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

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

<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

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

A scan for FVG ions in crude filtrate from WT and null-FVG mutant WH6 strains.

<p>Laser ablation electrospray ionization-mass spectrometry was used to scan crude culture filtrates for the ion corresponding to 4-formylaminooxyvinylglycine (FVG). Each duplicate well of a 96-well plate contained crude culture filtrate from wild-type <i>Pseudomonas fluorescens</i> WH6, a null FVG-mutant strain [WH6-30G (Δ<i>gvgH</i>) or WH6-31G (Δ<i>gvgI</i>)], or non-inoculated filtrate. Data were collected from 20 laser pulses per sample well, and the peak trace corresponds to the extracted ion chromatogram for sodiated FVG, <i>m/z</i> /183.0372.</p

Detection of 4-formylaminooxyvinylglycine in culture filtrates of <i>Pseudomonas fluorescens</i> WH6 and <i>Pantoea ananatis</i> BRT175 by laser ablation electrospray ionization-mass spectrometry

<div><p>The oxyvinylglycine 4-formylaminooxyvinylglycine (FVG) arrests the germination of weedy grasses and inhibits the growth of the bacterial plant pathogen <i>Erwinia amylovora</i>. Both biological and analytical methods have previously been used to detect the presence of FVG in crude and extracted culture filtrates of several <i>Pseudomonas fluorescens</i> strains. Although a combination of these techniques is adequate to detect FVG, none is amenable to high-throughput analysis. Likewise, filtrates often contain complex metabolite mixtures that prevent the detection of FVG using established chromatographic techniques. Here, we report the development of a new method that directly detects FVG in crude filtrates using laser ablation electrospray ionization-mass spectrometry (LAESI-MS). This approach overcomes limitations with our existing methodology and allows for the rapid analysis of complex crude culture filtrates. To validate the utility of the LAESI-MS method, we examined crude filtrates from <i>Pantoea ananatis</i> BRT175 and found that this strain also produces FVG. These findings are consistent with the antimicrobial activity of <i>P</i>. <i>ananatis</i> BRT175 and indicate that the spectrum of bacteria that produce FVG stretches beyond rhizosphere-associated <i>Pseudomonas fluorescens</i>.</p></div

Results of biological assays and LAESI-MS detection of FVG for a dilution series of culture filtrate from wild-type <i>Pseudomonas fluorescens</i> WH6.

<p>Results of biological assays and LAESI-MS detection of FVG for a dilution series of culture filtrate from wild-type <i>Pseudomonas fluorescens</i> WH6.</p