Identification and characterization of a new soybean promoter induced by Phakopsora pachyrhizi, the causal agent of Asian soybean rust.

BACKGROUND: Phakopsora pachyrhizi is a biotrophic fungal pathogen responsible for the Asian soybean rust disease causing important yield losses in tropical and subtropical soybean-producing countries. P. pachyrhizi triggers important transcriptional changes in soybean plants during infection, with several hundreds of genes being either up- or downregulated. RESULTS: Based on published transcriptomic data, we identified a predicted chitinase gene, referred to as GmCHIT1, that was upregulated in the first hours of infection. We first confirmed this early induction and showed that this gene was expressed as early as 8 h after P. pachyrhizi inoculation. To investigate the promoter of GmCHIT1, transgenic soybean plants expressing the green fluorescence protein (GFP) under the control of the GmCHIT1 promoter were generated. Following inoculation of these transgenic plants with P. pachyrhizi, GFP fluorescence was detected in a limited area located around appressoria, the fungal penetration structures. Fluorescence was also observed after mechanical wounding whereas no variation in fluorescence of pGmCHIT1:GFP transgenic plants was detected after a treatment with an ethylene precursor or a methyl jasmonate analogue. CONCLUSION: We identified a soybean chitinase promoter exhibiting an early induction by P. pachyrhizi located in the first infected soybean leaf cells. Our results on the induction of GmCHIT1 promoter by P. pachyrhizi contribute to the identification of a new pathogen inducible promoter in soybean and beyond to the development of a strategy for the Asian soybean rust disease control using biotechnological approaches.

Ambient alkaline pH prevents maturation but not synthesis of ASPA, the aspartyl protease from Penicillium roqueforti.

Penicillium roqueforti secretes an aspartyl protease, ASPA, which represents the main extracellular proteolytic activity. Alkaline pH of the medium plays a major role by inhibiting the enzymatic activity and stopping aspA expression in the presence of casein, an inducing protein. However, casein degradation by the mature enzyme produces peptides which can induce aspA expression at acidic and alkaline pH. ASPA synthesized as a proenzyme is processed at an acidic pH but not at an alkaline pH. The data indicate that, in P. roqueforti, alkaline pH has an indirect repressive effect by inhibiting ASPA maturation and the release of inducers. At an acidic pH, the mature enzyme degrades extracellular proteins and peptides are released to induce aspA. In contrast, at an alkaline pH, the proenzyme remains inactive, the inducing substances are consequently not produced and aspA is no longer expressed.

aspS encoding an unusual aspartyl protease from Sclerotinia sclerotiorum is expressed during phytopathogenesis.

The gene aspS encoding an aspartyl protease has been cloned from Sclerotinia sclerotiorum by screening a genomic library with a PCR-amplified fragment of the gene. The open reading frame of 1368 bp interrupted by one intron would encode a preproprotein of 435 amino acids. The catalytic aspartyl residues characteristic of aspartyl proteases are conserved; however, the active-site motif (DSG) in the N-terminal lobe is unusual in that Ser replaced Thr used in the active-site motif (DTG) of the C-terminal lobe and in all other fungal aspartyl proteases. RT-PCR revealed that aspS expression in axenic culture is not subjected to catabolite repression and demonstrated that aspS is expressed from the beginning of infection of sunflower cotyledons.

Isolation and expression of a nitrogen regulatory gene, nmc, of Penicillium roqueforti.

The nmc gene, encoding a global nitrogen regulator, has been cloned and characterized from Penicillium roqueforti, a fungus used in the dairy industry. The deduced amino acid sequence predicts a protein of 860 amino acids in length whose zinc finger DNA binding domain is at least 94% identical to those of the homologous fungal proteins. Northern blot analysis showed that nmc expression is induced by nitrogen starvation and not repressed by variation of the external pH.

Controls of the expression of aspA, the aspartyl protease gene from Penicillium roqueforti.

The gene (aspA) encoding the extracellular aspartyl protease from Penicillium roqueforti was cloned and characterized. Northern hybridization analyses and beta-casein degradation assays revealed that aspA was strongly induced by casein in the medium and efficiently repressed by ammonia. External alkaline pH overrides casein induction, resulting in aspA repression. Cis-acting motifs known to mediate nitrogen and pH regulation of fungal gene expression are present in the aspA promoter and protein-DNA binding experiments showed that mycelial proteins interact with various regions of the promoter. Due to the efficient environmental controls on aspA expression, the promoter of aspA is an attractive candidate for the development of a controllable gene expression system in P. roqueforti.

Characterization of a protease deficient strain of Penicillium roqueforti generated by heterologous plasmid integration: potential use for protein production.

A strain of Penicillium roqueforti was transformed with a plasmid which confers resistance to phleomycin. Stable transformants were selected. They all present a high resistance to the antibiotic. Their proteolytic activity was tested. One transformant is a proteolytic deficient strain unable to degrade casein. It is characterized by a tandem integration of the transformant vector in one site of the genome. The extracellular proteins profile of the strain reveals the absence of a 43 kDa polypeptide which probably corresponds to the aspartyl protease of Penicillium roqueforti. The aspA gene which encodes aspartyl protease is not expressed in the transformant and Southern analyses show that the aspA gene is not disrupted by the transformation vector.