Fig. S1 (a) Genome organization of Bean pod mottle virus (BPMV) RNA2 and vector construction strategy. BPMV RNA2 is translated into two overlapping carboxy co-terminal polyproteins. CR, RNA2 replication cofactor; MP, movement protein; L-CP, large coat protein; S-CP, small coat protein. The vector pG7R2V (Zhang and Ghabrial, 2006) contains a green fluorescent protein (GFP) fragment (ΔGFP) inserted between the coding regions of MP and L-CP, and also contains additional restriction sites (BamHI and MscI) for cloning of the gene of interest (GOI). In constructing the vector, the Q/M cleavage site sequence between MP and L-CP (the dipeptide QM plus flanking sequences) was duplicated. A T7 RNA polymerase promoter sequence was engineered upstream of the modified full-length RNA2 cDNA and cloned into plasmid pGEM T easy to generate pG7R2V. The plasmid pG7R2V can be linearized by digestion with SalI prior to transcription. (b) Percentage sequence identity between the five known GmCaM genes. Values above the diagonal are based on full-length sequences and values below the diagonal are based on coding region sequences.


Fig. S2 Immunoblot analysis of total protein from soybean plants infected with empty and recombinant Bean pod mottle virus (BPMV) vectors. Samples of 20 μg of total protein extracted from mock soybean plants or soybean plants previously inoculated with vector, recombinant GmCaM4-overexpressing (GmCaM4-OE) and GmCaM4-silenced (GmCaM4-SI) transcripts were subjected to sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using an anti-BPMV coat protein (CP) antiserum. The positions of large (L-CP) and small (S-CP) CPs are indicated to the right. An arrowhead points to a minor host protein detectable in the Western blots using the BPMV-CP antiserum. Extracts from the same plants as used for Western blot analysis were also tested by double-antibody sandwich enzyme-linked immunosorbent analysis (ELISA), as described previously (Ghabrial and Schultz, 1983), and the corresponding ELISA values for the different treatments are listed below the Western blot.


Fig. S3 Reverse transcription-polymerase chain reaction (RT-PCR) analysis and quantification by densitometry. (a) Semi-quantitative RT-PCR to assess the GmCaM4 transcript levels in GmCaM4-SI and empty vector soybean plants using a pair of primers designed to amplify the entire coding sequence of GmCaM4 (450 bp). (b) Quantification of bands generated by RT-PCR was performed using a Bio-Rad Gel-Doc XR+ system and Quantity One software (Bio-Rad Laboratory, Hercules, CA, USA). Briefly, intensity profiles for selected bands are determined and the area under the profile curve to the baseline is integrated, resulting in units of intensity × millimetres.


Table S1 Primers used in this study.

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