Viral RNase3 co-localizes and interacts with the antiviral defense protein SGS3 in plant cells

RESEARCH ARTICLE Vi al RNase3 Co-Localizes and In e ac s wi h he An i i al De ense P o ein SGS3 in Plan Cells Isabel Weinheime 1¤a , Tuuli Haikonen 1¤b , Ma jo Ala-Poikela 1¤c , Mi ko Mose 2 , Janne S eng 1 , Minna-Liisa Rajamäki 1 *, Ja i P. T. Valkonen 1 1Depa men o Ag icul u al Sciences, Uni e si y o Helsinki, Helsinki, Finland, 2Founda ion Edmund Mach, San Michele, I aly ¤a Cu en Add ess: Roche Diagnos ics GmbH, Penzbe g, Ge many ¤b Cu en Add ess: Na u al Resou ces Ins i u e Finland (Luke), Ho icul u al p oduc ion esea ch, Piikkiö, Finland ¤c Cu en Add ess: Depa men o Plan , Soil and En omological Sciences, Uni e si y o Idaho, Moscow, Idaho, Uni ed S a es o Ame ica *minna. ajamaki@helsinki. i Abs ac Swee po a o chlo o ic s un i us (SPCSV; amily Clos e o i idae) encodes a Class 1 RNase III endo ibonuclease (RNase3) ha supp esses pos - ansc ip ional RNA in e e - ence (RNAi) and elimina es an i i al de ense in swee po a o plan s (Ipomoea ba a as). Fo RNAi supp ession, RNase3 clea es double-s anded small in e e ing RNAs (ds-siRNA) and long dsRNA o agmen s ha a e oo sho o be u ilized in RNAi. Howe e , RNase3 can supp ess only RNAi induced by sense RNA. Sense-media ed RNAi in ol es hos sup- p esso o gene silencing 3 (SGS3) and RNA–dependen RNA polyme ase 6 (RDR6). In his s udy, subcellula localiza ion and hos in e ac ions o RNase3 we e s udied in plan cells. RNase3 was ound o in e ac wi h SGS3 o swee po a o and A abidopsis haliana when exp essed in lea es, and i localized o SGS3/RDR6 bodies in he cy oplasm o lea cells and p o oplas s. RNase3 was also de ec ed in he nucleus. Co-exp ession o RNase3 and SGS3 in lea issue enhanced he supp ession o RNAi, as compa ed wi h exp ession o RNase3 alone. These esul s sugges addi ional mechanisms needed o e icien RNase3-media ed supp ession o RNAi and p o ide new in o ma ion abou he subcellula con ex and phase o he RNAi pa hway in which RNase3 ealizes RNAi supp ession. In oduc ion Swee po a o chlo o ic s un i us (SPCSV, genus C ini i us; Clos e o i idae) p edisposes swee po a o (Ipomoea ba a as) o hea y yield losses by elimina ing he basal an i i al de ense based on pos - ansc ip ional RNA in e e ence (RNAi), which also ende s plan s suscep ible o o he un ela ed i uses [1]. The dsRNA-speci ic Class 1 RNase III endo ibonuclease (RNase3) encoded by SPCSV is su icien o elimina ing an i i al de ense when exp essed om a ansgene in swee po a o plan s [2]. RNase3 can supp ess sense RNA–media ed RNAi PLOS ONE | DOI:10.1371/jou nal.pone.0159080 July 8, 2016 1/16 a11111 OPEN ACCESS Ci a ion: Weinheime I, Haikonen T, Ala-Poikela M, Mose M, S eng J, Rajamäki M-L, e al. (2016) Vi al RNase3 Co-Localizes and In e ac s wi h he An i i al De ense P o ein SGS3 in Plan Cells. PLoS ONE 11 (7): e0159080. doi:10.1371/jou nal.pone.0159080 Edi o : Rui Lu, Louisiana S a e Uni e si y, UNITED STATES Recei ed: Ma ch 8, 2016 Accep ed: June 27, 2016 Published: July 8, 2016 Copy igh : © 2016 Weinheime e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y S a emen : All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. Funding: This wo k was unded by he Academy o Finland (g an s 1134335, 1253126 and 1276136 o JPTV), Viikki Doc o al P og am in Molecula Biosciences ( o IW), and he Finnish Doc o al P og am in Plan Sciences ( o TH). The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . (gene co-supp ession) bu no RNAi induced by an isense RNA o dsRNA [2–4]. RNase3 can p ocess long dsRNA and double-s anded sho in e e ing RNAs (siRNA) in a leng h- and sequence-independen manne [2,3], bu ce ain s uc u al an i-p ocessing de e minan s (asymme ic bulges) ypical o mic oRNA (miRNA) duplexes may in e e e wi h p ocessing [5]. Taken oge he , RNase3 can po en ially a ge he dsRNA subs a es o he Dice -like dsRNA-speci ic endo ibonucleases (DCL1-4), as well as he 21-, 22-, and 24-nucleo ide (n ) siRNA duplexes p oduced by DCLs in plan s and which guide he RNA-induced silencing complex (RISC) o a ge and clea e homologous RNAs [6]. RNA-dependen RNA polyme ases (RDRs) a e impo an in sense-media ed RNAi. They can copy ssRNA o dsRNA, which is subsequen ly p ocessed o siRNA by DCLs. In his p o- cess, seconda y siRNAs a e p oduced also om egions ou side he p ima y a ge ed a ea, which is called ansi i i y and enhances an i i al RNAi [7,8]. P e ious s udies ound no in lu- ence o RNase3 on ansi i i y [3]. SGS3 is ano he plan p o ein in ol ed in an i i al de ense, bu he mechanism is no well unde s ood. Fo example, a lowe ed le el o SGS3 mRNA enhances suscep ibili y o A abidopsis haliana (L.) Heynh. o Cucumbe mosaic i us (genus Cucumo i us) bu no Tu nip ein clea ing i us (genus Tobamo i us)o Tu nip mosaic i us (genus Po y i us)[9,10]. In po y i us in ec ion, SGS3-silencing educes accumula ion o i al RNA, as epo ed wi h Po a o i us A and Soybean mosaic i us s ain G7 [11,12]. The coo dina ed unc ions o SGS3 wi h RDR6 a e pi o al in ans-ac ing siRNA ( asiRNA) pa hways ha egula e plan gene exp ession. The i s s ep in he asiRNA pa hway is he miRNA-p og ammed clea age o asiRNA gene (TAS) ansc ip s, which a e s abilized by SGS3 [13]. Fo example, TAS3 is conse ed among plan species [14] and has wo miRNA390 a ge si es, o which he 3’si e is ecognized and clea ed speci ically by RISC con aining he RNase H–like endo ibonuclease A gonau e 7 (AGO7). Subsequen ly, RDR6 con e s he 5’- clea age agmen o TAS3 ansc ip s o dsRNA in SGS3/RDR6 bodies (also called siRNA bod- ies), and DCL4 p ocesses he dsRNA o 21-n siRNAs. Consis en wi h hese unc ions, AGO7 co-localizes wi h he SGS3/RDR6 bodies in he cy oplasm [15,16]. In ol emen o RDR6 and SGS3 in RNAi sugges s ha plan i uses may ha e e ol ed mechanisms o in e e e wi h hei unc ions. Indeed, he p o ein P6 o Rice yellow s un i us (genus Nucleo habdo i us) in e ac s wi h RDR6 o ice (O yza sa i a) and in e e es wi h sys- emic silencing o RNAi, bu canno supp ess RNAi locally in he lea es in which silencing is induced [17]. The e o e, i di e s om RNase3 ha supp esses RNAi locally [2]. On he o he hand, he iple gene block p o ein (TGBp1) o Plan ago asia ica mosaic i us (genus Po ex- i us) in e ac s wi h bo h RDR6 and SGS3 o media e hei agg ega ion and inhibi s SGS3/ RDR6-dependen dsRNA syn hesis and asiRNA accumula ion [18]. Simila ly, he P p o ein o Le uce nec o ic yellows i us (genus Cy o habdo i us) in e ac s wi h bo h RDR6 and SGS3 in la ge p o ein agg ega es, which inhibi s RNAi ampli ica ion [19]. Fu he mo e, he RNAi-sup- p essing p o eins V2 o Toma o yellow lea cu l i us (ssDNA genome, genus Begomo i us) [20], p2 o Rice s ipe i us (nega i e-sense ssRNA genome; genus Tenui i us)[21] and VPg o Po a o i us A (ssRNA genome, genus Po y i us)[11] in e ac wi h he SGS3 p o eins o oma o (Solanum lycope sicum L.), ice (O yza sa i a L.), and po a o (Solanum ube osum L.), espec i ely. RNase3 is a unique supp esso in e e ing wi h RNAi in an endo ibonuclease ac i i y- dependen manne . Howe e , li le is known abou he subcellula localiza ion and hos in e - ac ions o RNase3 in plan cells. RNase3 in e e es wi h sense-media ed RNAi bu is unable o supp ess RNAi induced by hai pin RNA [2], simila o he TGBp1, P, V2, p2 and VPg p o eins men ioned abo e [11,18–21]. The e o e, he aim o his s udy was o examine possible in e ac- ions o RNase3 wi h SGS3 and RDR6 and in e e ence wi h he RNAi pa hway in ol ing hese hos p o eins. RNase3 In e ac s wi h SGS3 PLOS ONE | DOI:10.1371/jou nal.pone.0159080 July 8, 2016 2/16 Resul s Subcellula localiza ion o RNase3 in cy oplasm and nucleus Ag oin il a ion was used o co-exp ess RNase3 (RNase3-dsRED; ed luo escen p o ein used o he C- e minus) in lea es o Nico iana ben hamiana Domin along wi h ib illa in (Fib2-GFP, g een luo escen p o ein used o he C- e minus), which is a majo nucleola p o- ein also p esen in Cajal bodies [22,23]. Con ocal mic oscopy o epide mal cells in he in il- a ed a eas a 2 days pos -in il a ion (dpi) e ealed dsRED signals in punc a e bodies in he cy oplasm (a owheads, Fig 1A and 1C) and in he nucleus, and also in subnuclea bodies ha di e ed om Cajal bodies (a ow in Fig 1A and 1C). The nucleolus and Cajal bodies did no show p onounced dsRED signals. Co-localiza ion o RNase3 and RDR6 The punc a e RNase3-con aining bodies de ec ed in N.ben hamiana epide mal cells esembled he SGS3/RDR6 bodies de ec ed in A. haliana and Nico iana abacum L. [15,16]. The e o e, RNase3-dsRED was co-exp essed wi h he RDR6 o A. haliana o N. abacum,bo hwi hGFP used o he C- e minus (A RDR6-GFP and N RDR6-GFP, espec i ely), in lea es o N. ben hamiana by ag oin il a ion. Con ocal mic oscopy showed ha signals o A RDR6-GFP (Fig 2A) and N RDR6-GFP (Fig 2B) we e obse ed in punc a e cy oplasmic bodies ha co-localized wi h he RNase3-dsRED-posi i e punc a e bodies. Co-localiza ion o A RDR6 and N RDR6 wi h RNase3 was also obse ed in p o oplas s p epa ed om he ag oin il a ed lea issues (S1 Fig). Subs i u ion o Asn37 and Glu44 wi h alanine in he ca aly ic si e o RNase3 esul s in an RNase3-Ala mu an lacking dsRNA clea age ac i i y [3]. Localiza ion s udies ca ied ou wi h RNase3-Ala used wi h dsRED e ealed signals in simila punc a e bodies and simila co-locali- za ion wi h RDR6 (S2 Fig) as ound wi h RNase3 (Fig 2). The RNase3-SGS3 complex co-localizes wi h SGS3/RDR6 bodies Co-localiza ion o RNase3 and RDR6 p omp ed us o es he possible in e ac ions o RNase3 wi h RDR6 o SGS3, which was done using bimolecula luo escence complemen a ion (BiFC) Fig 1. Subcellula localiza ion o RNase3 in epide mal cells o Nico iana ben hamiana ollowing exp ession by ag oin il a ion. (a) RNase3-dsRED ( ed signals) was de ec ed in cy oplasmic punc a e bodies by con ocal mic oscopy a 2 dpi. A ew o he many bodies a e poin ed ou wi h a owheads. I was also p esen in he nucleus and subnuclea bodies (a ow). (b) Fib illa in o A. haliana (Fib2) was exp essed as a usion wi h GFP (g een) and used as a ma ke o nucleolus (N) and Cajal bodies (C). (c) Me ged image e ealed localiza ion o RNase3 in subnuclea bodies (a ow) o he han nucleolus o Cajal bodies. Scale ba s, 10 μm. doi:10.1371/jou nal.pone.0159080.g001 RNase3 In e ac s wi h SGS3 PLOS ONE | DOI:10.1371/jou nal.pone.0159080 July 8, 2016 3/16 [24,25]. A RDR6 and A SGS3 o A. haliana and RNase3 we e in oduced o BiFC ec o s and exp essed in ansla ional usion wi h he N- o C- e minal hal es o yellow luo escen p o ein (YFP; YN o YC, espec i ely). BiFC assay o hese p o eins wi h eIF(iso)4E se ed as a nega i e con ol (Fig 3, igh panel). Co-exp ession o A RDR6 wi h A SGS3 se ed as a posi i e in e ac- ion con ol in he expe imen s and esul ed in yellow luo escence con ined o cy oplasmic punc a e bodies (Fig 3A), as expec ed [15]. RNase3 did no in e ac wi h A RDR6 (Fig 3C). In con as , in e ac ion o RNase3 wi h A SGS3 was eadily obse ed in cy oplasmic punc a e bodies (Fig 3E). Simila ly, RNase3-Ala in e ac ed wi h A SGS3 (Fig 3G). The SGS3 coding sequence o swee po a o (c . Huachano) (designa ed IbSGS3) was cloned u ilizing he ansc ip ome da a o swee po a o c . Xushu18 a ailable in NCBI Sequence Read A chi e (accession numbe SRX090758; [26]) (see Me hods and S3 Fig) and in oduced in o he BiFC ec o s. IbSGS3 in e ac ed wi h RNase3 in punc a e bodies (Fig 3I) simila o hose obse ed wi h he A SGS3-RNase3 in e ac ion (Fig 3E). Co-exp ession o YN-RNase3, A SGS3-YC, and A RDR6-mRFP e ealed ha cy oplasmic punc a e bodies exp essing BiFC signals (g een) o he RNase3-SGS3 in e ac ion co-localized wi h he signals ( ed) o RDR6-con aining bodies (Fig 4). Taken oge he , he esul s indica ed ha RNase3 in e ac s wi h SGS3 and hence co-localizes wi h SGS3/RDR6 bodies. The ca aly ic si e mu a ions in RNase3-Ala did no a ec hese in e ac ions and subcellula localiza ion. Co-exp ession o RNase3 and IbSGS3 enhances supp ession o RNAi RNase3 can supp ess sense-media ed RNAi, e.g., when silencing o he cons i u i ely exp essed g p ansgene is induced in N.ben hamiana 16c ollowing g p o e exp ession by ag oin il a- ion [2]. In luence o IbSGS3, RNase3 and RNase3-Ala (a mu an o RNase3 debili a ed o Fig 2. Co-localiza ion o RNase3 and RDR6 in epide mal cells o N.ben hamiana ollowing co-exp ession by ag oin il a ion. The ed signals o RNase3-dsRED and g een signals o (a) A RDR6-GFP (A. haliana) and (b) N RDR6-GFP (N. abacum) co-localized in cy oplasmic, punc a e bodies de ec ed by con ocal mic oscopy a 2 dpi. Images in (a) and (b) illus a e op ical planes in which many RDR6-con aining bodies we e obse ed. Scale ba s, 10 μm. doi:10.1371/jou nal.pone.0159080.g002 RNase3 In e ac s wi h SGS3 PLOS ONE | DOI:10.1371/jou nal.pone.0159080 July 8, 2016 4/16 Fig 3. In e ac ions o SGS3 wi h RDR6 and RNase3 in plan a.P o ein in e ac ions we e es ed by BiFC and moni o ed in epide mal cells o N.ben hamiana using epi luo escence mic oscopy. The name o he p o ein indica es whe he he YFP hal was used o he N- o C- e minus o he es p o ein (e.g., YN-RNase3 and IbSGS3-YC, espec i ely). Co-exp ession o he es p o eins wi h eIF(iso)4E o po a o [27] was included as a nega i e con ol o in e ac ion (panels b, d, , h, j and k). (a) In e ac ion o A RDR6 and A SGS3 esul ed in SGS3/RDR6 bodies (posi i e con ol); (b) nega i e con ol. (c) RNase3 did no in e ac wi h A RDR6, bu RNase3 In e ac s wi h SGS3 PLOS ONE | DOI:10.1371/jou nal.pone.0159080 July 8, 2016 5/16 ca aly ic ac i i y on dsRNA), as well as he e ec o IbSGS3 co-exp essed wi h RNase3 o RNa- se3-Ala, on sense-media ed g p silencing was es ed in N.ben hamiana 16c. I RNase3 o RNa- se3-Ala was no used, he co esponding Ag obac e ium s ain was eplaced wi h a s ain exp essing β-glucu onidase (GUS, nega i e con ol). Because he YN- agged IbSGS3 (SGS3-YN) was used in hese expe imen s, i was eplaced, when no used, by an Ag obac e - ium s ain exp essing YN, so o main ain he sense-media ed g p silencing p essu e simila in all ea men s. Following co-exp ession o g p,YN and GUS, GFP luo escence ini ially inc eased, bu hen dec eased subs an ially by 6 dpi (Fig 5A and 5B), indica ing no signi ican supp ession o silencing. Simila esul s we e ob ained ollowing co-exp ession o g p and IbSGS3. These esul s we e consis en wi h de ec able accumula ion o g p-de i ed siRNA, as es ed by no he n analysis (Fig 5C). Howe e , when lea issues we e co-in il a ed wi h Ag o- bac e ium s ains o exp ession o GFP and RNase3, GFP luo escence emained highe han he backg ound luo escence esul ing om exp ession o he cons i u i ely exp essed g p ansgene o he 16c line (no e he g een luo escen eins in Fig 5A and 5B) and he addi ional exp ession o he in il a ed g p-exp essing cons uc . GFP luo escence was u he enhanced in lea issue co-exp essing GFP, IbSGS3, and RNase3 (Fig 5A and 5B). g p mRNA le els we e no much inc eased in lea issues co-in il a ed wi h g p and RNase3, bu hey we e clea ly ele- a ed in lea es co-in il a ed wi h g p,IbSGS3 and RNase3, as compa ed wi h lea issues in il- a ed wi h g p and GUS,o g p,GUS and IbSGS3 (Fig 5C). Accumula ion o g p-speci ic siRNAs was nega i ely co ela ed wi h g p mRNA accumula ion (Fig 5C) and was high in lea issues co-exp essing g p and RNase3-Ala (Fig 5C). In con as , li le g p–de i ed siRNA was obse ed in lea issue co-exp essing g p,RNase3-Ala and IbSGS3 al hough supp ession o silencing could no be obse ed isually in he lea es (Fig 5C). (e) in e ac ed wi h A SGS3 (A. haliana) and (i) IbSGS3 (I.ba a as). (g) Ca aly ically inac i e RNase3-Ala was also able o in e ac wi h SGS3. Da a we e collec ed a 2 dpi. All images we e acqui ed a he same magni ica ion. Scale ba , 20 μm. doi:10.1371/jou nal.pone.0159080.g003 Fig 4. In e ac ions o RNase3 and A SGS3, as assessed wi h BiFC and con ocal mic oscopy, co-localize wi h cy oplasmic punc a e bodies con aining A RDR6 in epide mal cells o N.ben hamiana.The h ee p o eins we e co-exp essed by ag oin il a ion. Images we e acqui ed a an op ical plane in which many RDR6-con aining bodies we e obse ed a 2 dpi. The dashed ci cles poin ou some o he cy oplasmic punc a e bodies ha show signals o he RNase3-SGS3 in e ac ion and co-localize wi h RDR6. Scale ba , 10 μm. doi:10.1371/jou nal.pone.0159080.g004 RNase3 In e ac s wi h SGS3 PLOS ONE | DOI:10.1371/jou nal.pone.0159080 July 8, 2016 6/16 RNase3 In e ac s wi h SGS3 PLOS ONE | DOI:10.1371/jou nal.pone.0159080 July 8, 2016 7/16 RNase3 and IbSGS3 we e eadily exp essed in he issues, as de ec ed by wes e n analysis (Fig 5D). These esul s we e ep oduced in i e independen expe imen s, indica ing enhanced supp ession o gene silencing in he p esence o bo h IbSGS3 and RNase3. asiRNA p oduc ion is no a ec ed by RNase3 RNase3 has no been ound o in e e e wi h ansi i i y [3], bu possible in e e ence o RNase3 wi h he asiRNA pa hway has no been es ed. The e o e, TAS3 ansc ip –de i ed siRNA we e analyzed in ansgenic swee po a o plan s (c . Huachano) exp essing RNase3 [2]. Compu a ional analysis o he Sequence Read A chi e da abase sequences o swee po a o c . Xushu18 (SRX090758) e ealed ha he A. haliana TAS3 5’D7(+) ansc ip -speci ic asiRNA p obe [28] aligned well wi h a numbe o eads, which allowed pa ial econs uc ion o he pu a i e IbTAS3 ansc ip sha ing high sequence iden i y wi h known TAS3 ansc ip s. The A. haliana D7(+)-speci ic p obe was used o de ec asiRNA in swee po a o c . Huachano and wo RNase3- ansgenic lines, bu no di e ences in he asiRNA amoun s we e de ec ed (S4 Fig). Discussion Ou esul s show ha RNase3 co-localizes and is associa ed wi h SGS3/RDR6 bodies impli- ca ed in plan gene egula ion and an i i al RNAi [9,29]. The associa ion is likely media ed by he in e ac ion o RNase3 wi h SGS3, as indica ed by BiFC assay. Dep i a ion o SGS3 can educe RNAi-based i us esis ance and enhance accumula ion o cucumo i uses in plan s [9], whe eas accumula ion o po y i uses co ela es posi i ely wi h SGS3 accumula ion [11,12]. In his espec i is no ewo hy ha co-exp ession o SGS3 wi h RNase3 sligh ly (bu ep oducibly) enhanced supp ession o sense RNA–induced RNAi by RNase3. Yoshikawa e al.[13] ha e shown ha in he mic o-RNA (miRNA) di ec ed p ocess o ans-ac ing small in e e ing RNA ( asiRNAs) p oduc ion om TAS2 gene ansc ip by RISC, he 3' clea age agmen o he TAS2 ansc ip is p o ec ed om deg ada ion. In his p ocess, SGS3 binds o AGO1-RISC ia he dsRNA o med by in e ac ion o he miRNA wi h he a ge RNA. The au ho s also Fig 5. Co-exp ession o RNase3 and IbSGS3 enhances supp ession o sense-media ed gene silencing (gene co-supp ession). (a) and (b) Fou sec o s (1 o 4) o a lea o N.ben hamiana 16c cons i u i ely exp essing g p we e ag oin il a ed o co-exp ess GFP and (1) GUS (nega i e con ol), (2) IbSGS3 and RNase3, (3) RNase3, o (4) IbSGS3. I RNase3 was no used, he co esponding Ag obac e ium s ain was eplaced wi h a s ain exp essing β-glucu onidase (GUS, nega i e con ol). IbSGS3 was exp essed wi h he N-p oximal pa o YFP used o he C- e minus. I i was no used, i was eplaced wi h an Ag obac e ium s ain exp essing YN (N-p oximal hal o y p) o main ain simila sense-media ed silencing p essu e. The ea men s a e posi ioned di e en ly in he wo lea es in e ms o he younge (basal) and olde ( ip) pa o he lea . Silencing o g p was obse ed by he disappea ance o GFP luo escence (sec o s 1 and 4), whe eas GFP luo escence abo e he backg ound le el indica ed supp ession o g p silencing (sec o s 2 and 3). The lea was pho og aphed unde UV ligh a 6 dpi. Simila esul s we e ob ained in i e independen expe imen s. (c) No he n analysis o g p mRNA and g p mRNA-de i ed siRNA in he ag oin il a ed lea issues. Co-exp ession o GUS o IbSGS3 wi h GFP by ag oin il a ion in g p- ansgenic lea es esul ed in g p silencing, as shown by he eadily de ec able accumula ion o g p-de i ed siRNA (Fig 5C). In con as , co- exp ession o GFP and RNase3 esul ed only in low accumula ion o g p siRNA, and no g p siRNA could be de ec ed ollowing co-exp ession o GFP, RNase3 and SGS3; howe e , accumula ion o g p mRNA was enhanced (Fig 5C). Co-exp ession o he RNase3-Ala mu an (disabled om ca aly ic ac i i y on dsRNA) wi h GFP esul ed in eadily de ec able accumula ion o g p siRNA, whe eas co-exp ession o RNase3-Ala, SGS3 and GFP esul ed in low accumula ion o g p siRNA (Fig 5C). 25S and 5S ibosomal RNA is shown as a loading con ol, espec i ely. (d) Wes e n analysis o RNase3 and IbSGS3-YN in he ag oin il a ed lea sec o s illus a ed in (a) by immunoblo ing using an i-RNase3 and an i-GFP an ibodies, espec i ely. Molecula masses o he de ec ed p o eins (kDa) we e es ima ed by compa ison wi h he p o ein ma ke un in he gel. doi:10.1371/jou nal.pone.0159080.g005 RNase3 In e ac s wi h SGS3 PLOS ONE | DOI:10.1371/jou nal.pone.0159080 July 8, 2016 8/16 concluded ha he SGS3-dependen s abiliza ion o he 3' agmen o TAS2 RNA is c ucial o asiRNA p oduc ion [13]. Howe e , he esul s o ou s udy may sugges ha in e ac ion o RNase3 wi h SGS3 is pa o he p o ec i e unc ions o SGS3, o assis s RNase3 in a ge ing and clea ing ds-siRNA o seconda y i al dsRNA syn hesized by RDR6. The ca aly ic ac i i y on dsRNA is pi o al o supp ession o RNAi by RNase3 and esul s in sho agmen s o ~14 base pai s ha a e oo sho o be inco po a ed in RISC and a e inac i e in RNAi [2,4]. I is no ewo hy ha RNase3 was ound o in e ac wi h he SGS3 homologs o wo un ela ed plan species, A. haliana and swee po a o, o which he la e is he na u al hos o SPCSV. These indings imply ha RNase3-SGS3 in e ac ions could be a common and an impo an mecha- nism o SPCSV o con ol an i i al RNAi in i s hos plan s. SGS3 and RDR6 a e in ol ed in asiRNA pa hways, bu mu a ions in SGS3 o RDR6 do no always yield a disce nable pheno ypic change in A. haliana [29,30]. Hence, also in e e ence o RNase3 wi h he asiRNA pa hways would no necessa ily cause any pheno ype, whe eas pe u ba ion o miRNA homeos asis mo e o en is associa ed wi h mo phological changes [31]. The ansgenic swee po a o plan s exp essing RNase3 do no show any pheno ype di e - en om wild- ype plan s, excep mo e p onounced pu ple pigmen a ion in lea es, simila o SPCSV-in ec ed plan s. The e o e, we es ed he possible in luence o RNase3 on TAS3 an- sc ip –based asiRNA p oduc ion, which ep esen s he mos conse ed asiRNA gene in plan s [14], bu esul s we e nega i e. O he asiRNA pa hways could be elucida ed in swee po a o and in luence o RNase3 on hem es ed in u u e s udies. The punc a e bodies con aining RNase3, SGS3 and RDR6 we e cy oplasmic, which is con- sis en wi h p e ious s udies epo ing occu ence o he SGS3/RDR6 bodies in cy oplasm wi hou any speci ic associa ion wi h o he cellula bodies o o ganelles [15]. In addi ion, RNase3 was de ec ed in he nucleus. RDR6 is also ound in he nucleus, in con as o he SGS3/RDR6 bodies [32]. The size o RNase3-dsRED (ca. 51 kDa) could allow passi e di usion o he nucleus [25], bu he nuclea RNase3-con aining bodies, which we e dis inc om Cajal bodies, a e in iguing and sugges ha RNase3 migh ha e a unc ional ole in he nucleus. In gene al, ecen s udies ques ion he iew ha pos - ansc ip ional RNAi would occu only in he cy oplasm. dsRNA di ec ed o an in on can silence a gene [32] and DCL4 esponsible o p oduc ion o he majo i y o he i us-de i ed siRNAs in A. haliana [33] is de ec ed only in he nucleus [15,16,32]. Fu he mo e, nuclea impo o he P6 p o ein o Cauli lowe mosaic i us is equi ed o he supp ession o an i i al RNAi [34]. The e o e, i is likely ha an i i al RNAi occu s in bo h he cy oplasmic and nuclea compa men s [32]. Finally, he s udies o Ga cia-Ruiz e al.[33] ha e shown ha basal le els o an i i al RNAi and siRNA biogenesis emain in A abidopsis mu an s lacking RDR1, RDR2 and RDR6, which sugges s ha he e is an addi ional, unknown pa hway p oducing dsRNA. Hence, he possible in e e ence o RNase3 wi h RNAi in he nucleus and he p oduc ion o RNase3 ia he pos ula ed new pa h- way emains an in e es ing subjec o u he s udy. Taken oge he , he e is limi ed bu inc easing p ecedence o in e ac ion be ween i al RNAi supp esso s wi h SGS3 and i s impac on RNAi. V2 o Toma o yellow lea cu l i us ( amily Gemini i idae) [20,35], p2 o Rice s ipe i us [21], TGBp1 o Plan ago asia ica mosaic i us [18], p o ein P o Le uce nec o ic yellows i us [19], VPg o Po a o i us A [11] and RNase3 o SPCSV ep esen examples epo ed om un ela ed i uses. The uni ying ac o is ha all six i al p o eins a e able o supp ess only sense RNA–induced RNAi, indica ing ha hey in e e e wi h he s eps o RNAi in ol ing ac i i ies o RDR6 and SGS3. Hence, he esul s p esen ed he e ad ance unde s anding o he mechanisms needed o e icien RNase3-me- dia ed supp ession o RNAi and p o ide new in o ma ion abou he subcellula con ex and phase o he RNAi pa hway in which RNase3 ealizes RNAi supp ession. RNase3 In e ac s wi h SGS3 PLOS ONE | DOI:10.1371/jou nal.pone.0159080 July 8, 2016 9/16 28. Allen E, Xie Z, Gus a son AM, Ca ing on JC. Mic oRNA-di ec ed phasing du ing ans-ac ing siRNA biogenesis in plan s. Cell. 2005; 121: 207–221. PMID: 15851028 29. Pe agine A, Yoshikawa M, Wu G, Alb ech HL, Poe hig RS. SGS3 and SGS2/SDE1/RDR6 a e equi ed o ju enile de elopmen and he p oduc ion o ans-ac ing siRNAs in A abidopsis. Genes De . 2004; 18: 2368–2379. PMID: 15466488 30. Adeno X, Elmayan T, Lau esse gues D, Bou e S, Bouche N, Gasciolli V e al. DRB4-dependen TAS3 ans-ac ing siRNAs con ol lea mo phology h ough AGO7. Cu Biol. 2006; 16: 927–32. PMID: 16682354 31. Voinne O. Induc ion and supp ession o RNA silencing: insigh s om i al in ec ions. Na Re Gene . 2005; 6: 206–220. PMID: 15703763 32. Ho e P, I ashu a S, Pon es O, Vi ins A, Pikaa d C, M oczka A, e al. Pos anc ip ional gene silencing in nuclei. P oc Na l Acad Sci U S A. 2011; 108: 409–414. doi: 10.1073/pnas.1009805108 PMID: 21173264 33. Ga cia-Ruiz H, Takeda A, Chapman EJ, Sulli an CM, Fahlg en N, B empelis KJ, e al. A abidopsis RNA-dependen RNA polyme ases and Dice -like p o eins in an i i al de ense and small in e e ing RNA biogenesis du ing Tu nip mosaic i us in ec ion. Plan Cell. 2010; 22: 481–496. doi: 10.1105/ pc. 109.073056 PMID: 20190077 34. Haas G, Aze edo J, Moissia d G, Geld eich A, Himbe C, Bu eau M, e al. Nuclea impo o CaMV P6 is equi ed o in ec ion and supp ession o he RNA silencing ac o DRB4. 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