Expression of green fluorescent protein in Aureobasidium pullulans and quantification of the fungus on leaf surfaces.

A red-shifted, mutated form of the jelly-fish green fluorescent protein (GFP) under control of a TEF promoter was expressed at high levels in the filamentous fungus Aureobasidium pullulans. In the three transformants studied, all morphotypes of the fungus, including pigmented chlamydospores, expressed GFP and fluoresced brightly. Confocal microscopy showed that the intra-cellular distribution of GFP was nonuniform. When applied to leaf surfaces, the transformants were readily visible and amenable to quantification by image analysis. Thus, GFP expression, together with quantitative image analysis, may provide a powerful method for ecological studies of plant-microbe relationships in nature.

Quantitative sense-specific determination of murine coronavirus RNA by reverse transcription polymerase chain reaction.

In many applications, it is useful to know the sense and amount of viral RNAs present in a sample. In theory, sense-specific measurement of viral RNAs may be achieved by reverse transcription polymerase chain reaction (RT-PCR) assays which utilize primers of defined polarity during the RT step. However, in practice, it has been shown that such assays are prone to artifacts, such as non-specific priming, which drastically diminish their reliability. Using murine coronavirus MHV-4 as a model, we describe and validate several modifications of the RT-PCR procedure which eliminate these artifacts. Key RT-PCR parameters which were optimized include the design of tagged primers, DNase treatment of in vitro transcribed RNA standards, specification of temperature differences between RT and PCR annealing steps, and use of competitive RNA templates for quantitative assays. The assays described may be used to determine the sense and abundance of any viral or host RNA of interest in complex biological specimens.

Expression of lip genes during growth in soil and oxidation of anthracene by Phanerochaete chrysosporium.

mRNA extraction from soil and quantitation by competitive reverse transcription-PCR were combined to study the expression of the 10 known lignin peroxidase (lip) genes in anthracene-transforming soil cultures of Phanerochaete chrysosporium. Levels of extractable lipA transcript and protein (LiP H8) were well correlated, although they were separated by a 2-day lag period. The patterns of transcript abundance over time in soil-grown P. chrysosporium varied among the nine lip mRNAs detected; comparison with lip gene expression under different liquid culture conditions suggested an early phase of carbon limitation for the cultures as a whole, which was followed by a transition to nitrogen starvation. Anthracene transformation occurred throughout the 25-day course of the experiment and, therefore, likely involves mechanisms distinct from those involved in oxidation of non-LiP substrate polycyclic aromatic hydrocarbons.

Manganese peroxidase mRNA and enzyme activity levels during bioremediation of polycyclic aromatic hydrocarbon-contaminated soil with Phanerochaete chrysosporium.

mRNA extraction from soil and quantitation by competitive reverse transcription-PCR were combined to study the expression of three manganese peroxidase (MnP) genes during removal of polycyclic aromatic hydrocarbons from cultures of Phanerochaete chrysosporium grown in presterilized soil. Periods of high mnp transcript levels and extractable MnP enzyme activity were temporally correlated, although separated by a short (1- to 2-day) lag period. This time frame also coincided with maximal rates of fluorene oxidation and chrysene disappearance in soil cultures, supporting the hypothesis that high ionization potential polycyclic aromatic hydrocarbons are oxidized in soil via MnP-dependent mechanisms. The patterns of transcript abundance over time in soil-grown P. chrysosporium were similar for all three of the mnp mRNAs studied, indicating that transcription of this gene family may be coordinately regulated under these growth conditions.

Quantitation of fungal mRNAs in complex substrates by reverse transcription PCR and its application to Phanerochaete chrysosporium-colonized soil.

Thorough analysis of fungi in complex substrates has been hampered by inadequate experimental tools for assessing physiological activity and estimating biomass. We report a method for the quantitative assessment of specific fungal mRNAs in soil. The method was applied to complex gene families of Phanerochaete chrysosporium, a white-rot fungus widely used in studies of organopollutant degradation. Among the genes implicated in pollutant degradation, two closely related lignin peroxidase transcripts were detected in soil. The pattern of lignin peroxidase gene expression was unexpected; certain transcripts abundant in defined cultures were not detected in soil cultures. Transcripts encoding cellobiohydrolases and beta-tubulin were also detected. The method will aid in defining the roles of specific genes in complex biological processes such as organopollutant degradation, developing strategies for strain improvement, and identifying specific fungi in environmental samples.