Cytoskeleton reorganization/disorganization is a key feature of induced inaccessibility for defence to successive pathogen attacks

Abstract

In this work, we investigated the involvement of the long-term dynamics of cytoskeletal reorganization on the induced inaccessibility phenomenon by which cells that successfully defend against a previous fungal attack become highly resistant to subsequent attacks. This was performed on pea through double inoculation experiments using inappropriate (Blumeria graminis f. sp. avenae, Bga) and appropriate (Erysiphe pisi, Ep) powdery mildew fungi. Pea leaves previously inoculated with Bga showed a significant reduction of later Ep infection relative to leaves inoculated only with Ep, indicating that cells had developed induced inaccessibility. This reduction in Ep infection was higher when the time interval between Bga and Ep inoculation ranged between 18 and 24 h, although increased penetration resistance in co-infected cells was observed even with time intervals of 24 days between inoculations. Interestingly, this increase in resistance to Ep following successful defence to the inappropriate Bga was associated with an increase in actin microfilament density that reached a maximum at 18-24 h after Bga inoculation and very slowly decreased afterwards. The putative role of cytoskeleton reorganization/disorganization leading to inaccessibility is supported by the suppression of the induced resistance mediated by specific actin (cytochalasin D, latrunculin B) or general protein (cycloheximide) inhibitors.

Keywords: cell memory; cytoskeleton; disease resistance; inaccessibility; papilla; pea; powdery mildew.

Figure 1

Assessment of induced inaccessibility in pea cv. Messire. (a) Percentage of leaves covered by Erysiphe pisi (Ep) mycelium at 5 days after inoculation. Leaves were inoculated with Blumeria graminis f. sp. avenae (Bga) 24 h before Ep inoculation. (b) Micrographs showing a Bga/Ep co‐attacked epidermal cell, a Bga/Ep adjacent infected epidermal cell and an Ep‐infected epidermal cell distant from any Bga infection. In the Bga/Ep co‐attacked and Bga/Ep adjacent examples, Ep was not successful in infecting the epidermal cell and no secondary hyphae were visible at 48 h after inoculation. In the Bga/Ep distant example, Ep successfully infected the pea epidermal cell and three secondary hyphae and a secondary appressorium (App) were visible. AGT, appressorial germ tube; PGT, primary germ tube. (c) Percentage of successful Ep penetration on co‐infected, adjacent and distant epidermal cells of pea previously attacked by Bga. Significant differences at **P < 0.01 and ***P < 0.001 between double Bga/Ep‐inoculated and Ep single‐inoculated leaves.

Figure 2

Effect of the time interval between Blumeria graminis f. sp. avenae (Bga) and Erysiphe pisi (Ep) inoculations on successful Ep penetration on co‐attacked (black squares), adjacent (grey triangles) and distant (black diamonds) attacked pea epidermal cells compared with controls, i.e. Ep single‐inoculated cells (white squares). Data are mean of five replicates ± standard errors.

Figure 3

Confocal laser scanning microscopy images of actin filament reorganization in pea epidermal cells in response to Blumeria graminis f. sp. avenae (Bga) inoculation. Confocal images were taken at 12, 18 and 24 h and 3, 4, 6 and 24 days after Bga inoculation following actin staining by Alexa Fluor® phalloidin. Microfilaments are shown in green. Bars, 20 µm.

Figure 4

Quantification of localized fluorescence of actin microfilaments in pea epidermal cells in response to Blumeria graminis f. sp. avenae (Bga) inoculation. Fluorescence intensities were quantified by image analysis over different time intervals during the period 12 h to 24 days after inoculation. Data are the means of at least five replications. Significant differences at *P < 0.05, **P < 0.01 and ***P < 0.001 with respect to non‐inoculated controls.

Figure 5

Effects of different concentrations of cytochalasin D (a) and latrunculin B (b) on induced inaccessibility. Leaves were inoculated with Blumeria graminis f. sp. avenae (Bga) and then, after 24 h, subjected to chemical treatment or water during a further 24 h, followed by inoculation with Erysiphe pisi (Ep). Controls were Ep single‐inoculated leaves for comparison (white squares). The effects of the inhibitors were recorded in Bga/Ep co‐infected (black squares), adjacent (grey triangles) and distant (black diamonds) epidermal cells.

Figure 6

Effects of 0.1 µm cycloheximide (Chx) on induced inaccessibility. Leaves were inoculated with Blumeria graminis f. sp. avenae (Bga) and then, after 24 h, subjected to chemical treatment or water during a further 24 h, followed by inoculation with Erysiphe pisi (Ep). Controls were Ep single‐inoculated leaves for comparison. The effects of the inhibitors were recorded in Bga/Ep co‐infected (Co‐inf), adjacent (Adjac) and distant epidermal cells. Significant differences at *P < 0.05, with respect to water‐treated leaves.