A 5 P degradation hot spot influences molecular farming of anticancerogenic nuclease TBN1 in tobacco cells

Plant Cell, Tissue and Organ Culture (PCTOC) A 5 P degradation hot spot influences molecular farming of anticancerogenic nuclease TBN1 in tobacco cells Anna Týcová a,b, Rajen J. J. Piernikarczyk c, Michael Kugler d, Petra Lipovová d, Tomáš Podzimek d, Gerhard Steger c, Jaroslav Matoušek a * a Biology Centre of the CAS, v. v. i., Institute of Plant Molecular Biology, Branišovská 31, 370 05 České Budějovice, Czech Republic b Faculty of Science, University of South Bohemia in České Budějovice, Branišovská 1760, 370 05 České Budějovice, Czech Republic c Institute of Physical Biology, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany d University of Chemistry and Technology Prague, Technická 3, 166 28 Prague, Czech Republic * Corresponding author: jmat@umbr.cas.cz, tel. +420 387 775 937, fax +420 385 310 356 Online Resource 1

Fig. S1 Schematic drawing of the TBN1 expression cassette (a) and TBN1-GFP psoup vector (b). (a) the scheme represents a T-DNA part of the plant vector plv07 bearing 35Sdriven TBN1 terminated by CaMV terminator. BR and BL indicate the right and left T-DNA borders, respectively.

Fig. S2 Procedure for analysis of TBN1-specific 5 degradome. Polyadenylated RNAs (mrnas and partly degraded mrnas) were purified using Dynabeads. 5 P mrna degradation products were ligated to RNA adapter, purified on mircat Performa Spin Column and precipitated according to mircat RNA Cloning Kit (IDT). RT step was performed using SuperScript III Reverse Transcriptase (Invitrogen) and specific 3E TBN1 primer at 50 C for 60 min. Samples were then purified on (EdgeBio) and processed using biotinylated 5 DNA adapter and 3 specific double-nested primers using TaqI polymerase. Fragments were then analysed on polyacrylamide gels and by molecular hybridization. Adapters and primers are described in Materials and methods.

Fig. S3 Western blot of TBN1 from N. benthamiana leaf sectors co-infiltrated with the PTGS suppressor p38. Crude extracts from N. benthamiana leaf sectors either infiltrated with the TBN1 vector (T), or co-infiltrated with the TBN1 vector and a vector for PTGS suppressor p38 (T+p38) were used in the Western blot assay. kd represents a part of prestained protein marker (Serva, Germany). The arrow indicates the position of the major specific nuclease band.

Fig. S4 Localization of TBN1 GFP. Confocal microscopy of leaves (a-b) and BY-2 cells (c-f) producing TBN1-GFP. The nucleus was stained with propidium iodide (red) (f). TBN1-GFP fusion protein is organized in filament-like structures indicated by the arrows. Figures in panels (a) and (b) are presented as 3D videos attached in Online Resource 2 - see files Video 1 and Video 2. Panels (c-e) show BY-2 cell producing TBN1-GFP bright field combined with GFP fluorescence (exposition 200 ms) and panel (f) BY-2 cell producing TBN1-GFP fluorescence GFP (exposition 200 ms) combined with propidium iodide (exposition 500 ms). For all images were used 60x magnification. Confocal microscopy was used for detection (see Materials and methods).

Fig. S5 TBN1 target validation by degradome analysis. (A) Analysis of degradome products on polyacrylamide gel. The bi-nested PCR products were run on 6% polyacrylamide gel and silver stained according to protocol described by Schumacher et al. (1986). Lane 1: 10 bp DNA ladder; Lane 2: Degradome products of TBN1; Lane 3: Degradome sample of TBN1 co-infiltrated with PTGS- suppressor (p19). Numbers at the band (column P) represent cleavage sites mapped at the TBN1 cdna. Position of cleavage sites (B) The putative positions of cleavage site were mapped according to the length of the PCR products relative to the position of reverse primer (black arrow near to 3'-end correspond to the region of designed reverse primer) on cdna of TBN1. The intensity of 5 P degradome products on polyacrylamide gel (i.e. intense bands and weak bands) are represented by a size of symbol P. The identified 5' P positions from the degradome library derived from TBN1-hybridizing fragments (see Fig. 3) are underlined. The length of cdna of TBN1 consist of 909-nt, the position of cleavage site is indicated by W. Positions encoding S-S bridges stabilizing the TBN1 protein structure are marked by S-S.

Fig. S6 Nucleotide alignment of nuclease NBBN1 from N. benthamiana and TBN1 from tomato. 5 P cleavage sites identified from the TBN1 degradome library are framed, regions of nucleotide identity between NBBN1 and TBN1 longer that 20 nucleotides are underlined. The long homology region (47 bp) close to the degradome hot spot is underlined twice. Cleavage positions of TBN1 within mismatches are indicated by asterisks.

Fig. S7 Domain composition of RDRs identified in N. benthamiana. Sequences were identified in the N. benthamiana genome database (solgenomics.net). Predicted proteins from corresponding cdnas were analyzed using Geneious R6 v.6.1.6. Domains were predicted using InterProScan database plugin option of this programme. (a) homologue of RDR2 of A. thaliana; (b) homologue of RDR6 of A. thaliana.

Fig. S8 qrt-pcr quantification of RDR polymerases in N. benthamiana wt leaf sectors infiltrated with plasmidless Agrobacterium. RDR2 (a) and RDR6 (b) mrna levels were normalized to the housekeeping gene actin. Samples were collected during 7 days post infiltration (DPI). 100% represents expression of RDR2 or RDR6 mrna at the beginning of investigation. Error bars represent a standard deviation. Asterisks indicates significant difference samples on late DPIs and 3 DPI (** P < 0.01).

Fig. S9 TBN1-GFP expression in suspension cultures. Western blot analysis of cells, desintegrated cells, and cultivation medium of BY-2 cells (a) and BY-2 transformed with TBN1 (b). (M) KaleidoscopeTM Prestained Standards; (1) desintegrated cells; (2) whole cells; (3) cultivation medium. The exposition was 5 s. Red arrow - TBN1_eGFP_HisTag (64 kda); Green arrow - egfp (27 kda). The Mouse anti GFP (ROCHE, USA) was used as primary antibody.

Fig. S10 Nuclease comparison and a potential mirna target complex. Comparison of nucleases on nucleotide levels (a) and an example of a potential mirna target complex at a position close to the long TBN1/NBBN1 identity region (b); cdna of recombinant nuclease TBN1 from tomato is compared using multiple alignment option of DNAsis v.2.6 (see Section 2.7.) with cdnas of homologues from N. benthamiana (NBBN1) and N. tabacum (NTBN1). The tree is rooted using the cdna of an anticancerogenic nuclease extracted from Arabis brassica (Podzimek et al. 2011). (b) a potential nuclease:gma-mir9742 target site with extensive sequence complementarity based on a RNAhybrid prediction is shown. Calculated stability levels for the hybrids reached -25.4 kcal mol -1 and -22.4 kcal mol -1 for NBBN1 and TBN1, respectively. The purines R and R translate to A and G for TBN1, respectively, and for NBBN1 vice versa. References Podzimek T, Matoušek J, Lipovová P, Poučková P, Spiwok V, Šantrůček J (2011) Biochemical properties of three plant nucleases with anticancer potential. Plant Sci. 180:343-351. doi: 10.1016/j.plantsci.2010.10.006 Schumacher J, Meyer N, Riesner D, Weidemann HL (1986) Diagnostic procedure for detection of viroids and viruses with circular RNAs by return -gel electrophoresis. J Phytopathol 115:332-43. doi: 10.1111/j.1439-0434.1986.tb04346.x