The EIN3 transcription factor GmEIL1 improves soybean resistance to Phytophthora sojae.

Phytophthora root and stem rot of soybean (Glycine max), caused by the oomycete Phytophthora sojae, is an extremely destructive disease worldwide. In this study, we identified GmEIL1, which encodes an ethylene-insensitive3 (EIN3) transcription factor. GmEIL1 was significantly induced following P. sojae infection of soybean plants. Compared to wild-type soybean plants, transgenic soybean plants overexpressing GmEIL1 showed enhanced resistance to P. sojae and GmEIL1-silenced RNA-interference lines showed more severe symptoms when infected with P. sojae. We screened for target genes of GmEIL1 and confirmed that GmEIL1 bound directly to the GmERF113 promoter and regulated GmERF113 expression. Moreover, GmEIL1 positively regulated the expression of the pathogenesis-related gene GmPR1. The GmEIL1-regulated defence response to P. sojae involved both ethylene biosynthesis and the ethylene signalling pathway. These findings suggest that the GmEIL1-GmERF113 module plays an important role in P. sojae resistance via the ethylene signalling pathway.

The MYB Transcription Factor GmMYB78 Negatively Regulates Phytophthora sojae Resistance in Soybean.

Phytophthora root rot is a devastating disease of soybean caused by Phytophthora sojae. However, the resistance mechanism is not yet clear. Our previous studies have shown that GmAP2 enhances sensitivity to P. sojae in soybean, and GmMYB78 is downregulated in the transcriptome analysis of GmAP2-overexpressing transgenic hairy roots. Here, GmMYB78 was significantly induced by P. sojae in susceptible soybean, and the overexpressing of GmMYB78 enhanced sensitivity to the pathogen, while silencing GmMYB78 enhances resistance to P. sojae, indicating that GmMYB78 is a negative regulator of P. sojae. Moreover, the jasmonic acid (JA) content and JA synthesis gene GmAOS1 was highly upregulated in GmMYB78-silencing roots and highly downregulated in overexpressing ones, suggesting that GmMYB78 could respond to P. sojae through the JA signaling pathway. Furthermore, the expression of several pathogenesis-related genes was significantly lower in GmMYB78-overexpressing roots and higher in GmMYB78-silencing ones. Additionally, we screened and identified the upstream regulator GmbHLH122 and downstream target gene GmbZIP25 of GmMYB78. GmbHLH122 was highly induced by P. sojae and could inhibit GmMYB78 expression in resistant soybean, and GmMYB78 was highly expressed to activate downstream target gene GmbZIP25 transcription in susceptible soybean. In conclusion, our data reveal that GmMYB78 triggers soybean sensitivity to P. sojae by inhibiting the JA signaling pathway and the expression of pathogenesis-related genes or through the effects of the GmbHLH122-GmMYB78-GmbZIP25 cascade pathway.

A transposon insertion in the promoter of OsUBC12 enhances cold tolerance during japonica rice germination.

Low-temperature germination (LTG) is an important agronomic trait for rice (Oryza sativa). Japonica rice generally has greater capacity for germination at low temperatures than the indica subpopulation. However, the genetic basis and molecular mechanisms underlying this complex trait are poorly understood. Here, we report that OsUBC12, encoding an E2 ubiquitin-conjugating enzyme, increases low-temperature germinability in japonica, owing to a transposon insertion in its promoter enhancing its expression. Natural variation analysis reveals that transposon insertion in the OsUBC12 promoter mainly occurs in the japonica lineage. The variation detected in eight representative two-line male sterile lines suggests the existence of this allele introgression by indica-japonica hybridization breeding, and varieties carrying the japonica OsUBC12 locus (transposon insertion) have higher low-temperature germinability than varieties without the locus. Further molecular analysis shows that OsUBC12 negatively regulate ABA signaling. OsUBC12-regulated seed germination and ABA signaling mainly depend on a conserved active site required for ubiquitin-conjugating enzyme activity. Furthermore, OsUBC12 directly associates with rice SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 1.1 (OsSnRK1.1), promoting its degradation. OsSnRK1.1 inhibits LTG by enhancing ABA signaling and acts downstream of OsUBC12. These findings shed light on the underlying mechanisms of UBC12 regulating LTG and provide genetic reference points for improving LTG in indica rice.

OsMAPK6 positively regulates rice cold tolerance at seedling stage via phosphorylating and stabilizing OsICE1 and OsIPA1.

Rice is a chilling-sensitive plant, and extremely low temperatures seriously decrease rice production. Several genes involved in chilling stress have been reported in rice; however, the chilling signaling in rice remains largely unknown. Here, we investigated the chilling tolerance phenotype of overexpression of constitutive active OsMAPK6 (CAMAPK6-OE) and OsMAPK6 mutant dsg1, and demonstrated that OsMAPK6 positively regulated rice chilling tolerance. It was shown that, under cold stress, the survival rate of dsg1 was significantly lower than that of WT, whereas CAMAPK6-OE display higher survival rate than WT. Physiological assays indicate that ion leakage and dead cell in dsg1 was much more severe than those in WT and CAMAPK6-OE. Consistently, expression of chilling responsive genes in dsg1, including OsCBFs and OsTPP1, was significantly lower than that of in WT and CAMAPK6-OE. Biochemical analyses revealed that chilling stress promotes phosphorylation of OsMAPK6. Besides, we found that OsMAPK6 interacts with and phosphorylates two key regulators in rice cold signaling, OsIPA1 and OsICE1, and then enhance their protein stability. Overall, our results revealed a cold-induced OsMAPK6-OsICE1/OsIPA1 signaling cascade by which OsMAPK6 was involved in rice chilling tolerance, which provides novel insights to understand rice cold response at seedling stage.

Adaptation to high latitudes through a novel allele of Hd3a strongly promoting heading date in rice.

KEY MESSAGE: A novel Hd3a allele strongly promoting rice heading date was identified, and it functions through florigen activation complex (FAC) and was selected during the spread of rice cultivation to high-latitude areas. Heading date is a critical agronomic trait for rice that determines the utilization of light and temperature conditions and thereby affects grain yield. Rice is a short day (SD) plant, and its photoperiodic information is processed by complex pathways and integrated by florigens to control flowering. In this study, we identified a novel allele for the florigen gene Heading date 3a (Hd3a), characterized by a C435G substitution in its coding region, by a genome-wide association study (GWAS) approach in a panel of 199 high-latitude japonica rice varieties. The C435G substitution induces plants to flower 10 days earlier in high-latitude area (long day condition). Then, we mutated C435 to G in Hd3a by prime editing and found the point mutation plants flowered 12 days earlier. Further molecular experiments showed the novel Hd3a protein can interact with GF14b protein and increase the expression of OsMADS14, the output gene of florigen activation complex (FAC). Molecular signatures of selection indicated that the novel Hd3a allele was selected during the process of rice cultivation expansion into high-latitude areas. Collectively, these results provide new insights into heading date regulation in high-latitude areas and advance improvements to rice adaptability to enhance crop yield.

Evaluation of Short-Season Soybean Genotypes for Resistance and Partial Resistance to Phytophthora sojae.

Phytophthora root and stem rot caused by Phytophthora sojae Kaufmann and Gerdemann is a soil-borne disease severely affecting soybean production worldwide. Losses caused by P. sojae can be controlled by both major genes and quantitative trait locus. Here, we tested 112 short-season soybean cultivars from Northeast China for resistance to P. sojae. A total of 58 germplasms were resistant to 7-11 P. sojae strains. Among these, Mengdou 28 and Kejiao 10-262 may harbor either Rps3a or multiple Rps genes conferring resistance to P. sojae. The remaining 110 germplasms produced 91 reaction types and may contain new resistance genes or gene combinations. Partial resistance evaluation using the inoculum layer method revealed that 34 soybean germplasms had high partial resistance, with a mean disease index lower than 30. Combining the results of resistance and partial resistance analyses, we identified 35 excellent germplasm resources as potential elite materials for resistance and tolerance in future breeding programs. In addition, we compared the radicle inoculation method with the inoculum layer method to screen for partial resistance to P. sojae. Our results demonstrate that the radicle inoculation method could potentially replace the inoculum layer method to identify partial resistance against P. sojae, and further verification with larger samples is required in the future.

Ubiquitin-Conjugating Enzyme OsUBC11 Affects the Development of Roots via Auxin Pathway.

Rice has 48 ubiquitin-conjugating enzymes, and the functions of most of these enzymes have not been elucidated. In the present study, a T-DNA insertional mutant named R164, which exhibited a significant decrease in the length of primary and lateral roots, was used as the experimental material to explore the potential function of OsUBC11. Analysis using the SEFA-PCR method showed that the T-DNA insertion was present in the promoter region of OsUBC11 gene, which encodes ubiquitin-conjugating enzyme (E2), and activates its expression. Biochemical experiments showed that OsUBC11 is a lysine-48-linked ubiquitin chain-forming conjugase. OsUBC11 overexpression lines showed the same root phenotypes. These results demonstrated that OsUBC11 was involved in root development. Further analyses showed that the IAA content of R164 mutant and OE3 line were significantly lower compared with wild-type Zhonghua11. Application of exogenous NAA restored the length of lateral and primary roots in R164 and OsUBC11 overexpression lines. Expression of the auxin synthesis regulating gene OsYUCCA4/6/7/9, the auxin transport gene OsAUX1, auxin/indole-3-acetic acid (Aux/IAA) family gene OsIAA31, auxin response factor OsARF16 and root regulator key genes, including OsWOX11, OsCRL1, OsCRL5 was significantly down-regulated in OsUBC11 overexpressing plants. Collectively, these results indicate that OsUBC11 modulates auxin signaling, ultimately affecting root development at the rice seedling stage.

GmWAK1, Novel Wall-Associated Protein Kinase, Positively Regulates Response of Soybean to Phytophthora sojae Infection.

Phytophthora root rot is a destructive soybean disease worldwide, which is caused by the oomycete pathogen Phytophthora sojae (P. sojae). Wall-associated protein kinase (WAK) genes, a family of the receptor-like protein kinase (RLK) genes, play important roles in the plant signaling pathways that regulate stress responses and pathogen resistance. In our study, we found a putative Glycine max wall-associated protein kinase, GmWAK1, which we identified by soybean GmLHP1 RNA-sequencing. The expression of GmWAK1 was significantly increased by P. sojae and salicylic acid (SA). Overexpression of GmWAK1 in soybean significantly improved resistance to P. sojae, and the levels of phenylalanine ammonia-lyase (PAL), SA, and SA-biosynthesis-related genes were markedly higher than in the wild-type (WT) soybean. The activities of enzymatic superoxide dismutase (SOD) and peroxidase (POD) antioxidants in GmWAK1-overexpressing (OE) plants were significantly higher than those in in WT plants treated with P. sojae; reactive oxygen species (ROS) and hydrogen peroxide (H2O2) accumulation was considerably lower in GmWAK1-OE after P. sojae infection. GmWAK1 interacted with annexin-like protein RJ, GmANNRJ4, which improved resistance to P. sojae and increased intracellular free-calcium accumulation. In GmANNRJ4-OE transgenic soybean, the calmodulin-dependent kinase gene GmMPK6 and several pathogenesis-related (PR) genes were constitutively activated. Collectively, these results indicated that GmWAK1 interacts with GmANNRJ4, and GmWAK1 plays a positive role in soybean resistance to P. sojae via a process that might be dependent on SA and involved in alleviating damage caused by oxidative stress.

Ca2+ deficiency triggers panicle degeneration in rice mediated by Ca2+ /H+ exchanger OsCAX1a.

Increasing rice yield has always been one of the primary objectives of rice breeding. However, panicle degeneration often occurs in rice-growing regions and severely curbs rice yield. In this study, we obtained a new apical panicle degeneration mutant, which induces a marked degeneration rate and diminishes the final grain yield. Cellular and physiological analyses revealed that the apical panicle undergoes programmed cell death, accompanied by excessive accumulations of peroxides. Following, the panicle degeneration gene OsCAX1a was identified in the mutant, which was involved in Ca2+ transport. Hydroponics assays and Ca2+ quantification confirmed that Ca2+ transport and distribution to apical tissues were restricted and over-accumulated in the mutant sheath. Ca2+ transport between cytoplasm and vacuole was affected, and the reduced Ca2+ content in the vacuole and cell wall of the apical panicle and the decreased Ca2+ absorption appeared in the mutant. RNA-Seq data indicated that the abnormal CBL (calcineurin b-like proteins) pathway mediated by deficient Ca2+ might occur in the mutant, resulting in the burst of ROS and programmed cell death in panicles. Our results explained the key role of OsCAX1a in Ca2+ transport and distribution and laid a foundation to further explore the genetic and molecular mechanisms of panicle degeneration in rice.

The AP2/ERF GmERF113 Positively Regulates the Drought Response by Activating GmPR10-1 in Soybean.

Ethylene response factors (ERFs) are involved in biotic and abiotic stress; however, the drought resistance mechanisms of many ERFs in soybeans have not been resolved. Previously, we proved that GmERF113 enhances resistance to the pathogen Phytophthora sojae in soybean. Here, we determined that GmERF113 is induced by 20% PEG-6000. Compared to the wild-type plants, soybean plants overexpressing GmERF113 (GmERF113-OE) displayed increased drought tolerance which was characterized by milder leaf wilting, less water loss from detached leaves, smaller stomatal aperture, lower Malondialdehyde (MDA) content, increased proline accumulation, and higher Superoxide dismutase (SOD) and Peroxidase (POD) activities under drought stress, whereas plants with GmERF113 silenced through RNA interference were the opposite. Chromatin immunoprecipitation and dual effector-reporter assays showed that GmERF113 binds to the GCC-box in the GmPR10-1 promoter, activating GmPR10-1 expression directly. Overexpressing GmPR10-1 improved drought resistance in the composite soybean plants with transgenic hairy roots. RNA-seq analysis revealed that GmERF113 downregulates abscisic acid 8'-hydroxylase 3 (GmABA8'-OH 3) and upregulates various drought-related genes. Overexpressing GmERF113 and GmPR10-1 increased the abscisic acid (ABA) content and reduced the expression of GmABA8'-OH3 in transgenic soybean plants and hairy roots, respectively. These results reveal that the GmERF113-GmPR10-1 pathway improves drought resistance and affects the ABA content in soybean, providing a theoretical basis for the molecular breeding of drought-tolerant soybean.

GmMKK4-activated GmMPK6 stimulates GmERF113 to trigger resistance to Phytophthora sojae in soybean.

Phytophthora root and stem rot is a worldwide soybean (Glycine max) disease caused by the soil-borne pathogen Phytophthora sojae. This disease is devastating to soybean production, so improvement of resistance to P. sojae is a major target in soybean breeding. Mitogen-activated protein kinase (MAPK) cascades are important signaling modules that convert environmental stimuli into cellular responses. Compared with extensive studies in Arabidopsis, the molecular mechanism of MAPK cascades in soybean disease resistance is barely elucidated. In this work, we found that the gene expression of mitogen-activated protein kinase 6 (GmMPK6) was potently induced by P. sojae infection in the disease-resistant soybean cultivar 'Suinong 10'. Overexpression of GmMPK6 in soybean resulted in enhanced resistance to P. sojae and silencing of GmMPK6 led to the opposite phenotype. In our attempt to dissect the role of GmMPK6 in soybean resistance to phytophthora disease, we found that MAPK kinase 4 (GmMKK4) and the ERF transcription factor GmERF113 physically interact with GmMPK6, and we determined that GmMKK4 could phosphorylate and activate GmMPK6, which could subsequently phosphorylate GmERF113 upon P. sojae infection, suggesting that P. sojae can stimulate the GmMKK4-GmMPK6-GmERF113 signaling pathway in soybean. Moreover, phosphorylation of GmERF113 by the GmMKK4-GmMPK6 module promoted GmERF113 stability, nuclear localization and transcriptional activity, which significantly enhanced expression of the defense-related genes GmPR1 and GmPR10-1 and hence improved disease resistance of the transgenic soybean seedlings. In all, our data reveal that the GmMKK4-GmMPK6-GmERF113 cascade triggers resistance to P. sojae in soybean and shed light on functions of MAPK kinases in plant disease resistance.

The BTB/POZ domain protein GmBTB/POZ promotes the ubiquitination and degradation of the soybean AP2/ERF-like transcription factor GmAP2 to regulate the defense response to Phytophthora sojae.

Phytophthora root and stem rot in soybean (Glycine max) is a destructive disease worldwide, and hence improving crop resistance to the causal pathogen, P. sojae, is a major target for breeders. However, it remains largely unclear how the pathogen regulates the various affected signaling pathways in the host, which consist of complex networks including key transcription factors and their targets. We have previously demonstrated that GmBTB/POZ enhances soybean resistance to P. sojae and the associated defense response. Here, we demonstrate that GmBTB/POZ interacts with the transcription factor GmAP2 and promotes its ubiquitination. GmAP2-RNAi transgenic soybean hairy roots exhibited enhanced resistance to P. sojae, whereas roots overexpressing GmAP2 showed hypersensitivity. GmWRKY33 was identified as a target of GmAP2, which represses its expression by directly binding to the promoter. GmWRKY33 acts as a positive regulator in the response of soybean to P. sojae. Overexpression of GmBTB/POZ released the GmAP2-regulated suppression of GmWRKY33 in hairy roots overexpressing GmAP2 and increased their resistance to P. sojae. Taken together, our results indicate that GmBTB/POZ-GmAP2 modulation of the P. sojae resistance response forms a novel regulatory mechanism, which putatively regulates the downstream target gene GmWRKY33 in soybean.

GmBTB/POZ promotes the ubiquitination and degradation of LHP1 to regulate the response of soybean to Phytophthora sojae.

Phytophthora sojae is a pathogen that causes stem and root rot in soybean (Glycine max [L.] Merr.). We previously demonstrated that GmBTB/POZ, a BTB/POZ domain-containing nuclear protein, enhances resistance to P. sojae in soybean, via a process that depends on salicylic acid (SA). Here, we demonstrate that GmBTB/POZ associates directly with soybean LIKE HETEROCHROMATIN PROTEIN1 (GmLHP1) in vitro and in vivo and promotes its ubiquitination and degradation. Both overexpression and RNA interference analysis of transgenic lines demonstrate that GmLHP1 negatively regulates the response of soybean to P. sojae by reducing SA levels and repressing GmPR1 expression. The WRKY transcription factor gene, GmWRKY40, a SA-induced gene in the SA signaling pathway, is targeted by GmLHP1, which represses its expression via at least two mechanisms (directly binding to its promoter and impairing SA accumulation). Furthermore, the nuclear localization of GmLHP1 is required for the GmLHP1-mediated negative regulation of immunity, SA levels and the suppression of GmWRKY40 expression. Finally, GmBTB/POZ releases GmLHP1-regulated GmWRKY40 suppression and increases resistance to P. sojae in GmLHP1-OE hairy roots. These findings uncover a regulatory mechanism by which GmBTB/POZ-GmLHP1 modulates resistance to P. sojae in soybean, likely by regulating the expression of downstream target gene GmWRKY40.

The 26S Proteasome Regulatory Subunit GmPSMD Promotes Resistance to Phytophthora sojae in Soybean.

Phytophthora root rot, caused by Phytophthora sojae is a destructive disease of soybean (Glycine max) worldwide. We previously confirmed that the bHLH transcription factor GmPIB1 (P. sojae-inducible bHLH transcription factor) reduces accumulation of reactive oxygen species (ROS) in cells by inhibiting expression of the peroxidase-related gene GmSPOD thus improving the resistance of hairy roots to P. sojae. To identify proteins interacting with GmPIB1 and assess their participation in the defense response to P. sojae, we obtained transgenic soybean hairy roots overexpressing GmPIB1 by Agrobacterium rhizogenes mediated transformation and examined GmPIB1 protein-protein interactions using immunoprecipitation combined with mass spectrometry. We identified 392 proteins likely interacting with GmPIB1 and selected 20 candidate genes, and only 26S proteasome regulatory subunit GmPSMD (Genbank accession no. XP_014631720) interacted with GmPIB1 in luciferase complementation and pull-down experiments and yeast two-hybrid assays. Overexpression of GmPSMD (GmPSMD-OE) in soybean hairy roots remarkably improved resistance to P. sojae and RNA interference of GmPSMD (GmPSMD -RNAi) increased susceptibility. In addition, accumulation of total ROS and hydrogen peroxide (H2O2) in GmPSMD-OE transgenic soybean hairy roots were remarkably lower than those of the control after P. sojae infection. Moreover, in GmPSMD-RNAi transgenic soybean hairy roots, H2O2 and the accumulation of total ROS exceeded those of the control. There was no obvious difference in superoxide anion (O2 -) content between control and transgenic hairy roots. Antioxidant enzymes include peroxidase (POD), glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT) are responsible for ROS scavenging in soybean. The activities of these antioxidant enzymes were remarkably higher in GmPSMD-OE transgenic soybean hairy roots than those in control, but were reduced in GmPSMD-RNAi transgenic soybean hairy roots. Moreover, the activity of 26S proteasome in GmPSMD-OE and GmPIB1-OE transgenic soybean hairy roots was significantly higher than that in control and was significantly lower in PSMD-RNAi soybean hairy roots after P. sojae infection. These data suggest that GmPSMD might reduce the production of ROS by improving the activity of antioxidant enzymes such as POD, SOD, GPX, CAT, and GmPSMD plays a significant role in the response of soybean to P. sojae. Our study reveals a valuable mechanism for regulation of the pathogen response by the 26S proteasome in soybean.

GmSnRK1.1, a Sucrose Non-fermenting-1(SNF1)-Related Protein Kinase, Promotes Soybean Resistance to Phytophthora sojae.

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.

GmBTB/POZ, a novel BTB/POZ domain-containing nuclear protein, positively regulates the response of soybean to Phytophthora sojae infection.

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.

Overexpression of a soybean 4-coumaric acid: coenzyme A ligase (GmPI4L) enhances resistance to Phytophthora sojae in soybean.

Phytophthora root and stem rot of soybean (Glycine max (L.) Merr.) caused by Phytophthora sojae is a destructive disease worldwide. The enzyme 4-coumarate: CoA ligase (4CL) has been extensively studied with regard to plant responses to pathogens. However, the molecular mechanism of the response of soybean 4CL to P. sojae remains unclear. In a previous study, a highly upregulated 4CL homologue was characterised through suppressive subtractive hybridisation library and cDNA microarrays, in the resistant soybean cultivar 'Suinong 10' after infection with P. sojae race 1. Here, we isolated the full-length EST, and designated as GmPI4L (P. sojae-inducible 4CL gene) in this study, which is a novel member of the soybean 4CL gene family. GmPI4L has 34-43% over all amino acid sequence identity with other plant 4CLs. Overexpression of GmPI4L enhances resistance to P. sojae in transgenic soybean plants. The GmPI4L is located in the cell membrane when transiently expressed in Arabidopsis protoplasts. Further analyses showed that the contents of daidzein, genistein, and the relative content of glyceollins are significantly increased in overexpression GmPI4L soybeans. Taken together, these results suggested that GmPI4L plays an important role in response to P. sojae infection, possibly by enhancing the content of glyceollins, daidzein, and genistein in soybean.

Phenylalanine ammonia-lyase2.1 contributes to the soybean response towards Phytophthora sojae infection.

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.

A Novel Soybean Dirigent Gene GmDIR22 Contributes to Promotion of Lignan Biosynthesis and Enhances Resistance to Phytophthora sojae.

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.