Overexpression of GmCaM4 in soybean enhances resistance to pathogens and tolerance to salt stress.

Plant diseases inflict heavy losses on soybean yield, necessitating an understanding of the molecular mechanisms underlying biotic/abiotic stress responses. Ca(2) (+) is an important universal messenger, and protein sensors, prominently calmodulins (CaMs), recognize cellular changes in Ca(2) (+) 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.

Identifying differentially expressed genes in leaves of Glycine tomentella in the presence of the fungal pathogen Phakopsora pachyrhizi.

To compare transcription profiles in genotypes of Glycine tomentella that are differentially sensitive to soybean rust, caused by the fungal pathogen Phakopsora pachyrhizi, four cDNA libraries were constructed using the suppression subtractive hybridization method. Libraries were constructed from rust-infected and non-infected leaves of resistant (PI509501) and susceptible (PI441101) genotypes of G. tomentella, and subjected to subtractive hybridization. A total of 1,536 sequences were obtained from these cDNA libraries from which 195 contigs and 865 singletons were identified. Of these sequenced cDNA clones, functions of 646 clones (61%) were determined. In addition, 160 clones (15%) had significant homology to hypothetical proteins; while the remaining 254 clones (24%) did not reveal any hits. Of those 646 clones with known functions, different genes encoding protein products involved in metabolism, cell defense, energy, protein synthesis, transcription, and cellular transport were identified. These findings were subsequently confirmed by real time RT-PCR and dot blot hybridization.

Non-antibiotic selection systems for soybean somatic embryos: the lysine analog aminoethyl-cysteine as a selection agent.

BACKGROUND: In soybean somatic embryo transformation, the standard selection agent currently used is hygromycin. It may be preferable to avoid use of antibiotic resistance genes in foods. The objective of these experiments was to develop a selection system for producing transgenic soybean somatic embryos without the use of antibiotics such as hygromycin. RESULTS: When tested against different alternate selection agents our studies show that 0.16 microg/mL glufosinate, 40 mg/L isopropylamine-glyphosate, 0.5 mg/mL (S-(2 aminoethyl)-L-cysteine) (AEC) and the acetolactate synthase (ALS) inhibitors Exceed and Synchrony both at 150 microg/mL inhibited soybean somatic embryo growth. Even at the concentration of 2 mg/mL, lysine+threonine (LT) were poor selection agents. The use of AEC may be preferable since it is a natural compound. Unlike the plant enzyme, dihydrodipicolinate synthase (DHPS) from E. coli is not feed-back inhibited by physiological concentrations of lysine. The dapA gene which codes for E. coli DHPS was expressed in soybean somatic embryos under the control of the CaMV 35S promoter. Following introduction of the construct into embryogenic tissue of soybean, transgenic events were recovered by incubating the tissue in liquid medium containing AEC at a concentration of 5 mM. Only transgenic soybeans were able to grow at this concentration of AEC; no escapes were observed. CONCLUSION: Genetically engineered soybeans expressing a lysine insensitive DHPS gene can be selected with the non-antibiotic selection agent AEC. We also report here the inhibitory effects of glufosinate, (isopropylamine-glyphosate) (Roundup), AEC and the ALS inhibitors Exceed and Synchrony against different tissues of soybean.

Changes in oil content of transgenic soybeans expressing the yeast SLC1 gene.

The wild type (Wt) and mutant form of yeast (sphingolipid compensation) genes, SLC1 and SLC1-1, have been shown to have lysophosphatidic acid acyltransferase (LPAT) activities (Nageic et al. in J Biol Chem 269:22156-22163, 1993). Expression of these LPAT genes was reported to increase oil content in transgenic Arabidopsis and Brassica napus. It is of interest to determine if the TAG content increase would also be seen in soybeans. Therefore, the wild type SLC1 was expressed in soybean somatic embryos under the control of seed specific phaseolin promoter. Some transgenic somatic embryos and in both T2 and T3 transgenic seeds showed higher oil contents. Compared to controls, the average increase in triglyceride values went up by 1.5% in transgenic somatic embryos. A maximum of 3.2% increase in seed oil content was observed in a T3 line. Expression of the yeast Wt LPAT gene did not alter the fatty acid composition of the seed oil.