Visualization of Growth and Morphology of Fungal Hyphae in planta  Using WGA-AF488 and Propidium Iodide Co-staining

Redkar, Amey; Jaeger, Elaine; Doehlemann, Gunther

doi:10.21769/bioprotoc.2942

Cited by 38 publications

(34 citation statements)

References 4 publications

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“…To visualise U. maydis infection progression, infected maize leaf samples were stained with WGA‐AF488 and propidium iodide as described in Redkar et al . (2018) and analysed using a Zeiss Axio Zoom V16 using the GFP filter for WGA‐AF488 and the DsRed filter for propidium iodide visualisation. Image processing was done using ImageJ.…”

Section: Methodsmentioning

confidence: 99%

Comparative transcriptome profiling identifies maize line specificity of fungal effectors in the maize–Ustilago maydis interaction

et al. 2021

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The biotrophic pathogen Ustilago maydis causes smut disease on maize (Zea mays) and induces the formation of tumours on all aerial parts of the plant. Unlike in other biotrophic interactions, no gene-forgene interactions have been identified in the maize-U. maydis pathosystem. Thus, maize resistance to U. maydis is considered a polygenic, quantitative trait. Here, we study the molecular mechanisms of quantitative disease resistance (QDR) in maize, and how U. maydis interferes with its components. Based on quantitative scoring of disease symptoms in 26 maize lines, we performed an RNA sequencing (RNA-Seq) analysis of six U. maydis-infected maize lines of highly distinct resistance levels. The different maize lines showed specific responses of diverse cellular processes to U. maydis infection. For U. maydis, our analysis identified 406 genes being differentially expressed between maize lines, of which 102 encode predicted effector proteins. Based on this analysis, we generated U. maydis CRISPR/Cas9 knock-out mutants for selected candidate effector sets. After infections of different maize lines with the fungal mutants, RNA-Seq analysis identified effectors with quantitative, maize line-specific virulence functions, and revealed auxin-related processes as a possible target for one of them. Thus, we show that both transcriptional activity and virulence function of fungal effector genes are modified according to the infected maize line, providing insights into the molecular mechanisms underlying QDR in the maize-U. maydis interaction.

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Section: Methodsmentioning

confidence: 99%

Comparative transcriptome profiling identifies maize line specificity of fungal effectors in the maize–Ustilago maydis interaction

et al. 2021

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“…The progression of infection by U. hordei teliospores on barley was visualized following a published protocol [ 45 ], with some modifications. A mixed staining solution consisting of 10 µg/mL of WGA-AF488 (Molecular Probes), 20 µg/mL of Propidium Iodide (PI; Sigma-Aldrich), 0.02% Tween 20 in phosphate-buffered saline (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 and 1.5 mM KH 2 PO 4 ), was used to vacuum infiltrate infected barley seedlings for 30 min.…”

Section: Methodsmentioning

confidence: 99%

UhAVR1, an HR-Triggering Avirulence Effector of Ustilago hordei, Is Secreted via the ER–Golgi Pathway, Localizes to the Cytosol of Barley Cells during in Planta-Expression, and Contributes to Virulence Early in Infection

Alonso

Ali

Song³

et al. 2020

JoF

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The basidiomycete Ustilago hordei causes covered smut disease of barley and oats. Virulence effectors promoting infection and supporting pathogen lifestyle have been described for this fungus. Genetically, six avirulence genes are known and one codes for UhAVR1, the only proven avirulence effector identified in smuts to date that triggers complete immunity in barley cultivars carrying resistance gene Ruh1. A prerequisite for resistance breeding is understanding the host targets and molecular function of UhAVR1. Analysis of this effector upon natural infection of barley coleoptiles using teliospores showed that UhAVR1 is expressed during the early stages of fungal infection where it leads to HR triggering in resistant cultivars or performs its virulence function in susceptible cultivars. Fungal secretion of UhAVR1 is directed by its signal peptide and occurs via the BrefeldinA-sensitive ER–Golgi pathway in cell culture away from its host. Transient in planta expression of UhAVR1 in barley and a nonhost, Nicotiana benthamiana, supports a cytosolic localization. Delivery of UhAVR1 via foxtail mosaic virus or Pseudomonas species in both barley and N. benthamiana reveals a role in suppressing components common to both plant systems of Effector- and Pattern-Triggered Immunity, including necrosis triggered by Agrobacterium-delivered cell death inducers.

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“…To visualise U. maydis infection progression, infected maize leaf samples were stained with WGA-AF488 and propidium iodide as described in Redkar et al (2018) and analysed using a Zeiss Axio Zoom V16 using the GFP filter for WGA-AF488 and the DsRed filter for propidium iodide visualization. Image processing was done using ImageJ.…”

Section: Methodsmentioning

confidence: 99%

Transcriptome analysis in the maize-Ustilago maydisinteraction identifies maize-line-specific activity of fungal effectors

Schurack

Depotter

Gupta

et al. 2020

Preprint

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The biotrophic pathogen The biotrophic pathogen Ustilago maydis causes smut disease on maize (Zea mays) and induces the formation of tumours on all aerial parts of the plant. Unlike in other biotrophic interactions, no gene-for-gene interactions have been identified in the maize-U. maydis pathosystem. Thus, maize resistance to Ustilago maydis is considered a polygenic, quantitative trait. Here, we study the molecular mechanisms of quantitative disease resistance (QDR) in maize, and how Ustilago maydis interferes with its components. Based on quantitative scoring of disease symptoms in 26 maize lines, we performed an RNA-Seq analysis of six Ustilago maydis -infected maize lines of highly distinct resistance levels. In accordance with the complex nature of QDR, the different maize lines showed specific responses of diverse cellular processes to Ustilago maydis infection. On the pathogen side, our analysis identified 406 Ustilago maydis genes being differentially expressed between maize lines, of which 102 encode predicted effector proteins. Based on this analysis, we generated Ustilago maydis CRISPR/Cas9 knockout mutants for selected candidate effector sets. Infections of different maize lines with the fungal mutants and subsequent RNA-sequencing identified effectors with quantitative, maize-line-specific virulence functions, and revealed auxin-related processes as a possible target for one of them. Thus, we show that both transcriptional activity and virulence function of fungal effector genes are modified according to the infected maize line, providing new insights into the molecular mechanisms underlying QDR in the maize-Ustilago maydis interaction.

show abstract

Visualization of Growth and Morphology of Fungal Hyphae in planta Using WGA-AF488 and Propidium Iodide Co-staining

Cited by 38 publications

References 4 publications

Comparative transcriptome profiling identifies maize line specificity of fungal effectors in the maize–Ustilago maydis interaction

Comparative transcriptome profiling identifies maize line specificity of fungal effectors in the maize–Ustilago maydis interaction

UhAVR1, an HR-Triggering Avirulence Effector of Ustilago hordei, Is Secreted via the ER–Golgi Pathway, Localizes to the Cytosol of Barley Cells during in Planta-Expression, and Contributes to Virulence Early in Infection

Transcriptome analysis in the maize-Ustilago maydisinteraction identifies maize-line-specific activity of fungal effectors

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