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.
Highlights
Phytophthora root rot is caused by the oomycete pathogen Phytophthora sojae Kaufmann and Gerdemann and destroys soybean crops worldwide (Schmitthenner, 1972, 1985; Tyler, 2007)
To verify interactions between proteins encoded by the candidate genes and GmPIB1, the full-length GmPIB1 gene was inserted into vector pGBKT7 and the candidate genes into vector pGADT7
Complex plant innate immune system networks have gradually developed with the evolution of plants and pathogens, and include the pathogen-associated molecular pattern-triggered immune response caused by pathogen-related molecular models and the effector-triggered immune response (Jones and Dangl, 2006)
Summary
Phytophthora root rot is caused by the oomycete pathogen Phytophthora sojae Kaufmann and Gerdemann and destroys soybean crops worldwide (Schmitthenner, 1972, 1985; Tyler, 2007). Breeding resistant cultivars is an effective and economical measure to control this disease; rapid changes in the pathogen population quickly overcome the resistance of new cultivars (Schmitthenner et al, 1996). Protein homeostasis facilitates cell senescence and protects cells from disease. As the main cellular protease complex, the 26S proteasome is at the core of maintaining protein homeostasis in cells (Smalle and Vierstra, 2004; Kurepa and Smalle, 2008). The 26S proteasome can be divided into two subcomplexes: the core particle (20S) and the regulatory particle (19S). The 19S regulatory particle consists of at least 19 proteins and the 20S core particle consists of 28 proteins. The 19S regulatory particle acts as a receptor, assisting in ubiquitination and unfolding of ubiquitinated protein substrates. Multiple catalytic sites degrade the substrate into short polypeptides, which are subsequently broken down by peptidases into peptides and amino acids that are recycled by cells (Tanaka et al, 1986; Hough et al, 1987)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
