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

Abstract

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.

Gene regulatory network inference in soybean upon infection byPhytophthora sojae

“…In the present study, K- means clustering of DEGs rendered nine gene co-expression clusters, seven of which were up- regulated and corresponded to defense-related functional annotations (e.g., MAPK signaling and secondary metabolism). Cooperatively, these clusters were enriched with statistically overrepresented TF families (i.e., MYB, WRKY, NAC, ERF, and C2H2) known to regulate plant secondary metabolism [22, 23] and reported in prior soybean- P. sojae studies [29,30,32,33,34,35,37,83]. Furthermore, WRKY and ERF were the most abundant TF families across the seven clusters.…”

“…While the abundance and diversity of TFs required for immunity vary across plant species and pathosystems [21], elucidated sensory regulatory networks tend to possess members of the bHLH, bZIP, ERF, MYB, NAC, and WRKY families [17,22,23] that collectively direct transcriptional reprogramming of downstream target genes [16]. Isolated studies have evidenced transcriptional reprogramming in soybean upon infection by P. sojae [24,25,26,27] and have identified various TFs associated with defense [28,29,30,31,32,33,34,35,36,37]; yet mechanistic insight regarding TF-target gene interactions and their organization within larger hierarchical networks is lacking. This systems-level information can be unraveled using gene regulatory networks (GRNs) [38], and regulatory hubs identified subsequently through analyses of network tunability and redundancy [21].…”

An updated assessment of the soybean–Phytophthora sojae pathosystem

“…sojae. Building upon this work, Liu, Wang, et al (2021) showed that GmPIB1 interacts with the 26S proteasome subunit PSMD11 in luciferase complementation and yeast two-hybrid assays. RNAi and over-expressing hairy root cultures revealed that PSMD11 mediates antioxidant activity upon P. sojae infection (Liu, Wang, et al, 2021).…”

“…RNAi and over‐expressing hairy root cultures revealed that PSMD11 mediates antioxidant activity upon P . sojae infection (Liu, Wang, et al, 2021). Similarly, RNAi‐mediated suppression of GmWRKY40 increased host susceptibility to P .…”

Gene regulatory network inference in soybean upon infection by Phytophthora sojae

“…While the abundance and diversity of TFs required for immunity vary across plant species and pathosystems [21], elucidated sensory regulatory networks tend to possess members of the bHLH, bZIP, ERF, MYB, NAC, and WRKY families [17,22,23] that collectively direct transcriptional reprogramming of downstream target genes [16]. Isolated studies have evidenced transcriptional reprogramming in soybean upon infection by P. sojae [24][25][26][27] and have identified various TFs associated with defense [28][29][30][31][32][33][34][35][36][37]; yet mechanistic insight regarding TF-target gene interactions and their organization within larger hierarchical networks is lacking [38]. This systems-level information can be unraveled using gene regulatory networks (GRNs) [38,39], and regulatory hubs identified subsequently through analyses of network tunability and redundancy [21].…”