Phytophthora root and stem rot of soybean [Glycine max (L.) Merr.], caused by Phytophthora sojae Kaufmann and Gerdemann, is a destructive disease throughout the soybean planting regions in the world. Here, we report insights into the function and underlying mechanisms of a novel ethylene response factor (ERF) in soybean, namely GmERF5, in host responses to P. sojae. GmERF5-overexpressing transgenic soybean exhibited significantly enhanced resistance to P. sojae and positively regulated the expression of the PR10, PR1-1, and PR10-1 genes. Sequence analysis suggested that GmERF5 contains an AP2/ERF domain of 58 aa and a conserved ERF-associated amphiphilic repression (EAR) motif in its C-terminal region. Following stress treatments, GmERF5 was significantly induced by P. sojae, ethylene (ET), abscisic acid (ABA), and salicylic acid (SA). The activity of the GmERF5 promoter (GmERF5P) was upregulated in tobacco leaves with ET, ABA, Phytophthora nicotianae, salt, and drought treatments, suggesting that GmERF5 could be involved not only in the induced defence response but also in the ABA-mediated pathway of salt and drought tolerance. GmERF5 could bind to the GCC-box element and act as a repressor of gene transcription. It was targeted to the nucleus when transiently expressed in Arabidopsis protoplasts. GmERF5 interacted with a basic helix–loop–helix transcription factor (GmbHLH) and eukaryotic translation initiation factor (GmEIF) both in yeast cells and in planta. To the best of our knowledge, GmERF5 is the first soybean EAR motif-containing ERF transcription factor demonstrated to be involved in the response to pathogen infection.

Isoflavone reductase (IFR) is an enzyme involved in the biosynthetic pathway of isoflavonoid phytoalexin in plants. IFRs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report the characterization of a novel member of the soybean isoflavone reductase gene family GmIFR. The cDNA of GmIFR was 1199 bp containing a 939 bp open reading frame encoding a polypeptide of 312 amino acids. Sequence analysis suggested that GmIFR contained a NAD(P) domain of 107 amino acids. Overexpression of GmIFR transgenic soybean exhibited enhanced resistance to Phytophthora sojae. Following stress treatments, GmIFR was significantly induced by P. sojae, ethephon (ET), abscisic acid (ABA), salicylic acid (SA). It is located in the cytoplasmic when transiently expressed in Arabidopsis protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while levels of genistein and glycitein had little change compared to that of control plants. Furthermore, we also found that the reactive oxygen species (ROS) content of transgenic soybean plants was significantly lower than that of control plants, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean.

Phytophthora sojae Kaufmann and Gerdemann causes Phytophthora root rot, a destructive soybean disease worldwide. A basic helix–loop–helix (bHLH) transcription factor is thought to be involved in the response to P. sojae infection in soybean, as revealed by RNA sequencing (RNA-seq). However, the molecular mechanism underlying this response is currently unclear. Here, we explored the function and underlying mechanisms of a bHLH transcription factor in soybean, designated GmPIB1 (P. sojae-inducible bHLH transcription factor), during host responses to P. sojae. GmPIB1 was significantly induced by P. sojae in the resistant soybean cultivar ‘L77-1863’. Analysis of transgenic soybean hairy roots with elevated or reduced expression of GmPIB1 demonstrated that GmPIB1 enhances resistance to P. sojae and reduces reactive oxygen species (ROS) accumulation. Quantitative reverse transcription PCR and chromatin immunoprecipitation–quantitative PCR assays revealed that GmPIB1 binds directly to the promoter of GmSPOD1 and represses its expression; this gene encodes a key enzyme in ROS production. Moreover, transgenic soybean hairy roots with GmSPOD1 silencing through RNA interference exhibited improved resistance to P. sojae and reduced ROS generation. These findings suggest that GmPIB1 enhances resistance to P. sojae by repressing the expression of GmSPOD1.

Phytophthora root and stem rot of soybean [Glycine max(L.) Merr.] caused byPhytophthora sojaeis a destructive disease worldwide. Phenylalanine ammonia-lyase (PAL) is one of the most extensively studied enzymes related to plant responses to biotic and abiotic stresses. However, the molecular mechanism of PAL in soybean in response toP.sojaeis largely unclear. Here, we characterize a novel member of the soybean PAL gene family,GmPAL2.1, which is significantly induced byP.sojae. Overexpression and RNA interference analysis demonstrates that GmPAL2.1 enhances resistance toP.sojaein transgenic soybean plants. In addition, the PAL activity inGmPAL2.1-OX transgenic soybean is significantly higher than that of non-transgenic plants after infection withP.sojae, while that inGmPAL2.1-RNAi soybean plants is lower. Further analyses show that the daidzein, genistein and salicylic acid (SA) levels and the relative content of glyceollins are markedly increased inGmPAL2.1-OX transgenic soybean. Taken together, these results suggest the important role of GmPAL2.1 functioning as a positive regulator in the soybean response toP.sojaeinfection, possibly by enhancing the content of glyceollins, daidzein, genistein and SA.

Phytophthora root and stem rot caused by the oomycete pathogen Phytophthora sojae is a destructive disease of soybean worldwide. Plant dirigent proteins (DIR) are proposed to have roles in biosynthesis of either lignan or lignin-like molecules, and are important for defense responses, secondary metabolism, and pathogen resistance. In the present work, a novel DIR gene expressed sequence tag (EST) is identified as up-regulated in the highly resistant soybean cultivar ‘Suinong 10’ inoculated with P. sojae. The full length cDNA is isolated using rapid amplification of cDNA ends (RACE), and designated GmDIR22 (GenBank accession no. HQ_993047). The full length GmDIR22 is 789 bp and contains a 567 bp open reading frame encoding a polypeptide of 188 amino acids. The sequence analysis indicated that GmDIR22 contains a conserved dirigent domain at amino acid residues 43–187. The quantitative real-time reverse transcription PCR demonstrated that soybean GmDIR22 mRNA is expressed most highly in stems, followed by roots and leaves. The treatments with stresses demonstrated that GmDIR22 is significantly induced by P. sojae and gibberellic acid (GA3), and also responds to salicylic acid (SA), methyl jasmonic acid (MeJA), and abscisic acid (ABA). The GmDIR22 is targeted to the cytomembrane when transiently expressed in Arabidopsis protoplasts. Moreover, The GmDIR22 recombinant protein purified from Escherichia coli could effectively direct E-coniferyl alcohol coupling into lignan (+)-pinoresinol. Accordingly, the overexpression of GmDIR22 in transgenic soybean increased total lignan accumulation. Moreover, the lignan extracts from GmDIR22 transgenic plants effectively inhibits P. sojae hyphal growth. Furthermore, the transgenic overexpression of GmDIR22 in the susceptible soybean cultivar ‘Dongnong 50’ enhances its resistance to P. sojae. Collectively, these data suggested that the primary role of GmDIR22 is probably involved in the regulation of lignan biosynthesis, and which contributes to resistance to P. sojae.

Pathogenesis-related proteins (PR proteins) play crucial roles in the plant defense system. A novel PRP gene was isolated from highly resistant soybean infected with Phytophthora sojae (P. sojae) and was named GmPRP (GenBank accession number: KM506762). The amino acid sequences of GmPRP showed identities of 74%, 73%, 72% and 69% with PRP proteins from Vitis vinifera, Populus trichocarpa, Citrus sinensis and Theobroma cacao, respectively. Quantitative real-time reverse transcription PCR (qRT-PCR) data showed that the expression of GmPRP was highest in roots, followed by the stems and leaves. GmPRP expression was upregulated in soybean leaves infected with P. sojae. Similarly, GmPRP expression also responded to defense/stress signaling molecules, including salicylic acid (SA), ethylene (ET), abscisic acid (ABA) and jasmonic acid (JA). GmPRP was localized in the cell plasma membrane and cytoplasm. Recombinant GmPRP protein exhibited ribonuclease activity and significant inhibition of hyphal growth of P. sojae 1 in vitro. Overexpression of the GmPRP gene in T₂ transgenic tobacco and T₂ soybean plants resulted in enhanced resistance to Phytophthora nicotianae (P. nicotianae) and P. sojae race 1, respectively. These results indicated that the GmPRP protein played an important role in the defense of soybean against P. sojae infection.