The cytoskeleton is disrupted by the bacterial effector HrpZ, but not by the bacterial PAMP flg22, in tobacco BY-2 cells

Abstract

Plant innate immunity is composed of two layers. Basal immunity is triggered by pathogen-associated molecular patterns (PAMPs) such as the flagellin-peptide flg22 and is termed PAMP-triggered immunity (PTI). In addition, effector-triggered immunity (ETI) linked with programmed cell death and cytoskeletal reorganization can be induced by pathogen-derived factors, such as the Harpin proteins originating from phytopathogenic bacteria. To get insight into the link between cytoskeleton and PTI or ETI, this study followed the responses of actin filaments and microtubules to flg22 and HrpZ in vivo by spinning-disc confocal microscopy in GFP-tagged marker lines of tobacco BY-2. At a concentration that clearly impairs mitosis, flg22 can induce only subtle cytoskeletal responses. In contrast, HrpZ causes a rapid and massive bundling of actin microfilaments (completed in ~20min, i.e. almost simultaneously with extracellular alkalinization), which is followed by progressive disintegration of actin cables and cytoplasmic microtubules, a loss of cytoplasmic structure, and vacuolar disintegration. Cytoskeletal disruption is proposed as an early event that discriminates HrpZ-triggered ETI-like defence from flg22-triggered PTI.

ACTIN DEPOLYMERIZING FACTOR4 Regulates Actin Dynamics during Innate Immune Signaling in Arabidopsis

“…In contrast with increased actin abundance during host-microbe interactions, certain signals from pathogenic fungi like the Verticillium dahlia toxin or the bacterial Harpin elicitor can stimulate the dose-dependent destruction of cortical actin filaments (Yuan et al, 2006;Qiao et al, 2010). Moreover, a study with flg22 treatment of tobacco (Nicotiana tabacum) Bright Yellow-2 cells failed to demonstrate any quantitative changes in actin organization, perhaps due to cell type-specific signaling cascades (Guan et al, 2013). Gross rearrangements in actin cytoskeletal architecture often correlate with the altered dynamics of single actin filaments Li et al, 2012;Tóth et al, 2012;Henty-Ridilla et al, 2013b).…”

Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein

“…Whereas cryptogein induces a well-characterized HR in tobacco plants and cell death in suspension cells, flagellin acts as a nonnecrotic elicitor (Denoux et al, 2008). Moreover, cryptogein induces a dramatic depolymerization of cortical microtubules in tobacco cells after 1 h of treatment (Binet et al, 2001), whereas flg22 did not cause any such changes in the radial or cortical microtubules (Guan et al, 2013). If the modification of the global level of PM order could be part of the conserved basal defense responses, the transient modification of membrane fluidity might correspond to a more specific response.…”

Tubulin marker line of grapevine suspension cells as a tool to follow early stress responses

“…10 11 Phenotyping cellular responses and treatments. PCV as indicator for growth, mitotic 12 index, and viability were measured as described by Guan et al (2013a). Based on our 13 previous work (Guan et al 2013a), flg22 and HrpZ were chosen as elicitors to trigger either 14 basal innate immunity (PTI, flg22), or cell-death related ETI-like responses (HrpZ).…”

“…PCV as indicator for growth, mitotic 12 index, and viability were measured as described by Guan et al (2013a). Based on our 13 previous work (Guan et al 2013a), flg22 and HrpZ were chosen as elicitors to trigger either 14 basal innate immunity (PTI, flg22), or cell-death related ETI-like responses (HrpZ). Aliquots 15 of 20 µl from the suspension were placed on a slide and covered with a coverslip for 16 microscopy after 3 days of cocultivation with 100 nM flg22 (as mock control, water was 17 used), 1.7 µM HrpZ (as mock control, 5 mM MES were used), or with 500 µM (±)-Jasmonic 18 acid (JA) (Sigma-Aldrich, Germany) (100 mM of a stock solution in absolute ethanol were 19 diluted with distilled water as mock control, water was used).…”

“…3). The concentrations and incubation time had been derived from 9 previous studies in the non-transformed cell line (Guan et al 2013a;Ismail et al 2012;10 Hamayun et al 2010). All treatments affected cell growth as reflected by a reduced increase 11 of PCV.…”