Induction of Distinct Defense-Associated Protein Patterns in Aphanomyces euteiches (Oomycota)–Elicited and –Inoculated Medicago truncatula Cell-Suspension Cultures: A Proteome and Phosphoproteome Approach

Abstract

A comprehensive proteomic approach was applied to investigate molecular events occurring upon inoculation of Medicago truncatula cell-suspension cultures with the oomycete root pathogen Aphanomyces euteiches. Establishment of an inoculation assay in the cell cultures allowed a direct comparison between proteins induced by elicitation with a crude culture extract of the oomycete and by inoculation with A. euteiches zoospores representing the natural infection carrier. Oxidative burst assays revealed responsiveness of the cell cultures for perception of elicitation and inoculation signals. The plant "elicitation proteome" resembles the "inoculation proteome" in early incubation stages and includes proteins induced following initial oxidative burst and defense reactions, but also proteins involved in the antioxidative system. However, approximately 2 days after incubation, the inoculation proteome differs drastically from the proteome of elicited cultures, where a cessation of responses assignable to A. euteiches elicitation occurred. The specific protein induction patterns of zoospore-inoculated cells appeared consistent with the protein induction identified in recent studies for an A. euteiches infection in planta and consist of three functional groups: i) pathogenesis-related proteins, ii) proteins associated with secondary phenylpropanoid or phytoalexin metabolism, and, particularly, iii) proteins assigned to carbohydrate metabolism and energy-related cellular processes. Phosphoproteomic analyses revealed consistent and specific activation of these defense-related pathways already at very early timepoints of inoculation, providing evidence that the identified protein profiles are representative for an established A. euteiches infection of M. truncatula.

Silencing of the Rac1 GTPaseMtROP9inMedicago truncatulaStimulates Early Mycorrhizal and Oomycete Root Colonizations But Negatively Affects Rhizobial Infection

“…Therefore, we examined whether the suppression of MtROP9 alters ROS levels in MtROP9i roots. ROS accumulation was measured in root segments of MtROP9i, Mtvector, and Mtwt after inoculation by a luminol-based chemiluminescence assay to determine the production of H 2 O 2 during a period of 1 h after the application of two spore inocula: (1) 500 mL of lake water containing 250,000 Aphanomyces euteiches vital zoospores, as described previously (Trapphoff et al, 2009); and (2) 500 mL of water inoculum containing 10,000 viable Glomus intraradices spores. These measurements revealed clearly reduced ROS production in the MtROP9i transgenic roots as compared with the Mtwt and Mtvector root segments (Fig.…”

“…Oxidative burst measurements were performed using a luminol-based chemiluminescence assay to determine the production of H 2 O 2 during a period of 1 h after application of microbial fractions to root fragments of MtROP9i, Mtvector, and Mtwt as described previously (Trapphoff et al, 2009). For the measurements, 20 root pieces of 0.5 cm length were collected from MtROP9i, Mtvector, and Mtwt each and transferred to 200 mL of tap water in illuminometer cuvettes.…”

“…They were kept for 4 h in the dark at room temperature. Afterward, the tap water was removed and root fragments were subjected to different experimental treatments: (1) 500 mL of lake water containing 250,000 A. euteiches vital zoospores as described previously (Trapphoff et al, 2009); (2) 500 mL of water inoculum containing 10,000 viable G. intraradices spores (Premier Tech); (3) 500 mL of S. meliloti bacterial culture (optical density at 600 nm of 1) in PS buffer; (4) 500 mL of S. meliloti NF (10 27 M) in PS buffer; and, as controls, (5) an equal volume of lake water (control for 1); (6) deionized water (control for 2); and (7) PS buffer (controls for 3 and 4). In addition, we applied a G. intraradices spore inoculum that had been incubated for 2 d at 25°C in the dark.…”

Large-Scale Phosphoprotein Analysis inMedicago truncatulaRoots Provides Insight into in Vivo Kinase Activity in Legumes

“…Several characterized protein kinases are required for symbiosis signal transduction in M. truncatula roots (Lévy et al, 2004;Yoshida and Parniske, 2005;Smit et al, 2007). A recent antibodybased study of cultured M. truncatula cells observed protein phosphorylation changes at the proteomic level in response to fungal infection (Trapphoff et al, 2009); however, the target residues of the phosphorylation events were not determined. A variety of studies have determined in vitro phosphorylation sites on legume proteins and demonstrated the biological importance of the target residues by mutagenesis (Yoshida and Parniske, 2005;Arrighi et al, 2006;Lima et al, 2006;Miyahara et al, 2008;Yano et al, 2008).…”

From callus to embryo: a proteomic view on the development and maturation of somatic embryos in Cyclamen persicum

“…(3) Is there a Cyclamenspecific auxin accumulation profile? To resolve the new questions concerning post-translational modifications a gelor mass spectrometry-based approach like performed by Trapphoff et al (2009) or Grimsrud et al (2010 could be helpful. A metabolic profiling of auxin levels in the different stages of embryogenesis of Cyclamen could elucidate the dynamics of this plant hormone and its correlation to auxin-amidohydrolase and auxin-storage conjugates.…”