Post-harvest light treatment increases expression levels of recombinant proteins in transformed plastids of potato tubers.

Plastid genetic engineering represents an attractive system for the production of foreign proteins in plants. Although high expression levels can be achieved in leaf chloroplasts, the results for non-photosynthetic plastids are generally discouraging. Here, we report the expression of two thioredoxin genes (trx f and trx m) from the potato plastid genome to study transgene expression in amyloplasts. As expected, the highest transgene expression was detected in the leaf (up to 4.2% of TSP). The Trx protein content in the tuber was approximately two to three orders of magnitude lower than in the leaf. However, we demonstrate that a simple post-harvest light treatment of microtubers developed in vitro or soil-grown tubers induces up to 55 times higher accumulation of the recombinant protein in just seven to ten days. After the applied treatment, the Trx f levels in microtubers and soil-grown tubers increased to 0.14% and 0.11% of TSP, respectively. Moreover, tubers stored for eight months maintained the capacity of increasing the foreign protein levels after the light treatment. Post-harvest cold induction (up to five times) at 4°C was also detected in microtubers. We conclude that plastid transformation and post-harvest light treatment could be an interesting approach for the production of foreign proteins in potato.

Serial analysis of gene expression reveals conserved links between protein kinase A, ribosome biogenesis, and phosphate metabolism in Ustilago maydis.

The switch from budding to filamentous growth is a key aspect of invasive growth and virulence for the fungal phytopathogen Ustilago maydis. The cyclic AMP (cAMP) signaling pathway regulates dimorphism in U. maydis, as demonstrated by the phenotypes of mutants with defects in protein kinase A (PKA). Specifically, a mutant lacking the regulatory subunit of PKA encoded by the ubc1 gene displays a multiple-budded phenotype and fails to incite disease symptoms, although proliferation does occur in the plant host. A mutant with a defect in a catalytic subunit of PKA, encoded by adr1, has a constitutively filamentous phenotype and is nonpathogenic. We employed serial analysis of gene expression to examine the transcriptomes of a wild-type strain and the ubc1 and adr1 mutants to further define the role of PKA in U. maydis. The mutants displayed changes in the transcript levels for genes encoding ribosomal proteins, genes regulated by the b mating-type proteins, and genes for metabolic functions. Importantly, the ubc1 mutant displayed elevated transcript levels for genes involved in phosphate acquisition and storage, thus revealing a connection between cAMP and phosphate metabolism. Further experimentation indicated a phosphate storage defect and elevated acid phosphatase activity for the ubc1 mutant. Elevated phosphate levels in culture media also enhanced the filamentous growth of wild-type cells in response to lipids, a finding consistent with PKA regulation of morphogenesis in U. maydis. Overall, these findings extend our understanding of cAMP signaling in U. maydis and reveal a link between phosphate metabolism and morphogenesis.

Mapping of genomic regions (quantitative trait loci) controlling production and quality in industrial cultures of the edible basidiomycete Pleurotus ostreatus.

Industrial production of the edible basidiomycete Pleurotus ostreatus (oyster mushroom) is based on a solid fermentation process in which a limited number of selected strains are used. Optimization of industrial mushroom production depends on improving the culture process and breeding new strains with higher yields and productivities. Traditionally, fungal breeding has been carried out by an empirical trial and error process. In this study, we used a different approach by mapping quantitative trait loci (QTLs) controlling culture production and quality within the framework of the genetic linkage map of P. ostreatus. Ten production traits and four quality traits were studied and mapped. The production QTLs identified explain nearly one-half of the production variation. More interestingly, a single QTL mapping to the highly polymorphic chromosome VII appears to be involved in control of all the productivity traits studied. Quality QTLs appear to be scattered across the genome and to have less effect on the variation of the corresponding traits. Moreover, some of the new hybrid strains constructed in the course of our experiments had production or quality values higher than those of the parents or other commercial strains. This approach opens the possibility of marker-assisted selection and breeding of new industrial strains of this fungus.

Quantitative trait loci controlling vegetative growth rate in the edible basidiomycete Pleurotus ostreatus.

Mycelium growth rate is a quantitative characteristic that exhibits continuous variation. This trait has applied interest, as growth rate is correlated with production yield and increased advantage against competitors. In this work, we studied growth rate variation in the edible basidiomycete Pleurotus ostreatus growing as monokaryotic or dikaryotic mycelium on Eger medium or on wheat straw. Our analysis resulted in identification of several genomic regions (quantitative trait loci [QTLs]) involved in the control of growth rate that can be mapped on the genetic linkage map of this fungus. In some cases monokaryotic and dikaryotic QTLs clustered at the same map position, indicating that there are principal genomic areas responsible for growth rate control. The availability of this linkage map of growth rate QTLs can help in the design of rational strain breeding programs based on genomic information.

Differentially regulated, vegetative-mycelium-specific hydrophobins of the edible basidiomycete Pleurotus ostreatus.

Three different hydrophobins (Vmh1, Vmh2, and Vmh3) were isolated from monokaryotic and dikaryotic vegetative cultures of the edible fungus Pleurotus ostreatus. Their corresponding genes have a number of introns different from those of other P. ostreatus hydrophobins previously described. Two genes (vmh1 and vmh2) were expressed only at the vegetative stage, whereas vmh3 expression was also found in the fruit bodies. Furthermore, the expression of the three hydrophobins varied significantly with culture time and nutritional conditions. The three genes were mapped in the genomic linkage map of P. ostreatus, and evidence is presented for the allelic nature of vmh2 and POH3 and for the different locations of the genes coding for the glycosylated hydrophobins Vmh3 and POH2. The glycosylated nature of Vmh3 and its expression during vegetative growth and in fruit bodies suggest that it should play a role in development similar to that proposed for SC3 in Schizophyllum commune.