scholarly journals Green Fluorescent Protein Transformation Sheds More Light on a Widespread Mycoparasitic Interaction

2019 ◽  
Vol 109 (8) ◽  
pp. 1404-1416
Author(s):  
Márk Z. Németh ◽  
Alexandra Pintye ◽  
Áron N. Horváth ◽  
Pál Vági ◽  
Gábor M. Kovács ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Powdery Mildew ◽  
Plant Pathogens ◽  
Fluorescent Protein ◽  
Environmental Fate ◽  
Molecular Genetic ◽  
Powdery Mildews ◽  
Before And After ◽  
Mycoparasitic Fungi ◽  
Green Fluorescent

Powdery mildews, ubiquitous obligate biotrophic plant pathogens, are often attacked in the field by mycoparasitic fungi belonging to the genus Ampelomyces. Some Ampelomyces strains are commercialized biocontrol agents of crop pathogenic powdery mildews. Using Agrobacterium tumefaciens-mediated transformation (ATMT), we produced stable Ampelomyces transformants that constitutively expressed green fluorescent protein (GFP) to (i) improve the visualization of the mildew–Ampelomyces interaction and (ii) decipher the environmental fate of Ampelomyces fungi before and after acting as a mycoparasite. Detection of Ampelomyces structures, and especially hyphae, was greatly enhanced when diverse powdery mildew, leaf, and soil samples containing GFP transformants were examined with fluorescence microscopy compared with brightfield and differential interference contrast optics. We showed for the first time, to our knowledge, that Ampelomyces strains can persist up to 21 days on mildew-free host plant surfaces, where they can attack powdery mildew structures as soon as these appear after this period. As saprobes in decomposing, powdery mildew-infected leaves on the ground and also in autoclaved soil, Ampelomyces strains developed new hyphae but did not sporulate. These results indicate that Ampelomyces strains occupy a niche in the phyllosphere where they act primarily as mycoparasites of powdery mildews. Our work has established a framework for a molecular genetic toolbox for the genus Ampelomyces using ATMT.

scholarly journals GFP transformation sheds more light on a widespread mycoparasitic interaction

2019 ◽  
Author(s):  
Márk Z. Németh ◽  
Alexandra Pintye ◽  
Áron N. Horváth ◽  
Pál Vági ◽  
Gábor M. Kovács ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Fluorescent Protein ◽  
Environmental Fate ◽  
Molecular Genetic ◽  
Powdery Mildews ◽  
Before And After ◽  
Mycoparasitic Fungi ◽  
Green Fluorescent ◽  
First Time ◽  
Plant Surfaces

ABSTRACTPowdery mildews (PMs), ubiquitous obligate biotrophic plant pathogens, are often attacked in the field by mycoparasitic fungi belonging to the genus Ampelomyces. Some Ampelomyces strains are commercialized biocontrol agents of crop pathogenic PMs. Using Agrobacterium tumefaciens-mediated transformation (ATMT), we produced stable Ampelomyces transformants that constitutively expressed the green fluorescent protein (GFP), to (i) improve the visualization of the PM-Ampelomyces interaction; and (ii) decipher the environmental fate of Ampelomyces before and after acting as a mycoparasite. Detection of Ampelomyces structures, and especially hyphae, was greatly enhanced when diverse PM, leaf and soil samples containing GFP transformants were examined with fluorescence microscopy compared to brightfield and DIC optics. We showed for the first time that Ampelomyces can persist up to 21 days on PM-free host plant surfaces, where it can attack PM structures as soon as these appear after this period. As a saprobe in decomposing, PM-infected leaves on the ground, and also in autoclaved soil, Ampelomyces developed new hyphae, but did not sporulate. These results indicate that Ampelomyces occupies a niche in the phyllosphere where it acts primarily as a mycoparasite of PMs. Our work has established a framework for a molecular genetic toolbox for Ampelomyces using ATMT.

scholarly journals Ecological Basis of the Interaction betweenPseudozyma flocculosaand Powdery Mildew Fungi

2010 ◽  
Vol 77 (3) ◽  
pp. 926-933 ◽  
Author(s):  
Walid Hammami ◽  
Candy Quiroga Castro ◽  
Wilfried Rémus-Borel ◽  
Caroline Labbé ◽  
Richard R. Bélanger
Keyword(s):  
Powdery Mildew ◽  
Fluorescent Protein ◽  
Close Relative ◽  
Tomato Plants ◽  
Powdery Mildews ◽  
Biocontrol Activity ◽  
Cellobiose Lipids ◽  
Powdery Mildew Fungi ◽  
Green Fluorescent ◽  
Ecological Basis

ABSTRACTIn this work, we sought to understand how glycolipid production and the availability of nutrients could explain the ecology ofPseudozyma flocculosaand its biocontrol activity. For this purpose, we compared the development ofP. flocculosato that of a close relative, the plant pathogenUstilago maydis, under different environmental conditions. This approach was further supported by measuring the expression ofcyp1, a pivotal gene in the synthesis of unique antifungal cellobiose lipids of both fungi. On healthy cucumber and tomato plants, the expression ofcyp1remained unchanged over time inP. flocculosaand was undetected inU. maydis. At the same time, green fluorescent protein (GFP) strains of both fungi showed only limited green fluorescence on control leaves. On powdery mildew-infected cucumber leaves,P. flocculosainduced a complete collapse of the pathogen colonies, but glycolipid production, as studied bycyp1expression, was still comparable to that of controls. In complete contrast,cyp1was upregulated nine times whenP. flocculosawas applied toBotrytis cinerea-infected leaves, but the biocontrol fungus did not develop very well on the pathogen. Analysis of the possible nutrients that could stimulate the growth ofP. flocculosaon powdery mildew structures revealed that the complex Zn/Mn played a key role in the interaction. Other related fungi such asU. maydisdo not appear to have the same nutritional requirements and hence lack the ability to colonize powdery mildews. Whether production of antifungal glycolipids contributes to the release of nutrients from powdery mildew colonies is unclear, but the specificity of the biocontrol activity ofP. flocculosatoward Erysiphales does appear to be more complex than simple antibiosis.

scholarly journals In Vivo Study of Trichoderma-Pathogen-Plant Interactions, Using Constitutive and Inducible Green Fluorescent Protein Reporter Systems

2004 ◽  
Vol 70 (5) ◽  
pp. 3073-3081 ◽  
Author(s):  
Zexun Lu ◽  
Riccardo Tombolini ◽  
Sheridan Woo ◽  
Susanne Zeilinger ◽  
Matteo Lorito ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Plant Pathogens ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Critical Role ◽  
Pythium Ultimum ◽  
Confocal Scanning Laser Microscopy ◽  
Green Fluorescent

ABSTRACT Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.

Use of the green fluorescent protein to locate α-amylase gene expression in barley grains

2002 ◽  
Vol 29 (9) ◽  
pp. 1037 ◽  
Author(s):  
Peter R. Matthews ◽  
Sarah Thornton ◽  
Frank Gubler ◽  
Rosemary White ◽  
John V. Jacobsen
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Confocal Laser ◽  
Aleurone Layer ◽  
Amylase Gene ◽  
Gfp Fluorescence ◽  
Amylase Production ◽  
Before And After ◽  
Barley Grains ◽  
Green Fluorescent

A green fluorescent protein (GFP) gene was cloned between the promoter and 3� regions from a barley high isoelectric point (pI) α-amylase gene, then inserted into barley. GFP fluorescence was used to locate and quantify expression of the transgene in barley grains following hydration. Light and confocal laser microscopy revealed fluorescence in the known regions of α-amylase synthesis in the scutellar epithelium, aleurone layer and embryonic axis. Fluorescence was quantified using a simple fluorescence assay, which showed induction of the transgene to mirror the induction of α-amylase in aleurone exposed to gibberellic acid. Expression from the transgene was also shown to be inhibited by abscisic acid, in the same way as expression of endogenous α-amylase genes. Overall, the transgenic grain revealed patterns of α-amylase expression before and after germination, and showed strong potential for further studies investigating both α-amylase production and transport of gibberellin in malting grain.

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