scholarly journals Exploring Infection of Wheat and Carbohydrate Metabolism in Mycosphaerella graminicola Transformants with Differentially Regulated Green Fluorescent Protein Expression

2001 ◽  
Vol 14 (2) ◽  
pp. 156-163 ◽  
Author(s):  
Eric A. Rohel ◽  
Andrew C. Payne ◽  
Bart A. Fraaije ◽  
Derek W. Hollomon
Keyword(s):  
Green Fluorescent Protein ◽  
Carbon Source ◽  
Promoter Region ◽  
Fluorescent Protein ◽  
Isocitrate Lyase ◽  
Mycosphaerella Graminicola ◽  
Cell Junctions ◽  
Constitutive Promoter ◽  
Wheat Leaves ◽  
Green Fluorescent

A Mycosphaerella graminicola strain transformed with the green fluorescent protein (GFP) downstream of either a carbon source-repressed promoter or a constitutive promoter was used to investigate in situ carbohydrate uptake during penetration of the fungus in wheat leaves. The promoter region of the acu-3 gene from Neurospora crassa encoding isocitrate lyase was used as a carbon source-repressed promoter. The promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase was used as a constitutive promoter. Fluorometric measurement of GFP gene expression in liquid cultures of acu-3-regulated transformants indicated that the N. crassa acu-3 promoter functions in M. graminicola as it does in N. crassa, i.e., acetate induced and carbon source repressed. Glucose, fructose, and saccharose triggered the repression, whereas mannitol, xylose, and cell wall polysaccharides did not. Monitoring the GFP level during fungal infection of wheat leaves revealed that acu-3 promoter repression occurred after penetration until sporulation, when newly differentiated pycnidiospores fluoresced. The use of GFP transformants also allowed clear visualization of M. graminicola pathogenesis. No appressoria were formed, but penetration at cell junctions was observed. These results give new insight into the biotrophic status of M. graminicola.

scholarly journals CgDN3: An Essential Pathogenicity Gene of Colletotrichum gloeosporioides Necessary to Avert a Hypersensitive-Like Response in the Host Stylosanthes guianensis

2000 ◽  
Vol 13 (9) ◽  
pp. 929-941 ◽  
Author(s):  
Sally-Anne Stephenson ◽  
Jodie Hatfield ◽  
Anca G. Rusu ◽  
Donald J. Maclean ◽  
John M. Manners
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Promoter Region ◽  
Primary Infection ◽  
Colletotrichum Gloeosporioides ◽  
Fluorescent Protein ◽  
Axenic Culture ◽  
Host Cells ◽  
Stylosanthes Guianensis ◽  
Green Fluorescent

A gene of Colletotrichum gloeosporioides that is induced by nitrogen starvation in axenic culture and is expressed at the early stages of infection of the host Stylosanthes guianensis has been identified and its role in pathogenicity tested. The sequence of this gene, named CgDN3, indicated that it encodes a protein of 74 amino acids that contains a predicted 18 amino acid signal sequence for secretion of a basic 54 amino acid mature protein with weak homology to an internal region of plant wall-associated receptor kinases. Mutants of C. gloeosporioides were produced by homologous recombination in which part of the coding sequence and promoter region of the CgDN3 gene was replaced with a hygromycin-resistance gene cassette. Mutations in the CgDN3 gene were confirmed in two independent transformants and Northern (RNA) analysis demonstrated the disrupted CgDN3 gene was not expressed. The mutants had faster mycelial growth rates in vitro but produced spores that germinated to form appressoria normally on the leaf surface. However, the CgDN3 mutants were unable to infect and reproduce on intact host leaves. Microscopic analysis revealed small clusters of necrotic host cells at inoculation sites on leaves, suggesting that these mutants elicited a localized, host hypersensitive-like response. The mutants were able to grow necrotrophically and reproduce on leaves when conidia were inoculated directly onto wound sites. The putative promoter region of the CgDN3 gene was fused to a gene encoding a modified jellyfish green fluorescent protein and introduced into the fungus. Following inoculation, strong expression of green fluorescent protein was observed in primary infection vesicles in infected epidermal cells with weaker expression evident in hyphae growing within infected leaf tissue. These findings indicate that CgDN3 encodes a novel pathogenicity determinant associated with the biotrophic phase of primary infection and required to avert a hypersensitive-like response by a compatible host.

scholarly journals Immunolocalization of Dinitrogenase Reductase Produced by Klebsiella pneumoniae in Association withZea mays L

2000 ◽  
Vol 66 (2) ◽  
pp. 783-787 ◽  
Author(s):  
Marisa K. Chelius ◽  
Eric W. Triplett
Keyword(s):  
Green Fluorescent Protein ◽  
Carbon Source ◽  
Klebsiella Pneumoniae ◽  
Fluorescent Protein ◽  
Suitable Habitat ◽  
Cultivation Conditions ◽  
Plant Cultivation ◽  
Dinitrogenase Reductase ◽  
Green Fluorescent ◽  
Exogenous Carbon Source

ABSTRACT The endophytic lifestyle of Klebsiella pneumoniae is described, including the production of dinitrogenase reductase by bacteria residing in maize root tissue. The green fluorescent protein (GFP) was used to detect the colonization of maize by K. pneumoniae strains 2028 and 342. These strains were found to reside in intercortical layers of the stem and within the region of maturation in the root. The production of dinitrogenase reductase by GFP-tagged bacteria was visualized using immunolocalization. This activity was only apparent when bacteria were supplied with an exogenous carbon source. The results suggest that maize provides a suitable habitat for K. pneumoniae and that this species is capable of producing nitrogenase under the appropriate plant cultivation conditions.

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