Overexpression of GmCaM4 in soybean enhances resistance to pathogens and tolerance to salt stress
Summary Plant diseases inflict heavy losses on soybean yield, necessitating an understanding of the molecular mechanisms underlying biotic/abiotic stress responses. Ca2+ is an important universal messenger, and protein sensors, prominently calmodulins (CaMs), recognize cellular changes in Ca2+ in re...
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| Published in | Molecular plant pathology Vol. 15; no. 2; pp. 145 - 160 |
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| Main Authors | , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
England
Blackwell Publishing Ltd
01.02.2014
John Wiley & Sons, Inc John Wiley and Sons Inc |
| Subjects | |
| Online Access | Get full text |
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| Summary: | Summary
Plant diseases inflict heavy losses on soybean yield, necessitating an understanding of the molecular mechanisms underlying biotic/abiotic stress responses. Ca2+ is an important universal messenger, and protein sensors, prominently calmodulins (CaMs), recognize cellular changes in Ca2+ in response to diverse signals. Because the development of stable transgenic soybeans is laborious and time consuming, we used the Bean pod mottle virus (BPMV)‐based vector for rapid and efficient protein expression and gene silencing. The present study focuses on the functional roles of the gene encoding the soybean CaM isoform GmCaM4. Overexpression of GmCaM4 in soybean resulted in enhanced resistance to three plant pathogens and increased tolerance to high salt conditions. To gain an understanding of the underlying mechanisms, we examined the potential defence pathways involved. Our studies revealed activation/increased expression levels of pathogenesis‐related (PR) genes in GmCaM4‐overexpressing plants and the accumulation of jasmonic acid (JA). Silencing of GmCaM4, however, markedly repressed the expression of PR genes. We confirmed the in vivo interaction between GmCaM4 and the CaM binding transcription factor Myb2, which regulates the expression of salt‐responsive genes, using the yeast two‐hybrid (Y2H) system and bimolecular fluorescence complementation assays. GmCaM4 and Glycine max CaM binding receptor‐like kinase (GmCBRLK) did not interact in the Y2H assays, but the interaction between GmCaM2 and GmCBRLK was confirmed. Thus, a GmCaM2–GmCBRLK‐mediated salt tolerance mechanism, similar to that reported in Glycine soja, may also be functional in soybean. Confocal microscopy showed subcellular localization of the green fluorescent protein (GFP)‐GmCaM4 fusion protein in the nucleus and cytoplasm. |
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| Bibliography: | istex:E66272282DB080AD1D3F1AE281C3C87AFA0AFB19 United Soybean Board - No. 0262 ark:/67375/WNG-2ZT8GL27-C ArticleID:MPP12075 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. Kentucky Science and Engineering Foundation ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
| ISSN: | 1464-6722 1364-3703 |
| DOI: | 10.1111/mpp.12075 |