologous to AtFib2 (GenBank accession AAG10153) andHsaFib (GenBank accession AAH19260) They share morethan 74 amino acid sequence identity (Figure 1(a)) Theprotein has 314 amino acid (aa) residues and consists of threefunctional regions including a GAR region an RNA bindingregion and an 120572-helical region (Figure 1(b))TheGAR regionhas 61 aas (aa8ndash68) and is located in the N-terminal of theproteinTheRNAbinding region is seated in themiddle of theprotein (aa131ndash221) in which the most possible interactionsites with RNA are from aa176 to aa183 The 120572-helical regionis located near to the C-terminal (aa231ndash279) followed by anuclear localization signal (NLS aa307ndash313) motif It is worthmentioning that proline encoded by aa131 is predicted tobe involved in most protein activities of NbFib2 The RNAbinding region 120572-helical region and NLS together form aMTase domain implying that NbFib2 will localize in nucleus

In addition the web server I-TASSER [17] was adoptedto predict the 3D structure of NbFib2 using PDB ID 1g8sA(Pyrococcus horikoshii fibrillarin) as template (Figure 1(c))The analysis predicted that NbFib2 belongs to 120572120573 proteins aclass of structural domains in which the secondary structureis composed of alternating 120572-helices and 120573-sheets along thebackbone The 120573-sheet is an external structure while mostpart of 120572-helices is internal The GAR region mostly consistsof hairpin-120573 motif The RNA binding region forms a 120573-120572-120573motif and is randomly coiled (Figure 1(c)) The C-terminalregion is 120572-helical and the subcellular localization signalpeptide is exposed outside

Furthermore the quality and reliability of protein struc-ture predictionwere evaluated by several assessmentmethodsincluding the C-score TM-score and root-mean-squaredeviation (RMSD) A high C-score indicates a high confi-dence in prediction and vice versa and a protein structureprediction is considered as reliable if its C-score is in the rangebetween minus5 and 2 Similarly the TM-score and RMSD areoften used to measure the accuracy of a structure modelingif a reference structure is known For NbFib2 the C-scoreof the predicted structure is minus234 and the estimated TM-score and RMSD are 044plusmn014 and 118plusmn45 A respectivelyThese results suggest that the predicted structure of NbFib2 isreliable and closely matched to the topology of the referenceprotein (eg PDB ID 1g8sA)

We then used the COFACTOR server [18] to predict thefunctions of NbFib2 including the Enzyme Classification(EC) number Gene Ontology (GO) and protein-ligandbinding sites using the protein structure (PDB ID 2ipxA)as template The analysis found that NbFib2 and humanfibrillarin share high similarity in ligand-binding sites with anEC value of 0437 which is in the range of reliable scores forthe EC prediction Thus the NbFib2 protein might also havea similar active site at residue 131 like human fibrillarin Thefunction prediction also revealed that NbFib2 has a NLS andseveral regions targeting nucleolus andCajal body suggestingthat the protein may localize in the two organelles Thesepredictions were confirmed by DAPI staining

22 Subcellular Localization of NbFib2 and Western BlotAnalysis NbFib2 was found in the nucleolus and Cajal bodyof N benthamiana epidermis cells by DAPI fluorescencestaining (Figure 2)Western blot analysis was used to confirmthe protein expression In the leaves inoculated withAgrobac-terium carrying 35S-GFP a protein with a molecular weightidentical to GFP (30 kD) was detected while in the leavesinoculated with Agrobacterium carrying pEarley101-NbFib2a protein with a molecular weight identical to NbFib2-YFPfusion protein (75 kD) was detected The two proteins werenot detected in the negative control leaves (Figure 3)

23 Verifying the Function of NbFib2 Using VIGS Tendays after infiltrating plants with different genes silencedstarted to develop different phenotypes At about three weekspostinoculation (dpi) the obvious and typical symptomsappeared The leaves in the NbPDS-silenced plants (positive


TT TTT TTT TT






TT TT TT




TT TT



1205721

1205731 1205732 1205733 1205734 1205722 1205735

1205723 1205736 1205724 1205781 1205737 1205725 1205738 1205726

1205739 1205727 1205728 12057310 12057311



TTT

737783

(a)

GAR


131Phe RBS NLS



120572

(b) (c)




3 Discussion




(a) (b)

(c) (d)

(e) (f)



72

65

34

26

(kD)

1 2 3M















2 and 150120583M


(a) (b)

(c) (d)

(e) (f)








1 2 3

(a)

(b)

(c)

(d)

(e)

(f)












Acknowledgments


References

































TT TTT TTT TT






TT TT TT




TT TT



1205721

1205731 1205732 1205733 1205734 1205722 1205735

1205723 1205736 1205724 1205781 1205737 1205725 1205738 1205726

1205739 1205727 1205728 12057310 12057311



TTT

737783

(a)

GAR


131Phe RBS NLS



120572

(b) (c)




3 Discussion




(a) (b)

(c) (d)

(e) (f)



72

65

34

26

(kD)

1 2 3M















2 and 150120583M


(a) (b)

(c) (d)

(e) (f)








1 2 3

(a)

(b)

(c)

(d)

(e)

(f)












Acknowledgments


References

































(a) (b)

(c) (d)

(e) (f)



72

65

34

26

(kD)

1 2 3M















2 and 150120583M


(a) (b)

(c) (d)

(e) (f)








1 2 3

(a)

(b)

(c)

(d)

(e)

(f)












Acknowledgments


References

































72

65

34

26

(kD)

1 2 3M















2 and 150120583M


(a) (b)

(c) (d)

(e) (f)








1 2 3

(a)

(b)

(c)

(d)

(e)

(f)












Acknowledgments


References

































(a) (b)

(c) (d)

(e) (f)








1 2 3

(a)

(b)

(c)

(d)

(e)

(f)












Acknowledgments


References






































1 2 3

(a)

(b)

(c)

(d)

(e)

(f)












Acknowledgments


References






































Acknowledgments


References
































the subcellular localization and functional analysis of ... · short sequences: onesequence formsan...

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