Figure S1 U1:GFP + Fib2:RFP Sun1:RFP a f b g c h d i e j 10µm 10µm

Figure S1 Transient co-expression of U1:GFP and RFP-tagged marker proteins for the inner nuclear membrane or nucleolus/Cajal bodies in N. benthamiana epidermis cells. a-e) U1:GFP co-expressed with AtFib2:RFP, a nucleolus and Cajal body marker; f-j) U1:GFP co-expressed with AtSun1:RFP, a marker for inner nuclear membrane. a,f) GFP channel; b,g) RFP channel; c,h) autofluorescence of chloroplasts shown in blue; d,i) DIC channel and e, j) merged channels. Note that the nucleus shown in f-j represents the same nucleus as in in Fig. 1f. Bar represents 10 µm.

Figure S2 a b c d e f g h i j k l U2:GFP ER marker merged 20µm 10µm Endosome marker g h i 20µm j k l 10µm

Figure S2 Transient co-expression of U2:GFP and RFP-tagged ER marker or endosome protein in N. benthamiana epidermis cells. a), d), g) and j) show U2:GFP, whereas b) and e) show the RFP fluorescence of the ER marker protein; h) and k) the RFP-tagged endosome marker protein. c), f), i) and l) merged pictures. Co-localization of U2:GFP and marker proteins appear yellow-orange. All pictures are projections of a z-stack, except pictures a-c) and g-i). Bar represents either 20 µm (a-c, g-i) or 10 µm (d-f, j-l).

Figure S3 a b c d e f g h i Figure S3 merged U4::GFP ER marker a b c d e f 10µm g h i 20µm Figure S3 Transient co-expression of U4:GFP and RFP-tagged ER marker protein in N. benthamiana epidermis cells. a), d) and g) merged pictures, b), e) and h) show U4:GFP, whereas c), f) and i) show the RFP fluorescence of the ER marker protein. Note that picture in g-i) represent a magnification of squared area in d) to show the web-like structure of the ER. Co-localization of U4:GFP and ER marker protein appear yellow-orange. All pictures are projections of a z-stack, except pictures g-i). Bar represents 10 µm (a-f) or 20 µm (g-i).

Figure S4 a b c αHA αMyc Figure S4   M-Rep CP Clink MP NSP U1 U2 U4 Predicted size[kDa] 32 19 13 17 11.5 Predicted size of nanoviral protein fused to YFPC and HA-tag[kDa] 42.6 29.6 23.6 27.6 22.1 Predicted size of nanoviral protein fused to YFPN and Myc-tag[kDa] 55.3 42.3 36.3 40.3 34.8 Figure S4 Western blot analysis of PNYDV protein BiFC constructs. Nanoviral protein:cMyc:YFPN and nanoviral protein:HA:YFPC were expressed transiently in leaves of N. benthamiana plants and analyzed at 2 dpai by western blot analysis using either (a) anti-HA- or (b) anti-cMyc-specific antibodies for detection. Predicted molecular mass of nanovirus proteins and as BiFC constructs are summarized in panel c. Please note that U2-VenusC-HA is larger, and CP-VenusN-myc smaller than expected, which can only be explained by degradation and/or post-translational modifications. Expression constructs of U2-VenusC and CP-VenusN were sequenced several times and expressed several times with no different result as shown here.

Figure S5 Figure S5 Yeast-2-hybrid assay to confirm NSP & NSP interaction (GBT9 N + GAD N). Interaction of pSV40 + p53 serves as a positive control in this assay.

Figure S6 a b Chp N N Chp Nu Nu 10µm 10µm M-Rep+NSP with nucleolus marker b M-Rep+CP with nucleolus marker Chp N N Chp Nu Nu 10µm 10µm Figure S6 Co-expression of PNYDV a)M-Rep & NSP and b) M-Rep & CP BiFC constructs together with AtFib2 fused to RFP (nucleolus and cajal bodies marker). Note the re-localization of M-Rep in complex with CP to the nucleolus rim and speckles within the nucleus. White arrows pinpoint to the nucleolus (Nu) whereas (N) is the nucleus. Chloroplasts (Chp) are shown in blue. Bars represent 10µm.