Characterization of growth and toxin production in BFP and BFPΔ<i>toxA</i> isolates.

<p>(A) Colonial morphology of isolates grown for 6 days on V8-PDA agar. SDS-PAGE of 20 μl of crude culture filtrates either (B) silver-stained or (C) on western blot with anti-ToxA antisera. The molecular mass of standards and their position are indicated on the left of each gel. The arrowhead indicates the mobility of ToxA in the BFP crude culture filtrate. (D) Inoculation of isolates on the ToxC-sensitive cultivar ‘6B365’. Leaves were harvested 5 days post inoculation.</p

Symptom development induced by D308 and D308 transformants on ToxA-sensitive and -insensitive cultivars.

<p>The ToxA-insensitive and -sensitive cultivars, ‘Auburn’ and ‘TAM 105’, respectively, were inoculated and the symptoms (A) monitored and (B) symptoms on ‘TAM 105’ quantified (chlorosis and necrosis). Bars represent means of three leaves per experiment from three independent experiments (nine total), error bars represent standard error, and * indicates a statistical difference from D308 as measured by a Student’s t-test (<i>P</i> < 0.05). (C) Symptom development on the ToxA-sensitive cultivar, ‘Katepwa’. Leaves were harvested 6 days post inoculation.</p

Necrotrophic Effector Epistasis in the <i>Pyrenophora tritici-repentis</i>-Wheat Interaction

<div><p><i>Pyrenophora tritici-repentis</i>, the causal agent of tan spot disease of wheat, mediates disease by the production of host-selective toxins (HST). The known toxins are recognized in an ‘inverse’ gene-for-gene manner, where each is perceived by the product of a unique locus in the host and recognition leads to disease susceptibility. Given the importance of HSTs in disease development, we would predict that the loss of any of these major pathogenicity factors would result in reduced virulence and disease development. However, after either deletion of the gene encoding the HST ToxA or, reciprocally, heterologous expression of <i>ToxA</i> in a race that does not normally produce the toxin followed by inoculation of ToxA-sensitive and insensitive wheat cultivars, we demonstrate that ToxA symptom development can be epistatic to other HST-induced symptoms. ToxA epistasis on certain ToxA-sensitive wheat cultivars leads to genotype-specific increases in total leaf area affected by disease. These data indicate a complex interplay between host responses to HSTs in some genotypes and underscore the challenge of identifying additional HSTs whose activity may be masked by other toxins. Also, through mycelial staining, we acquire preliminary evidence that ToxA may provide additional benefits to fungal growth <i>in planta</i> in the absence of its cognate recognition partner in the host.</p></div

Mycelial growth <i>in planta</i> is restricted to lesions in BFP and BFPΔ<i>toxA</i> inoculated ‘TAM 105’.

<p>One centimeter leaf sections harvested from BFP and BFPΔ<i>toxA</i> inoculated leaves 6 days post inoculation were scanned (Leaf section), chlorophyll concentration estimated (Chl.), and mycelia stained with WGA-FITC. Stained leaves were imaged with a fluorescent microscope and the whole leaf image is a montage of 6 separate panels that cover the entire 1 cm leaf section. The bottom row represents increased magnification images of select (numbered) lesions.</p

<i>ToxA</i> gene replacement with a hygromycin resistance cassette in the <i>P</i>. <i>tritici-repentis</i> isolate, BFP.

<p>(A) Schematic of the <i>ToxA</i>-containing genomic region of the <i>P</i>. <i>tritici-repentis</i> reference genome (top) and the gene replacement construct containing 5’ and 3’ <i>ToxA</i> flanking regions and the hygromycin resistance gene (<i>hph</i>) driven by the <i>trpC</i> promoter, <i>trpC</i>::<i>hph</i> (bottom). Black arrowheads indicate the position of PCR primers used for cloning and positional screening and the numbers above the top schematic indicate primer sequence position on supercontig 1.4. Genomic DNA from BFP and transformants was (B) PCR amplified to test for <i>ToxA</i> replacement (left) and proper orientation of the <i>trpC</i>::<i>hph</i> replacement construct (right) and (C), subjected to qPCR to predict the copy number of the <i>trpC</i>::<i>hph</i> fragment by calculating the ratio of the concentration of <i>hph</i> to the concentration of the single copy gene <i>chitin synthase A</i> (<i>CSA</i>). The molecular mass of standards in the molecular mass ladder (M) is indicated on the left of panel B.</p

Mycelial growth <i>in planta</i> of D308 and D308 transformants.

<p>Isolates were inoculated on the ToxA- and ToxC-sensitive cultivars (A) ‘TAM 105’ and (B) ‘6B365’, respectively. One centimeter leaf sections harvested from inoculated leaves 6 days post inoculation were scanned (Leaf section), chlorophyll concentration estimated (Chl.), and mycelia stained with WGA-FITC. Stained leaves were imaged with a fluorescent microscope and the whole leaf image is a montage of 6 separate panels that cover the entire 1 cm leaf section. The bottom row represents increased magnification of the same size of select (numbered) lesions. Arrows point to multiple conidia.</p

Symptom development induced by BFP and BFPΔ<i>toxA</i> isolates on ToxA-sensitive and -insensitive cultivars.

<p>The ToxA-insensitive and -sensitive cultivars, ‘Auburn’ and ‘TAM 105’, respectively, were inoculated and the symptoms (A) monitored and (B) quantified (chlorosis and necrosis). Bars represent means of values from three leaves per experiment from three independent experiments (nine total), error bars represent standard error, and * indicates a statistical difference from BFP as measured by a Student’s t-test (<i>P</i> < 0.05). (C) Symptom development on two additional ToxA-sensitive cultivars, ‘Katepwa’ and ‘Glenlea’. Leaves were harvested 5 days post inoculation.</p

Host-Selective Toxins of <em>Pyrenophora tritici-repentis</em> Induce Common Responses Associated with Host Susceptibility

<div><p><em>Pyrenophora tritici-repentis</em> (<em>Ptr</em>), a necrotrophic fungus and the causal agent of tan spot of wheat, produces one or a combination of host-selective toxins (HSTs) necessary for disease development. The two most studied toxins produced by <em>Ptr,</em> Ptr ToxA (ToxA) and Ptr ToxB (ToxB), are proteins that cause necrotic or chlorotic symptoms respectively. Investigation of host responses induced by HSTs provides better insight into the nature of the host susceptibility. Microarray analysis of ToxA has provided evidence that it can elicit responses similar to those associated with defense. In order to evaluate whether there are consistent host responses associated with susceptibility, a similar analysis of ToxB-induced changes in the same sensitive cultivar was conducted. Comparative analysis of ToxA- and ToxB-induced transcriptional changes showed that similar groups of genes encoding WRKY transcription factors, RLKs, PRs, components of the phenylpropanoid and jasmonic acid pathways are activated. ROS accumulation and photosystem dysfunction proved to be common mechanism-of-action for these toxins. Despite similarities in defense responses, transcriptional and biochemical responses as well as symptom development occur more rapidly for ToxA compared to ToxB, which could be explained by differences in perception as well as by differences in activation of a specific process, for example, ethylene biosynthesis in ToxA treatment. Results of this study suggest that perception of HSTs will result in activation of defense responses as part of a susceptible interaction and further supports the hypothesis that necrotrophic fungi exploit defense responses in order to induce cell death.</p> </div

ToxB- and ToxA-induced regulation of receptor and receptor-associated families over time.

<p>Differences between control and toxin treatment presented as: line graphs for fold change (left axis) and bar graphs for the number of genes (right axis) that are significantly up- (lines above zero and grey bars) or down-regulated (lines below zero and white bars) at indicated hours post infiltration (hpi) (bottom axis). Receptor family names are to the top right of each graph, and include: brassinosteroid-associated receptor kinase (BAK1), gibberellin (GID1), and wall-associated kinase (WAK).</p

ToxB- and ToxA-induced regulation of genes involved in oxidative stress and the effect of ToxB on ROS accumulation.

<p>(A) Differences between control and toxin-treatment presented as: line graphs for fold change (left axis) and bar graphs for the number of genes (right axis) that are significantly up- (lines above zero and grey bars) or down-regulated (lines below zero and white bars) at indicated hours post infiltration (hpi) (bottom axis). The gene family names are to the top right of each graph, and include: superoxide dismutase (SOD), glutathione peroxidase (GPX), and ascorbate peroxidase (APX). (B) Light microscopy of leaves infiltrated with either H₂O or ToxB and stained with nitroblue tetrazolium at 24 and 48 hpi. Arrowheads indicate examples of NBT stain associated with chloroplasts.</p