Search for Partner Proteins of A. thaliana Immunophilins Involved in the Control of Plant Immunity

The involvement of plant immunophilins in multiple essential processes such as development, various ways of adapting to biotic and abiotic stresses, and photosynthesis has already been established. Previously, research has demonstrated the involvement of three immunophilin genes (AtCYP19-1/ROC3, AtFKBP65/ROF2, and AtCYP57) in the control of plant response to invasion by various pathogens. Current research attempts to identify host target proteins for each of the selected immunophilins. As a result, candidate interactors have been determined and confirmed using a yeast 2-hybrid (Y2H) system for protein–protein interaction assays. The generation of mutant isoforms of ROC3 and AtCYP57 harboring substituted amino acids in the in silico-predicted active sites became essential to achieving significant binding to its target partners. This data shows that ROF2 targets calcium-dependent lipid-binding domain-containing protein (At1g70790; AT1) and putative protein phosphatase (At2g30020; АТ2), whereas ROC3 interacts with GTP-binding protein (At1g30580; ENGD-1) and RmlC-like cupin (At5g39120). The immunophilin AtCYP57 binds to putative pyruvate decarboxylase-1 (Pdc1) and clathrin adaptor complex-related protein (At5g05010). Identified interactors confirm our previous findings that immunophilins ROC3, ROF2, and AtCYP57 are directly involved with stress response control. Further, these findings extend our understanding of the molecular functional pathways of these immunophilins

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

Impaired Chloroplast Biogenesis in Immutans, an Arabidopsis Variegation Mutant, Modifies Developmental Programming, Cell Wall Composition and Resistance to Pseudomonas syringae.

The immutans (im) variegation mutation of Arabidopsis has green- and white- sectored leaves due to action of a nuclear recessive gene. IM codes for PTOX, a plastoquinol oxidase in plastid membranes. Previous studies have revealed that the green and white sectors develop into sources (green tissues) and sinks (white tissues) early in leaf development. In this report we focus on white sectors, and show that their transformation into effective sinks involves a sharp reduction in plastid number and size. Despite these reductions, cells in the white sectors have near-normal amounts of plastid RNA and protein, and surprisingly, a marked amplification of chloroplast DNA. The maintenance of protein synthesis capacity in the white sectors might poise plastids for their development into other plastid types. The green and white im sectors have different cell wall compositions: whereas cell walls in the green sectors resemble those in wild type, cell walls in the white sectors have reduced lignin and cellulose microfibrils, as well as alterations in galactomannans and the decoration of xyloglucan. These changes promote susceptibility to the pathogen Pseudomonas syringae. Enhanced susceptibility can also be explained by repressed expression of some, but not all, defense genes. We suggest that differences in morphology, physiology and biochemistry between the green and white sectors is caused by a reprogramming of leaf development that is coordinated, in part, by mechanisms of retrograde (plastid-to-nucleus) signaling, perhaps mediated by ROS. We conclude that variegation mutants offer a novel system to study leaf developmental programming, cell wall metabolism and host-pathogen interactions

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

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Not AvailableThe plant cell wall has many significant structural and physiological roles, but the contributions of the various components to these roles remain unclear. Modification of cell wall properties can affect key agronomic traits such as disease resistance and plant growth. The plant cell wall is composed of diverse polysaccharides often decorated with methyl, acetyl, and feruloyl groups linked to the sugar subunits. In this study, we examined the effect of perturbing cell wall acetylation by making transgenic Arabidopsis (Arabidopsis thaliana) and Brachypodium (Brachypodium distachyon) plants expressing hemicellulose- and pectin-specific fungal acetylesterases. All transgenic plants carried highly expressed active Aspergillus nidulans acetylesterases localized to the apoplast and had significant reduction of cell wall acetylation compared with wild-type plants. Partial deacetylation of polysaccharides caused compensatory up-regulation of three known acetyltransferases and increased polysaccharide accessibility to glycosyl hydrolases. Transgenic plants showed increased resistance to the fungal pathogens Botrytis cinerea and Bipolaris sorokiniana but not to the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. These results demonstrate a role, in both monocot and dicot plants, of hemicellulose and pectin acetylation in plant defense against fungal pathogens.Not Availabl

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

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

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