Decreased Polysaccharide Feruloylation Compromises Plant Cell Wall Integrity and Increases Susceptibility to Necrotrophic Fungal Pathogens

The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity (CWI) and function remains unclear. Modifications of cell wall composition can induce plant responses known as CWI control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, decreased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant CWI, which contributes to plant resistance to necrotrophic pathogens. Keywords: ferulic acid, cell wal

Post-Synthetic Defucosylation of AGP by Aspergillus nidulans α-1,2-Fucosidase Expressed in Arabidopsis Apoplast Induces Compensatory Upregulation of α-1,2-Fucosyltransferases.

Cell walls are essential components of plant cells which perform a variety of important functions for the different cell types, tissues and organs of a plant. Besides mechanical function providing cell shape, cell walls participate in intercellular communication, defense during plant-microbe interactions, and plant growth. The plant cell wall consists predominantly of polysaccharides with the addition of structural glycoproteins, phenolic esters, minerals, lignin, and associated enzymes. Alterations in the cell wall composition created through either changes in biosynthesis of specific constituents or their post-synthetic modifications in the apoplast compromise cell wall integrity and frequently induce plant compensatory responses as a result of these alterations. Here we report that post-synthetic removal of fucose residues specifically from arabinogalactan proteins in the Arabidopsis plant cell wall induces differential expression of fucosyltransferases and leads to the root and hypocotyl elongation changes. These results demonstrate that the post-synthetic modification of cell wall components presents a valuable approach to investigate the potential signaling pathways induced during plant responses to such modifications that usually occur during plant development and stress responses

Real-time qPCR analysis of <i>FUT</i> gene expression in transgenic lines AnF and wild type plants.

<p>Relative expression levels were calculated as comparison to the <i>ACTIN-2</i> reference gene, whose expression was not affected. 2^−ΔΔCt method was used for determining difference between transcripts copy numbers in wild-type and transgenic plants. * Differences between transgenic lines and <i>Col</i>-0 are significant. Analysis of <i>AtFUT</i> genes expression level were done separately for: (A) Stems. (B) Leaves. (C) Roots.</p

Post-Synthetic Defucosylation of AGP by <i>Aspergillus nidulans</i> α-1,2-Fucosidase Expressed in <i>Arabidopsis</i> Apoplast Induces Compensatory Upregulation of α-1,2-Fucosyltransferases - Fig 1

<p>(A) The expression cassette of the vector developed for Arabidopsis transformation. Abbreviations: CaMV 35S –Tetramer of Cauliflower Mosaic virus 35S RNA Promoter, YFP, yellow fluorescent protein coding sequence. (B) PCR analysis of genomic DNA from transgenic Arabidopsis lines transformed with microbial <i>A</i>.<i>nidulans</i> α-fucosidase expression cassette and wild type plants. Herbicide resistant lines were confirmed to harbor the full construct using four pairs of primers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159757#pone.0159757.s003" target="_blank">S1 Table</a> for sequences). Lane 1—amplification of <i>AnF</i> genes from corresponding transgenic lines; Lane 5—amplification of the same genes from <i>Col</i>-0 wild type plant; Lane 2—amplification of hybrid fragment containing <i>AnF</i> gene linked to the <i>YFP</i> from mutant lines; Lane 6—amplification of the <i>AnF</i>-<i>YFP</i> fragment from <i>Col</i>-0 wild type plant; Lane 3—amplification of <i>YFP</i> gene from the AnF line; Lane 7 –amplification of <i>YFP</i> from <i>Col-0</i> wild type plant; Lane 4—amplification of <i>A</i>. <i>thaliana ACTIN</i>-2 gene fragment from AnF line, and Lane 8 –amplification of <i>ACTIN</i>-2 from <i>Col</i>-0 wild type plant. Analysis was done for three independent transgenic lines for each construction; picture shows results of PCR for single plant of each mutant line. (C) Western blot analysis of total proteins from apoplast of Arabidopsis AnF transgenic and wild type plants. The corresponding microbial fucosidase fused with YFP (116kDa) were found in transgenic lines and not in <i>Col</i>-0 control plants. Blots were produced using GFP monoclonal antibodies (1:5000 dilution).</p

Monosaccharide composition (mol%).

<p>(A) Monosaccharide composition of total cell walls extracted from the whole 4-week-old transgenic plants expressing AnF and wild type Col-0 plants. (B) Monosaccharide composition of cell wall fractions after pectin being removed. Analysis was done using stem, leaf and root tissues of 4-week-old transgenic Arabidopsis plants expressing AnF and wild type Col-0 plants. (C) Neutral monosaccharide composition (mol%) of AGP glycan. (D) Monosaccharide composition of cell wall fraction remaining after AGP removal. Analysis was done using stem, leaf, and root tissues of 4-week-old Arabidopsis plants. * Differences between transgenic lines and <i>Col</i>-0 are significant (n = 3, p<0.05).</p

Light and confocal microscopy images of different organs of 3-week old transgenic Arabidopsis plants expressing AnF.

<p>(A) Light microscopy image of AnF expressing plant root cells. (B) Localization of AnF protein fused with YFP in the root cells. (C) Light microscopy image of AnF expressing plant stem cells. (D) Localization of AnF protein fused with YFP in the stem cells. Bars = 0.2 mm.</p

Post-Synthetic Reduction of Pectin Methylesterification Causes Morphological Abnormalities and Alterations to Stress Response in Arabidopsis thaliana

Pectin is a critical component of the plant cell wall, supporting wall biomechanics and contributing to cell wall signaling in response to stress. The plant cell carefully regulates pectin methylesterification with endogenous pectin methylesterases (PMEs) and their inhibitors (PMEIs) to promote growth and protect against pathogens. We expressed Aspergillus nidulans pectin methylesterase (AnPME) in Arabidopsis thaliana plants to determine the impacts of methylesterification status on pectin function. Plants expressing AnPME had a roughly 50% reduction in methylester content compared with control plants. AnPME plants displayed a severe dwarf phenotype, including small, bushy rosettes and shorter roots. This phenotype was caused by a reduction in cell elongation. Cell wall composition was altered in AnPME plants, with significantly more arabinose and significantly less galacturonic acid, suggesting that plants actively monitor and compensate for altered pectin content. Cell walls of AnPME plants were more readily degraded by polygalacturonase (PG) alone but were less susceptible to treatment with a mixture of PG and PME. AnPME plants were insensitive to osmotic stress, and their susceptibility to Botrytis cinerea was comparable to wild type plants despite their compromised cell walls. This is likely due to upregulated expression of defense response genes observed in AnPME plants. These results demonstrate the importance of pectin in both normal growth and development, and in response to biotic and abiotic stresses.</p