Identification of transcriptional regulatory nodes in soybean defense networks using transient co-transactivation assays

Abstract

Plant responses to major environmental stressors, such as insect feeding, not only occur via the functions of defense genes but also involve a series of regulatory factors. Our previous transcriptome studies proposed that, in addition to two defense-related genes, GmVSPβ and GmN:IFR, a high proportion of transcription factors (TFs) participate in the incompatible soybean-common cutworm interaction networks. However, the regulatory mechanisms and effects of these TFs on those induced defense-related genes remain unknown. In the present work, we isolated and identified 12 genes encoding MYB, WRKY, NAC, bZIP, and DREB TFs from a common cutworm-induced cDNA library of a resistant soybean line. Sequence analysis of the promoters of three co-expressed genes, including GmVSPα, GmVSPβ, and GmN:IFR, revealed the enrichment of various TF-binding sites for defense and stress responses. To further identify the regulatory nodes composed of these TFs and defense gene promoters, we performed extensive transient co-transactivation assays to directly test the transcriptional activity of the 12 TFs binding at different levels to the three co-expressed gene promoters. The results showed that all 12 TFs were able to transactivate the GmVSPβ and GmN:IFR promoters. GmbZIP110 and GmMYB75 functioned as distinct regulators of GmVSPα/β and GmN:IFR expression, respectively, while GmWRKY39 acted as a common central regulator of GmVSPα/β and GmN:IFR expression. These corresponding TFs play crucial roles in coordinated plant defense regulation, which provides valuable information for understanding the molecular mechanisms involved in insect-induced transcriptional regulation in soybean. More importantly, the identified TFs and suitable promoters can be used to engineer insect-resistant plants in molecular breeding studies.

Genetic Architecture of Charcoal Rot (Macrophomina phaseolina) Resistance in Soybean Revealed Using a Diverse Panel

“…On chromosome 18 and 20, the APETALA2/ethylene-responsive element binding protein ( AP2 / EREBP ) plays various roles in plant growth and development, and in response to stresses as pathogen infection, drought, temperature, and salinity (Zhang et al, 2013 ; Tang et al, 2016 ). It has been reported that the ethylene pathway was activated in response to the pathogen attack in several crop species (Dong et al, 2015 ; Wang et al, 2015 ; Agarwal et al, 2016 ; Hong et al, 2016 ).…”

Knockdown of GmVQ58 encoding a VQ motif-containing protein enhances soybean resistance to the common cutworm (Spodoptera litura Fabricius)

“…GmN : IFR ( Glyma.01g172600 ) and GmVSPβ ( Glyma.08g200100 ), two CCW-responsive genes, positively regulate plant resistance to CCW in tobacco ( Wang et al , 2015 d ). Our previous studies showed that GmWRKY32 (Glyma.02g115200) could strongly activate the transcription of GmN : IFR (8.5-fold) and GmVSPβ (4.8-fold) in Arabidopsis protoplasts ( Wang et al , 2015 c ). Additionally, several members of the VQ family have been reported to physically interact with WRKY transcription factors to participate in plant defense reactions ( Cheng et al , 2012 ).…”

“…Through database analysis, GmVSPβ (encoding a vegetative storage protein), GmN:IFR (encoding an NADPH:isoflavone reductase) and GmWRKY32 (encoding a WRKY-type transcription factor) showed differential expression patterns between CCW-resistant and CCW-susceptible soybean lines. GmVSPβ and GmN:IFR positively regulate plant resistance to CCW in tobacco ( Wang et al , 2015 d ), and GmWRKY32 strongly activated the transcription of these genes in Arabidopsis protoplasts ( Wang et al , 2015 c ). In addition, many other differentially expressed genes were detected in our transcriptome database; these genes were involved in several important cellular processes, including primary signaling related to defense, transcriptional regulation, and secondary metabolism.…”

Identification of Two bZIP Transcription Factors Interacting with the Promoter of Soybean Rubisco Activase Gene (GmRCAα)

“…In higher plants, Arabidopsis protoplasts system was widely used to study the regulation between TFs and its binding promoter ( Yi et al, 2010 ; Figueiredo et al, 2012 ; Wang et al, 2015 ). A transient assay in Arabidopsis protoplasts was performed to investigate the effects of GmbZIP04g and GmbZIP07g on GmRCA α promoter activity, and the LUC/REN values of GmbZIP04g and GmbZIP07g were higher than that of the empty vector (Supplementary Figure S1 and Supplementary Table S3 ).…”

“…Arabidopsis protoplasts were isolated and transformed according to the method described by Wang et al (2015) . The effector, reporter and internal control were co-transformed into Arabidopsis protoplasts with 10, 5, and 0.5 μg, respectively.…”