Mutation of a Gene in the Fungus Leptosphaeria maculans Allows Increased Frequency of Penetration of Stomatal Apertures of Arabidopsis thaliana  Elliott Candace E. , Harjono, Howlett Barbara J.   Molecular Plant  Volume 1, Issue 3, Pages 471-481 (May 2008) DOI: 10.1093/mp/ssn014 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Trypan-Blue Staining of Intact Leaves of Arabidopsis thaliana Accessions and Mutants, and Brassica napus Inoculated with Leptosphaeria maculans Isolate IBCN18. Pycnidiospores (104) were placed on each half of each intact leaf or cotyledon of A. thaliana or B. napus respectively. Samples were harvested at 3, 5, 7, and 14 dpi and stained with lactophenol-trypan-blue. Pycnidiospores applied to A. thaliana leaves did not germinate when re-suspended in water (A), but did germinate upon the addition of 1% glucose on A. thaliana (B, D, and I) and on B. napus (N). After 5 d (5 dpi), hyphae entered stomatal apertures of all A. thaliana mutants (C, E, and J) and B. napus cotyledons (O) but proliferated only in the mesophyll layer of A. thaliana mutants (F and K) and B. napus (P). By 7 dpi, trypan-blue staining was present in A. thaliana mutant leaves (G and L) and B. napus cotyledons (Q), and, by 14 dpi, pycnidia were also observed (H, M, and R). × indicates that hyphal growth had been arrested and tissue was not colonized. Arrowheads, intercellular hyphae; arrows, pycnidia. Scale bars: 40 mm. For each time point, six leaves or cotyledons were examined and representative photos are presented. Molecular Plant 2008 1, 471-481DOI: (10.1093/mp/ssn014) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Growth of Leptosphaeria maculans Isolate IBCN18 on Intact Leaves of Arabidopsis thaliana Mutants pen1-1 and pen2-1 and Accession Col-0, And Associated Plant Resistance Responses. Pycnidiospores (104 in 1% glucose) were placed on each half of each intact leaf. Samples were harvested at 3, 5, 7, and 14 dpi, as indicated and stained either with lactophenol-trypan-blue (A–C, E–I, and K–M) or with aniline-blue (5 dpi only; D, J, and N). Pycnidiospores had germinated on all leaves at 3 dpi (A, G, and K) and, by 5 dpi, some hyphae had penetrated stomatal apertures of pen1-1 and pen2-1 (B and H), but rarely those of Col-0 (L). Low magnification observation of infected leaves at 5 dpi showed increased trypan-blue staining of pen1-1 and pen2-1 (C and I) compared to that of Col-0 (M). Aniline-blue-staining showed increased fluorescence in pen1-1 and pen2-1 (D and J) compared with that of Col-0 (N). Intercellular hyphae were observed at 7 dpi (E) and pycnidia were observed at 14 dpi (F) on pen1-1 but hyphal growth was arrested on pen2-1 and Col-0 and tissues were not colonized (indicated by X). A black arrowhead marks intercellular hyphae, while white arrows mark pycnidia. Scale bars: 1 mm (C, I, and M), 40 μm (A, B, E–H, K, and L) and 20 μm (D, J, and N). For each time point, six leaves or cotyledons were examined and representative photos are presented. Molecular Plant 2008 1, 471-481DOI: (10.1093/mp/ssn014) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Penetration of Leptosphaeria maculans on Intact Leaves of Arabidopsis thaliana and Brassica napus. Whole-leaf mounts were harvested and fixed at 5 dpi and stained with trypan-blue. Stomatal apertures with associated hyphae (with or without penetration) were counted as infection sites. (A) to (C) Cross-sectional cartoons illustrating the assay for scoring penetration of L. maculans on intact leaves of A. thaliana or cotyledons of B. napus. Three types of infection sites, including an unsuccessful penetration (A) and two successful penetrations (B and C), are depicted. (D) The penetration frequency of L. maculans isolates IBCN18 and mutant A22 on A. thaliana Col-0 or Ler-0 or B. napus cv. Monty. Bars represent the mean percent penetration frequency of each isolate measured at 300 infection sites (50 sites collected on each of six leaves from three different plants) plus and minus the standard error of the mean. Bars labeled with different letters indicate values are significantly different by a student's t-test (P < 0.05). The experiment was repeated twice and similar results were obtained. Molecular Plant 2008 1, 471-481DOI: (10.1093/mp/ssn014) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 Defense Responses of Arabidopsis thaliana Col-0 and Ler-0 upon Inoculation with Leptosphaeria maculans Isolate IBCN18 or A22. Pycnidiospores were inoculated onto intact leaves of A. thaliana in the presence of 1% glucose. Leaves were harvested at 5 d post inoculation, and stained with trypan-blue (A to D) or aniline-blue (E to H) and observed using bright field and fluorescent microscopy. (A) and (E) Col-0 inoculated with L. maculans isolate IBCN18 (B) and (F) Ler-0 inoculated with L. maculans isolate IBCN18. (C) and (G) Col-0 inoculated with L. maculans isolate A22. (D) and (H) Ler-0 inoculated with L. maculans isolate A22. Dark-blue areas indicate a HR. Fluorescence indicates callose accumulation. Scale bars: 1 mm (A to D), 20 μm (E to H). Molecular Plant 2008 1, 471-481DOI: (10.1093/mp/ssn014) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 Diagrammatic Representation of Leptosphaeria maculans ipa Gene, Location of T-DNA Insertion and Expression of ipa Gene. (A) The ipa open reading frame (ORF) is represented by an open arrow and is 2109 bp. Arrows mark the location of primers used for RT–PCR. No introns are predicted. A grey triangle represents the position of the T-DNA insertion in ipa in isolate A22 after tryptophan414. (B) Reverse transcriptase PCR analysis of ipa and actin. L. maculans mycelia grown for 3 d in 10% Campbell's V8 juice and cDNA was diluted (neat, 1/5, 1/25, 1/125, and 1/625) and then amplified with primers P9 and P10 for 35 cycles for ipa and with primers LmActinF and LmActinR for 25 cycles for actin. A wedge represents decreasing concentration of cDNA template. Genomic DNA (g) was also amplified. Molecular Plant 2008 1, 471-481DOI: (10.1093/mp/ssn014) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 Hypersensitive Responses of Leaves of Arabidopsis thaliana Col-0 upon Inoculation with Wild-Type, A22 and Complemented A22 Isolates of Leptosphaeria maculans and Frequencies of Leaf Penetration by Fungal Hyphae. Pycnidiospore suspensions were supplemented with 1% glucose and placed on the surface of intact leaves. (A) Wild-type isolate IBCN18. (B) Mutant isolate A22. (C) A22c6. (D) A22c8. Isolates A22c6 and A22c8 are transformants of A22 isolate containing plasmid pNat–ipa c with the intact sequence of ipa. These transformants cause a similar degree of hypersensitivity to that caused by wild-type IBCN18. Scale bars: 0.5 cm. (E) Penetration frequencies were quantified on whole-mount samples. Bars represent the mean percent penetration frequency of each isolate measured at 300 infection sites (50 sites collected on each of six leaves from three different plants) plus and minus the standard error of the mean. Bars labeled with different letters indicate values are significantly different by a student's t-test (P < 0.05). The experiment was repeated and a similar result was obtained. Molecular Plant 2008 1, 471-481DOI: (10.1093/mp/ssn014) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions