Abstract
ROP-type GTPases of plants function as molecular switches within elementary signal transduction pathways such as the regulation of ROS synthesis via activation of NADPH oxidases (RBOH-respiratory burst oxidase homolog in plants). Previously, we reported that silencing of the Medicago truncatula GTPase MtROP9 led to reduced ROS production and suppressed induction of ROS-related enzymes in transgenic roots (MtROP9i) infected with pathogenic (Aphanomyces euteiches) and symbiotic microorganisms (Glomus intraradices, Sinorhizobium meliloti). While fungal infections were enhanced, S. meliloti infection was drastically impaired. In this study, we investigate the temporal proteome response of M. truncatula MtROP9i transgenic roots during the same microbial interactions under conditions of deprived potential to synthesize ROS. In comparison with control roots (Mtvector), we present a comprehensive proteomic analysis using sensitive MS protein identification. For four early infection time-points (1, 3, 5, 24 hpi), 733 spots were found to be different in abundance: 213 spots comprising 984 proteins (607 unique) were identified after S. meliloti infection, 230 spots comprising 796 proteins (580 unique) after G. intraradices infection, and 290 spots comprising 1240 proteins (828 unique) after A. euteiches infection. Data evaluation by GelMap in combination with a heatmap tool allowed recognition of key proteome changes during microbial interactions under conditions of hampered ROS synthesis. Overall, the number of induced proteins in MtROP9i was low as compared with controls, indicating a dual function of ROS in defense signaling as well as alternative response patterns activated during microbial infection. Qualitative analysis of induced proteins showed that enzymes linked to ROS production and scavenging were highly induced in control roots, while in MtROP9i the majority of proteins were involved in alternative defense pathways such as cell wall and protein degradation.
Highlights
RAC/ROP (Rho of plants) are plant-specific small GTPases that function as simple binary molecular switches within elementary signal transduction pathways by cycling between GTP-bound on modes and GDP-bound off modes
reactive oxygen species (ROS) generation is mainly related to activation of RBOH proteins which is achieved by protein interaction with cellular small GTPases, functioning as molecular switches
From the current investigations, we have shown that silencing of small GTPase MtROP9 in M. truncatula roots results in significant suppression of ROS-related enzymes at the early points of infection both for symbiotic and pathogenic interactions and a concomitant activation of alternative defense pathways by the host cell to counteract the infection progress
Summary
RAC/ROP (Rho of plants) are plant-specific small GTPases that function as simple binary molecular switches within elementary signal transduction pathways by cycling between GTP-bound on modes and GDP-bound off modes. ROPs are known to function in different developmental processes including polarized cell growth, pollen tube and root hair development, hormonal signaling as well as cell morphogenesis (Yang and Fu, 2007; Liu et al, 2010). They are implicated in regulating several cellular processes including vesicle trafficking, cytoskeleton organization and dynamics, auxin transport and response to pathogens (Nibau et al, 2006; Yalovsky et al, 2008; Lorek et al, 2010; Wu et al, 2011; Poraty-Gavra et al, 2013). In Arabidopsis thaliana, 11 ROPs are identified (Winge et al, 1997)
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