Identification of Candidate Ergosterol-Responsive Proteins Associated with the Plasma Membrane of Arabidopsis thaliana

Abstract

The impact of fungal diseases on crop production negatively reflects on sustainable food production and overall economic health. Ergosterol is the major sterol component in fungal membranes and regarded as a general elicitor or microbe-associated molecular pattern (MAMP) molecule. Although plant responses to ergosterol have been reported, the perception mechanism is still unknown. Here, Arabidopsis thaliana protein fractions were used to identify those differentially regulated following ergosterol treatment; additionally, they were subjected to affinity-based chromatography enrichment strategies to capture and categorize ergosterol-interacting candidate proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Mature plants were treated with 250 nM ergosterol over a 24 h period, and plasma membrane-associated fractions were isolated. In addition, ergosterol was immobilized on two different affinity-based systems to capture interacting proteins/complexes. This resulted in the identification of defense-related proteins such as chitin elicitor receptor kinase (CERK), non-race specific disease resistance/harpin-induced (NDR1/HIN1)-like protein, Ras-related proteins, aquaporins, remorin protein, leucine-rich repeat (LRR)- receptor like kinases (RLKs), G-type lectin S-receptor-like serine/threonine-protein kinase (GsSRK), and glycosylphosphatidylinositol (GPI)-anchored protein. Furthermore, the results elucidated unknown signaling responses to this MAMP, including endocytosis, and other similarities to those previously reported for bacterial flagellin, lipopolysaccharides, and fungal chitin.

Identification of MAMP-Responsive Plasma Membrane-Associated Proteins in Arabidopsis thaliana Following Challenge with Different LPS Chemotypes from Xanthomonas campestris

“…To validate the successful isolation procedure of the PM-associated fraction, MAP kinase (MAPK) Western blot analysis was performed [ 9 , 19 ]. A 12% SDS-PAGE gel (1-DE) coupled to densitometric analyses was performed with each of the said fractions (HM, MF, and PM) and was stained with Fairbanks Coomassie [ 59 ] as a further means of validation of the small-scale isolation method [ 60 ].…”

“…The precursor mass tolerance was 10 ppm and the fragment mass tolerance was 20 ppm. The protein false discovery rate (FDR) cut-off was 1% and the best score range was between 0–1000 where a score of greater than 300 is considered significant [ 60 , 61 ]. Once the mass spectra of the fragmented peptides were obtained, they were analyzed by the Byonic™ software and searched against the UniprotKB database, resulting in peptide spectrum matches (PSMs).…”

Adaptive evolution ofMoniliophthoraPR-1 proteins towards its pathogenic lifestyle

“…Given that, it is possible that MpPR-1 could have evolved different adaptive roles through neofunctionalization. Besides export of hydrophobic compounds of basal metabolism, they could be acting in the protection of the cell membrane against the disruption caused by antifungal compounds, in the detoxification of hydrophobic compounds like phytoalexins secreted by the host, or it could even be possible that those MpPR-1s expressed during infection are sequestering the membrane sterols of the fungus itself in order to prevent detection by a possible ergosterol recognition complex from the host (Khoza et al, 2019), thus compromising the elicitation of plant immunity in a similar fashion of MpChi, a chitinaselike effector that is highly expressed by M. perniciosa during the biotrophic stage of WDB (Fiorin et al, 2018).…”

Plant Proteomic Research 2.0: Trends and Perspectives

“…Five publications focus on biotic stress using various proteomic techniques. Khoza et al [4] used a proteomic technique to identify Arabidopsis plasma-membrane associated candidate proteins in response to fungal treatment as well as those possibly interacting with the microbe-associated molecular pattern as ligands. They identified defense-related proteins and elucidated unknown signaling responses to this microbe-associated molecular pattern, including endocytosis.…”

“…Subcellular proteomics has the potential to elucidate localized cellular responses and investigate communications among subcellular compartments during plant development and in response to biotic and abiotic stresses. This special issue includes the proteomic results in plasma membrane [4,30,31], chloroplast [11], and cell wall [17]. Additionally, the progress of proteomic research is understanding the post-translational modification such as phosphorylation [11,21,27].…”