A Novel Soybean Dirigent Gene GmDIR22 Contributes to Promotion of Lignan Biosynthesis and Enhances Resistance to Phytophthora sojae.
ABSTRACT: 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 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, 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, methyl jasmonic acid, and abscisic acid. 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.
Project description:Phytophthora root and stem rot of soybean caused by the oomycete Phytophthora sojae, is a destructive disease worldwide. Ethylene response factors (ERFs) play important roles in regulating plant biotic and abiotic stress tolerance. In this study, a new ERF gene, GmERF113, was isolated from the highly resistant soybean 'Suinong 10.' Sequence analysis suggested that the protein encoded by GmERF113 contained a conserved AP2/ERF domain of 58 amino acid and belonged to the B-4 subgroup of the ERF subfamily. Expression of GmERF113 was significantly induced by P. sojae, ethylene, and methyl jasmonate. GmERF113 protein localized to the nucleus when transiently expressed in Arabidopsis protoplasts, could bind to the GCC-box, and acted as a transcription activator. In addition, a region of the full-length GmERF113, GmERF113-II, interacted with a basic helix-loop-helix transcription factor (GmbHLH) in yeast cells. Full-length GmERF113 also interacted with GmbHLH in planta. GmERF113-overexpressing transgenic plants in susceptible cultivar 'Dongnong 50' soybean exhibited increased resistance to P. sojae and positively regulated the expression of the pathogenesis-related genes, PR1 and PR10-1. These results indicate that GmERF113 may play a crucial role in the defense of soybean against P. sojae infection.
Project description:Phytophthora root and stem rot, a destructive disease of soybean [Glycine max (L.) Merr.], is caused by the oomycete Phytophthora sojae. However, how the disease resistance mechanisms of soybean respond to P. sojae infection remains unclear. Previously, we showed that GmWRKY31, which interacts with a sucrose non-fermenting-1(SNF1)-related protein kinase (SnRK), enhances resistance to P. sojae in soybean. Here, we report that the membrane-localized SnRK GmSnRK1.1 is involved in the soybean host response to P. sojae. The overexpression of GmSnRK1.1 (GmSnRK1.1-OE) increased soybean resistance to P. sojae, and the RNA interference (RNAi)-mediated silencing of GmSnRK1.1 (GmSnRK1.1-R) reduced resistance to P. sojae. Moreover, the activities and transcript levels of the antioxidant enzymes superoxide dismutase and peroxidase were markedly higher in the GmSnRK1.1-OE transgenic soybean plants than in the wild type (WT), but were reduced in the GmSnRK1.1-R plants. Several isoflavonoid phytoalexins related genes GmPAL, GmIFR, Gm4CL and GmCHS were significantly higher in "Suinong 10" and GmSnRK1.1-OE lines than these in "Dongnong 50," and were significantly lower in GmSnRK1.1-R lines. In addition, the accumulation of salicylic acid (SA) and the expression level of the SA biosynthesis-related gene were significantly higher in the GmSnRK1.1-OE plants than in the WT and GmSnRK1.1-R plants, moreover, SA biosynthesis inhibitor treated GmSnRK1.1-R lines plants displayed clearly increased pathogen biomass compared with H2O-treated plants after 24 h post-inoculation. These results showed that GmSnRK1.1 positively regulates soybean resistance to P. sojae, potentially functioning via effects on the expression of SA-related genes and increased accumulation of SA.
Project description: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.
Project description:Phytophthora root and stem rot of soybean, caused by Phytophthora sojae (P. sojae), is a destructive disease in many soybean planting regions worldwide. In a previous study, an expressed sequence tag (EST) homolog of the major allergen Pru ar 1 in apricot (Prunus armeniaca) was identified up-regulated in the highly resistant soybean 'Suinong 10' infected with P. sojae. Here, the full length of the EST was isolated using rapid amplification of cDNA ends (RACE). It showed the highest homology of 53.46% with Gly m 4 after comparison with the eight soybean allergen families reported and was named Gly m 4-like (Gly m 4l, GenBank accession no. HQ913577.1). The cDNA full length of Gly m 4l was 707 bp containing a 474 bp open reading frame encoding a polypeptide of 157 amino acids. Sequence analysis suggests that Gly m 4l contains a conserved 'P-loop' (phosphate-binding loop) motif at residues 47-55 aa and a Bet v 1 domain at residues 87-120 aa. The transcript abundance of Gly m 4l was significantly induced by P. sojae, salicylic acid (SA), NaCl, and also responded to methyl jasmonic acid (MeJA) and ethylene (ET). The recombinant Gly m 4l protein showed RNase activity and displayed directly antimicrobial activity that inhibited hyphal growth and reduced zoospore release in P. sojae. Further analyses showed that the RNase activity of the recombinant protein to degrading tRNA was significantly affected in the presence of zeatin. Over-expression of Gly m 4l in susceptible 'Dongnong 50' soybean showed enhanced resistance to P. sojae. These results indicated that Gly m 4l protein played an important role in the defense of soybean against P. sojae infection.
Project description:Phytophthora root and stem rot of soybean [Glycine max (L.) Merr.] caused by Phytophthora sojae is 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 to P. sojae is largely unclear. Here, we characterize a novel member of the soybean PAL gene family, GmPAL2.1, which is significantly induced by P. sojae. Overexpression and RNA interference analysis demonstrates that GmPAL2.1 enhances resistance to P. sojae in transgenic soybean plants. In addition, the PAL activity in GmPAL2.1-OX transgenic soybean is significantly higher than that of non-transgenic plants after infection with P. sojae, while that in GmPAL2.1-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 in GmPAL2.1-OX transgenic soybean. Taken together, these results suggest the important role of GmPAL2.1 functioning as a positive regulator in the soybean response to P. sojae infection, possibly by enhancing the content of glyceollins, daidzein, genistein and SA.
Project description:Phytophthora root and stem rot of soybean [Glycine max (L.) Merr.] caused by the oomycete Phytophthora sojae, is a destructive disease worldwide. The molecular mechanism of the soybean response to P. sojae is largely unclear. We report a novel WRKY transcription factor (TF) in soybean, GmWRKY31, in the host response to P. sojae. Overexpression and RNA interference analysis demonstrated that GmWRKY31 enhanced resistance to P. sojae in transgenic soybean plants. GmWRKY31 was targeted to the nucleus, where it bound to the W-box and acted as an activator of gene transcription. Moreover, we determined that GmWRKY31 physically interacted with GmHDL56, which improved resistance to P. sojae in transgenic soybean roots. GmWRKY31 and GmHDL56 shared a common target GmNPR1 which was induced by P. sojae. Overexpression and RNA interference analysis demonstrated that GmNPR1 enhanced resistance to P. sojae in transgenic soybean plants. Several pathogenesis-related (PR) genes were constitutively activated, including GmPR1a, GmPR2, GmPR3, GmPR4, GmPR5a, and GmPR10, in soybean plants overexpressing GmNPR1 transcripts. By contrast, the induction of PR genes was compromised in transgenic GmNPR1-RNAi lines. Taken together, these findings suggested that the interaction between GmWRKY31 and GmHDL56 enhances resistance to P. sojae by regulating defense-related gene expression in soybean.
Project description: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.
Project description: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. 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 (placeCityABA), salicylic acid (SA). It is located in the cytoplasm when transiently expressed in soybean protoplasts. The daidzein levels reduced greatly for the seeds of transgenic plants, while the relative content of glyceollins in transgenic plants was significantly higher than that of non-transgenic plants. Furthermore, we found that the relative expression levels of reactive oxygen species (ROS) of transgenic soybean plants were significantly lower than those of non-transgenic plants after incubation with P. sojae, suggesting an important role of GmIFR might function as an antioxidant to reduce ROS in soybean. The enzyme activity assay suggested that GmIFR has isoflavone reductase activity.
Project description:Phytophthora sojae is a destructive pathogen of soybean [Glycine max (L.) Merr.] which causes stem and root rot on soybean plants worldwide. However, the pathogenesis and molecular mechanism of plant defence responses against P. sojae are largely unclear. Herein, we document the underlying mechanisms and function of a novel BTB/POZ protein, GmBTB/POZ, which contains a BTB/POZ domain found in certain animal transcriptional regulators, in host soybean plants in response to P. sojae. It is located in the cell nucleus and is transcriptionally up-regulated by P. sojae. Overexpression of GmBTB/POZ in soybean resulted in enhanced resistance to P. sojae. The activities and expression levels of enzymatic superoxide dismutase (SOD) and peroxidase (POD) antioxidants were significantly higher in GmBTB/POZ-overexpressing (GmBTB/POZ-OE) transgenic soybean plants than in wild-type (WT) plants treated with sterile water or infected with P. sojae. The transcript levels of defence-associated genes were also higher in overexpressing plants than in WT on infection. Moreover, salicylic acid (SA) levels and the transcript levels of SA biosynthesis-related genes were markedly higher in GmBTB/POZ-OE transgenic soybean than in WT, but there were almost no differences in jasmonic acid (JA) levels or JA biosynthesis-related gene expression between GmBTB/POZ-OE and WT soybean lines. Furthermore, exogenous SA application induced the expression of GmBTB/POZ and inhibited the increase in P. sojae biomass in both WT and GmBTB/POZ-OE transgenic soybean plants. Taken together, these results suggest that GmBTB/POZ plays a positive role in P. sojae resistance and the defence response in soybean via a process that might be dependent on SA.
Project description:Phytophthora root rot (PRR) caused by Phytophthora sojae is a serious disease of soybean. The most effective disease-control strategy is to deploy resistant cultivars carrying Rps genes. Soybean cultivar Yudou25 can effectively resist pathotypes of P. sojae in China. Previous studies have mapped the Rps gene in Yudou25, RpsYD25, on chromosome 3. In this study, at first RpsYD25 was located between SSR markers Satt1k3 (2.2 cM) and BARCSOYSSR_03_0253 (4.5 cM) by using an F2:3 population containing 165 families derived from Zaoshu18 and Yudou25. Then the recombination sites were identified in 1127 F3:4 families derived from Zaoshu18 and Yudou25 using the developed PCR-based SNP, InDel and SSR markers, and RpsYD25 was finely mapped in the a 101.3 kb genomic region. In this region, a zinc ion binding and nucleic acid binding gene Glyma.03g034700 and two NBS-LRR genes Glyma.03g034800 and Glyma.03g034900 were predicted as candidate genes of RpsYD25, and five co-segregated SSR markers with RpsYD25 were identified and validated to be diagnostic markers. Combined with the resistance reaction to multiple P. sojae isolates, seven of 178 soybean genotypes were detected to contain RpsYD25 using the five co-segregated SSR markers. The soybean genotypes carrying RpsYD25 and the developed co-segregated markers can be effectively applied in the breeding for P. sojae resistance in China.