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. 2020 Nov 6;11:603693. doi: 10.3389/fpls.2020.603693

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Copyright © 2020 Ghareeb, El-Sayed, Pound, Tetyuk, Hanika, Herrfurth, Feussner and Lipka.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Virulent (Noco2) and avirulent (Emwa1) isolates of H. arabidopsidis exhibit distinct invasion dynamics on A. thaliana Col-0 leaves. (A,B) Maximum projections of CLSM z-stack images showing plasma membrane (PM) marker (gray; A, left and middle panels; B, left panel) in a leaf of a 3-week-old transgenic A. thaliana line COLORFUL-PR1pro#1 at 1 day post inoculation (dpi) with Emwa1 spore (cyan overlay; A, left) and after staining with Fluorescent Brightener 28 (FB28). PM allows visualization of penetration between outer periclinal epidermal cell walls (yellow arrowhead) by Emwa1, intercellular growth into anticlinal cell walls (red arrowheads; A, middle; B, left), and extrahaustorial membrane (green arrowheads), and haustoria formation (asterisks) in pavement cell (A) and palisade mesophyll cells (B). Bright field images (right) show single plane sections. Scale bar, 10 μm. See also Videos S1 and S2. (C) Noco2 and Emwa1 penetration rates in leaves of 3-week-old COLORFUL lines. Penetration was determined as growth into anticlinal cell walls between epidermal cells (red arrowhead; A) at 1 and 2 day(s) post inoculation (dpi). (D,E) Frequency of haustoria establishment by Noco2 and Emwa1 determined as formation of extrahaustorial membrane (green arrowheads; A,B) in epidermal (D) and mesophyll cells (E) at 1 and 2 dpi. Measurements were obtained from three independent experiments, each containing 10 biological replicates. Data represent means ± SEM (F) Maximum projection of CLSM z-stack images showing overlays of reference (magenta) and PM (gray) markers in leaves of 3-week-old A. thaliana line COLORFUL-PR1pro#1 at 3 dpi after staining Noco2 (top) and Emwa1 (bottom) spores with FB28 (cyan). While Noco2 growth indicated by haustoria formation (green arrowheads) was extended in epidermis (left) and mesophyll (right) beyond the initial site of invasion, Emwa1 induced cell death (discontinuous lines) associated with disappearance of PM and reference fluorescence. Blue E and M indicate reference signals originating from epidermal and mesophyll cells. Scale bar, 25 μm (see also Supplementary Videos S3,S4 and Supplementary Figure S6A).