skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • Comparing methods for detection and quantification of plasmodesmal callose in Nicotiana benthamiana leaves during defense responses
Title: Comparing methods for detection and quantification of plasmodesmal callose in Nicotiana benthamiana leaves during defense responses
Callose, a beta-(1,3)-D-glucan polymer, is essential for regulating intercellular trafficking via plasmodesmata (PD). Pathogens manipulate PD-localized proteins to enable intercellular trafficking by removing callose at PD, or conversely by increasing callose accumulation at PD to limit intercellular trafficking during infection. Plant defense hormones like salicylic acid regulate PD-localized proteins to control PD and intercellular trafficking during immune defense responses such as systemic acquired resistance. Measuring callose deposition at PD in plants has therefore emerged as a popular parameter for assessing likely intercellular trafficking activity during plant immunity. Despite the popularity of this metric there is no standard for how these measurements should be made. In this study, we compared three commonly used methods for identifying and quantifying PD callose by aniline blue staining were evaluated to determine the most effective in the Nicotiana benthamiana leaf model. The results reveal that the most reliable method used aniline blue staining and fluorescent microscopy to measure callose deposition in fixed tissue. Manual or semi-automated workflows for image analysis were also compared and found to produce similar results although the semi-automated workflow produced a wider distribution of data points.  more » « less
Award ID(s):
2210127
PAR ID:
10497168
Author(s) / Creator(s):
; ; ;
Publisher / Repository:
The American Phytopathological Society
Date Published:
Journal Name:
Molecular Plant-Microbe Interactions®
ISSN:
0894-0282
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; (, Methods in cell biology)
    Anderson, CT; Elizabeth S. Haswell, ES; null (Ed.)
    Callose is a β-1,3-glucan polysaccharide that is deposited at discrete sites in the plant cell wall in response to microbial pathogens, likely contributing to protection against pathogen infection. Increased callose deposition also occurs in response to the 22-amino acid peptide flg22, a pathogen-associated molecular pattern (PAMP) derived from bacterial flagellin protein. Here, we provide protocols for callose staining using aniline blue in cotyledon and leaf tissue of the model plant Arabidopsis thaliana. Aniline blue stain utilizes a fluorochrome that complexes with callose for its visualization by microscopy using an ultraviolet (UV) filter. For robust quantification of callose deposits, we outline an automated image analysis workflow utilizing the freely available Fiji (Fiji Is Just ImageJ; NIH) software and a Trainable Weka Segmentation (TWS) plugin. Our methodology for automated analysis of large batches of images can be easily adapted to quantify callose in other tissues and plant species, as well as to quantify fluorescent structures other than callose. 
    more » « less
  2. ; ; ; ; ; ; ; ; ; (, New Phytologist)
    Summary Plasmodesmata (PD) allow direct communication across the cellulosic plant cell wall, facilitating the intercellular movement of metabolites and signaling molecules within the symplast. InArabidopsis thalianaembryos with reduced levels of the chloroplast RNA helicase ISE2, intercellular trafficking and the number of branched PD were increased. We therefore investigated the relationship between alteredISE2expression and intercellular trafficking.Gene expression analyses in Arabidopsis tissues whereISE2expression was increased or decreased identified genes associated with the metabolism of glucosinolates (GLSs) as highly affected.Concomitant with changes in the expression of GLS‐related genes, plants with abnormalISE2expression contained altered GLS metabolic profiles compared with wild‐type (WT) counterparts. Indeed, changes in the expression of GLS‐associated genes led to altered intercellular trafficking in Arabidopsis leaves. Exogenous application of GLSs but not their breakdown products also resulted in altered intercellular trafficking.These changes in trafficking may be mediated by callose levels at PD as exogenous GLS treatment was sufficient to modulate plasmodesmal callose in WT plants. Furthermore, auxin metabolism was perturbed in plants with increased indole‐type GLS levels. These findings suggest that GLSs, which are themselves transported between cells via PD, can act on PD to regulate plasmodesmal trafficking capacity. 
    more » « less
  3. ; ; ; ; ; ; ; ; ; ; et al (, Frontiers in Plant Science)
    IntroductionDuring proliferative plant cell division, the new cell wall, called the cell plate, is first built in the middle of the cell and then expands outward to complete cytokinesis. This dynamic process requires coordinated movement and arrangement of the cytoskeleton and organelles. MethodsHere we use live-cell markers to track the dynamic reorganization of microtubules, nuclei, endoplasmic reticulum, and endomembrane compartments during division and the formation of the cell plate in maize leaf epidermal cells. ResultsThe microtubule plus-end localized protein END BINDING1 (EB1) highlighted increasing microtubule dynamicity during mitosis to support rapid changes in microtubule structures. The localization of the cell-plate specific syntaxin KNOLLE, several RAB-GTPases, as well as two plasma membrane localized proteins was assessed after treatment with the cytokinesis-specific callose-deposition inhibitor Endosidin7 (ES7) and the microtubule-disrupting herbicide chlorpropham (CIPC). While ES7 caused cell plate defects inArabidopsis thaliana, it did not alter callose accumulation, or disrupt cell plate formation in maize. In contrast, CIPC treatment of maize epidermal cells occasionally produced irregular cell plates that split or fragmented, but did not otherwise disrupt the accumulation of cell-plate localized proteins. DiscussionTogether, these markers provide a robust suite of tools to examine subcellular trafficking and organellar organization during mitosis and cell plate formation in maize. 
    more » « less
  4. ; ; ; ; ; (, Journal of Cell Science)
    null (Ed.)
    Cytokinesis in land plants involves the formation of a cell plate that develops into the new cell wall. Callose, a β-1,3 glucan accumulates at later stages of cell plate development presumably to stabilize this delicate membrane network during expansion. Cytokinetic callose is considered specific to multicellular plant species, as it has not been detected in unicellular algae. Here we present callose at the cytokinesis junction of the unicellular charophyte, P. margaritaceum. Callose deposition at the division plane of P. margaritaceum showed distinct, spatiotemporal patterns likely representing distinct roles of this polymer in cytokinesis. Pharmacological inhibition by Endosidin 7 resulted in cytokinesis defects, consistent with the essential role for this polymer in P. margaritaceum cell division. Cell wall deposition at the isthmus zone was also affected by the absence of callose, demonstrating the dynamic nature of new wall assembly in P. margaritaceum. The identification of candidate callose synthase genes provides molecular evidence for callose biosynthesis in P. margaritaceum. The evolutionary implications of cytokinetic callose in this unicellular Zygnematopycean alga is discussed in the context of the conquest of land by plants. 
    more » « less
  5. ; ; ; (, Molecular Plant-Microbe Interactions®)
    Plant xylem colonization is the hallmark of vascular wilt diseases caused by phytopathogens within the Fusarium oxysporum species complex. Recently, xylem colonization has also been reported among endophytic F. oxysporum strains, resulting in some uncertainty. This study compares xylem colonization processes by pathogenic versus endophytic strains in Arabidopsis thaliana and Solanum lycopersicum, using Arabidopsis pathogen Fo5176, tomato pathogen Fol4287, and the endophyte Fo47, which can colonize both plant hosts. We observed that all strains were able to advance from epidermis to endodermis within 3 days postinoculation (dpi) and reached the root xylem at 4 dpi. However, this shared progression was restricted to lateral roots and the elongation zone of the primary root. Only pathogens reached the xylem above the primary-root maturation zone (PMZ). Related to the distinct colonization patterns, we also observed stronger induction of callose at the PMZ and lignin deposition at primary-lateral root junctions by the endophyte in both plants. This observation was further supported by stronger induction of Arabidopsis genes involved in callose and lignin biosynthesis during the endophytic colonization (Fo47) compared with the pathogenic interaction (Fo5176). Moreover, both pathogens encode more plant cell wall–degrading enzymes than the endophyte Fo47. Therefore, observed differences in callose and lignin deposition could be the combination of host production and the subsequent fungal degradation. In summary, this study demonstrates spatial differences between endophytic and pathogenic colonization, strongly suggesting that further investigations of molecular arm-races are needed to understand how plants differentiate friend from foe. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license . 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email