Overexpression ofGmCaM4in soybean enhances resistance to pathogens and tolerance to salt stress

Abstract

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.

CaM and CML emergence in the green lineage

“…Early transcriptomic analyses indicated that, in Arabidopsis (Arabidopsis thaliana) at least, many CML genes are stress responsive, suggesting a role in Ca 2+ -mediated stress signaling [15]. Subsequent functional analyses by reverse genetics have supported this hypothesis and, to date, several CMLs have been shown to be important during herbivore resistance [16,17], innate immunity [18][19][20][21], salt stress [22], or during both abiotic and biotic stresses, as observed for soybean GmCaM4 [23] and Arabidopsis CML9 [24]. In addition, functional roles in a variety of developmental processes have been uncovered, such as the involvement of Arabidopsis CML23 and 24 in flowering [25], CML42 in trichome branching [26], CML25 in pollen germination and tube elongation [27], CML7 in root hair elongation [28,29], CML39 in seedling establishment [30], and CML20 in microtubule organization [31].…”

Characterization and Functional Analysis of Calmodulin and Calmodulin-Like Genes in Fragaria vesca

“…In addition to biotic stress, CaMs and CMLs are also involved in abiotic stress responses. GmCaM4 , a salt-stress induced gene, has been reported to mediate Ca 2+ signaling and the downstream transcription factor (TF), MYB2 (Rao et al, 2014). In addition, a CaM3 knockout Arabidopsis mutant displayed impaired thermotolerance, whereas overexpression of AtCaM3 increased plant thermotolerance, via interactions with heat stress transcription factors (HSFs) (Zhang et al, 2009).…”

Recent Advances in In Planta Transient Expression and Silencing Systems for Soybean Using Viral Vectors

“…However, targeting the 5′ or 3′ untranslated regions that tend to be less conserved can be used to silence individual homologs or a homeologous pair of genes. Rao et al (2014) reported that targeting 198 nucleotides from the 5′ untranslated region of GmCaM4 silenced specifically this gene and resulted in approximately 50 % reduction in transcript levels. To target this sequence, these authors used a BPMV vector that required a single nucleotide change in the cloned sequence in order to change a stop codon into an amino acid-encoding codon.…”

“…BPMV has been used recently to overexpress the soybean calmodulin 4 protein ( GmCaM4) in soybean (Rao et al 2014). Overexpression of GmCaM4 resulted in enhanced resistance to salt stress and infection by the oomycete pathogen Phytophthora sojae and the necrotrophic fungal pathogens Alternaria tenuissima and Phomopsis longicolla.…”