Exploring infection of wheat and carbohydrate metabolism in Mycosphaerella graminicola transformants with differentially regulated green fluorescent protein expression

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.     

Expression of a plant protein by Neurospora crassa.

Heterologous expression of plant genes may serve as an important alternative for producing plant proteins. We have investigated the ability of the fungus Neurospora crassa to secrete zeamatin, a protein produced by Zea mays. Zeamatin was induced after being fused to glucoamylase, an extracellular hydrolase produced by N. crassa. Glucoamylase induction and other culture parameters were monitored in untransformed N. crassa grown in shaken liquid culture. A DNA plasmid, pGEZ, was constructed by inserting zeamatin-encoding cDNA into an expression cassette containing the promoter, a truncated open reading frame, and the terminator sequence of the N. crassa glucoamylase gene. Zeamatin-encoding cDNA was modified at the N terminus to include a kex-2 protease site, allowing cleavage of the chimeric product in the secretory pathway. Strains containing the chimeric gene construct were grown in liquid culture and induced for glucoamylase and zeamatin production. Zeamatin antibody detected a protein in a Western blot of concentrated culture supernatants that comigrated with authentic zeamatin. Secreted zeamatin was active in inhibiting the growth of Candida albicans in an agar diffusion assay, indicating that zeamatin had been correctly synthesized, processed, and secreted by N. crassa.     

Characterization of the promoter activity of a poxvirus conserved element

The conserved sequence element (CSE) is a highly conserved 42-bp poxvirus sequence that can function as a poxvirus promoter element. The CSE is composed of 2 repeats, each containing the highly conserved late poxvirus promoter sequence TAAAT. To define the location of the nucleotides critical for promoter function, polymerase chain reaction was carried out using primers that inserted modified versions of the CSE upstream of the green fluorescent protein (GFP), and the constructs were transiently transfected into cells by using GFP levels as a measure of promoter function. The results of this analysis revealed that the second TAAAT sequence, but not the first TAAAT sequence, is critical for promoter function of the CSE. Furthermore, deletion of half of the intervening sequence, i.e., from 10 to 5 nt, increases the promoter strength of the CSE as compared with the wild-type CSE. These results indicate the potential of this novel poxvirus promoter for driving high levels of gene expression.     

T cell-specific negative regulation of transcription of the human cytokine IL-4.

IL-4 secreted by activated T cells is a pleiotropic cytokine affecting growth and differentiation of diverse cell types such as T cells, B cells, and mast cells. We investigated the upstream regulatory elements of the human IL-4 promoter. A novel T cell-specific negative regulatory element (NRE) composed of two protein-binding sites were mapped in the 5' flanking region of the IL-4 gene: -311CTCCCTTCT-303 (NRE-I) and -288CTTTTTGCTT-TGC-300 (NRE-II). A T cell-specific protein Neg-1 and a ubiquitous protein Neg-2 binding to NRE-I and NRE-II, respectively, were identified. Furthermore, a positive regulatory element was found 45 bp downstream of the NRE. The enhancer activity of the PRE was completely suppressed when the NRE was present. These data suggest that IL-4 promoter activity is normally down-regulated by an NRE via repression of the enhancer positive regulatory element. These data may have implications for the stringent control of IL-4 expression in T cells.     

Site-specific recombination mediated by an adenovirus vector expressing the Cre recombinase protein: a molecular switch for control of gene expression.

We have constructed replication-defective human adenovirus (Ad) type 5 vectors containing the gene for the Cre recombinase from bacteriophage P1 under control of the human cytomegalovirus immediate-early promoter (AdCre). Expression of the protein was detected in replication-permissive (293) and in nonpermissive (MRC5) cell lines, and its biochemical activity was demonstrated in a cell-free recombination assay using a plasmid containing two loxP sites. To study Cre-mediated recombination in an intracellular system, we constructed an Ad vector (AdMA19) containing the luciferase cDNA under control of the human cytomegalovirus promoter but separated from it by an extraneous spacer sequence flanked by loxP sites which blocked luciferase expression. Upon coinfection of 293 or MRC5 cells with AdMA19 and AdCre, luciferase expression was specifically induced by Cre-mediated excision of the intervening sequence. The use of Ad vectors combined with the Cre-loxP system for regulation of gene expression and other possible applications is discussed.