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STREAMLINE PROTOCOL FOR ARABIDOPSIS APOPLASTIC FLUID ISOLATION ENABLES A DETAILED PROTEOMIC VIEW OF THE EXTRACELLULAR SPACE SURROUNDING PLANT CELLS

ABSTRACT: The apoplastic space surrounding plant cells, encompassing the cell well matrix, extracellular spaces and xylem, is the least understood compartment within plant tissues due to its lack of limiting membranes and its unavoidable damage upon tissue homogenization. Using a streamlined vacuum-infiltration/centrifugation protocol to enrich for the Arabidopsis apoplastic fluid (APF) combined with in-depth tandem mass spectrometry, we provide an improved view of its proteome that includes over 1,500 proteins possibly assigned to this compartment with minimized cytosolic contamination. Included are large and varied collections of hydrolytic activities devoted to cell wall metabolism, oxido-reductase reactions, cell-cell signaling, proteolysis, and pathogen protection via basal defense pathways. While numerous apoplast proteins were predicted to house N-terminal signal peptide sequences to direct extracellular secretion, a number did not, suggesting wide-spread use of non-classical export route(s). Among APF constituents are numerous pathogenesis-related proteins, glycosidases, subtilisin-type serine and aspartyl proteases, and the complement of subunits that assembles the core particle of the 26S proteasome. When this APF proteome is compared with those based on prior isolation methods, a consensus collection of 338 polypeptides emerges that offers a comprehensive view of the core APF proteome that manages the cell wall and interfaces with the environment.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Leaf

SUBMITTER: Kuo-En Chen

LAB HEAD: Richard D. Vierstra

PROVIDER: PXD060844 | Pride | 2025-03-28

REPOSITORIES: Pride

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Project description:A family of APSES transcription factor is known to be fungal-specific transcriptional regulators and play important roles in governing growth, differentiation, and virulence of diverse fungal pathogens. Yet none of APSES-like transcription factors have been identified and investigated in a basidiomycetous fungal pathogen, Cryptococcus neoformans. In the present study we discovered an APSES-like transcription factor, mbs1 (Mbp1/Swi4-like APSES protein 1), as one of novel flucytosine-responsive genes (total 194 genes) identified through comparative transcriptome analysis of C. neoformans hybrid sensor kinase mutants, tco1 and tco2 mutants, which displayed differential flucytosine-susceptibility. Supporting the microarray data, Northern blot and quantitative RT-PCR analysis confirmed that expression of mbs1 is rapidly induced in response to flucytosine in the wild-type strain, but not in the tco1 and tco2 mutants. Furthermore, C. neoformans with deletion of the mbs1 gene exhibited increased susceptibility to flucytosine. Intriguingly, mbs1 plays pleiotropic roles in diverse cellular process of C. neoformans. mbs1 positively regulates ergosterol biosynthesis and thereby its inhibition confers increased susceptibility and resistance to amphotericin B and azole drugs, respectively. mbs1 is also involved in DNA damage repair counteracting genotoxic stresses. During sexual differentiation mbs1 represses pheromone production, but promotes cell-cell fusion. Furthermore mbs1 is required for production of antioxidant melanin pigment and full virulence of C. neoformans. Finally we also performed DNA microarrray analysis to identify mbs1-regulated genes in C. neoformans. A majority of them were found to be involved in cell cycle regulation and DNA repair. Therefore, this study provides a novel antifungal therapeutic method for treatment of cryptococcosis. total RNAs are extracted 2 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), mbs1Δ), We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye.

			Action	DRS
	APF-M1-1-1.raw	Raw
	APF-M1-1-2.raw	Raw
	APF-M1-2-1.raw	Raw
	APF-M1-2-2.raw	Raw
	APF-M1-3-1.raw	Raw

Dataset Information

STREAMLINE PROTOCOL FOR ARABIDOPSIS APOPLASTIC FLUID ISOLATION ENABLES A DETAILED PROTEOMIC VIEW OF THE EXTRACELLULAR SPACE SURROUNDING PLANT CELLS

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