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Members of the GRAS gene family encode transcriptional regulators that have diverse functions in plant growth and development such as gibberellin signal transduction, root radial patterning, axillary meristem formation, phytochrome A signal transduction, and gametogenesis. Bioinformatic analysis identified 57 and 32 GRAS genes in rice and Arabidopsis, respectively. Here, we provide a complete overview of this gene family, describing the gene structure, gene expression, chromosome localization, protein motif organization, phylogenetic analysis, and comparative analysis between rice and Arabidopsis. Phylogenetic analysis divides the GRAS gene family into eight subfamilies, which have distinct conserved domains and functions. Both genome/segmental duplication and tandem duplication contributed to the expansion of the GRAS gene family in the rice and Arabidopsis genomes. The existence of GRAS-like genes in bryophytes suggests that GRAS is an ancient family of transcription factors, which arose before the appearance of land plants over 400 million years ago.

Rational engineering of filamentous fungi for improved cellulase production is hampered by our incomplete knowledge of transcriptional regulatory networks. We therefore used the model filamentous fungus Neurospora crassa to search for uncharacterized transcription factors associated with cellulose deconstruction. A screen of a N. crassa transcription factor deletion collection identified two uncharacterized zinc binuclear cluster transcription factors (clr-1 and clr-2) that were required for growth and enzymatic activity on cellulose, but were not required for growth or hemicellulase activity on xylan. Transcriptional profiling with nextgeneration sequencing methods refined our understanding of the N. crassa transcriptional response to cellulose and demonstrated that clr-1 and clr-2 were required for the bulk of that response, including induction of all major cellulase and some major hemicellulase genes. Functional CLR-1 was necessary for expression of clr-2 and efficient cellobiose utilization. Phylogenetic analyses showed that CLR-1 and CLR-2 are conserved in the genomes of most filamentous ascomycete fungi capable of degrading cellulose. In Aspergillus nidulans, a strain carrying a deletion of the clr-2 homolog (clrB) failed to induce cellulase gene expression and lacked cellulolytic activity on Avicel. Further manipulation of this control system in industrial production strains may significantly improve yields of cellulases for cellulosic biofuel production.

BackgroundOver the past 3 years, the CRISPR/Cas9 system has revolutionized the field of genome engineering. However, its application has not yet been validated in thermophilic fungi. Myceliophthora thermophila, an important thermophilic biomass-degrading fungus, has attracted industrial interest for the production of efficient thermostable enzymes. Genetic manipulation of Myceliophthora is crucial for metabolic engineering and to unravel the mechanism of lignocellulose deconstruction. The lack of a powerful, versatile genome-editing tool has impeded the broader exploitation of M. thermophila in biotechnology.ResultsIn this study, a CRISPR/Cas9 system for efficient multiplexed genome engineering was successfully developed in the thermophilic species M. thermophila and M. heterothallica. This CRISPR/Cas9 system could efficiently mutate the imported amdS gene in the genome via NHEJ-mediated events. As a proof of principle, the genes of the cellulase production pathway, including cre-1, res-1, gh1-1, and alp-1, were chosen as editing targets. Simultaneous multigene disruptions of up to four of these different loci were accomplished with neomycin selection marker integration via a single transformation using the CRISPR/Cas9 system. Using this genome-engineering tool, multiple strains exhibiting pronounced hyper-cellulase production were generated, in which the extracellular secreted protein and lignocellulase activities were significantly increased (up to 5- and 13-fold, respectively) compared with the parental strain.ConclusionsA genome-wide engineering system for thermophilic fungi was established based on CRISPR/Cas9. Successful expansion of this system without modification to M. heterothallica indicates it has wide adaptability and flexibility for use in other Myceliophthora species. This system could greatly accelerate strain engineering of thermophilic fungi for production of industrial enzymes, such as cellulases as shown in this study and possibly bio-based fuels and chemicals in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0693-9) contains supplementary material, which is available to authorized users.

The filamentous fungus Neurospora crassa is a model laboratory organism, but in nature is commonly found growing on dead plant material, particularly grasses. Using functional genomics resources available for N. crassa, which include a near-full genome deletion strain set and whole genome microarrays, we undertook a systemwide analysis of plant cell wall and cellulose degradation. We identified approximately 770 genes that showed expression differences when N. crassa was cultured on ground Miscanthus stems as a sole carbon source. An overlap set of 114 genes was identified from expression analysis of N. crassa grown on pure cellulose. Functional annotation of up-regulated genes showed enrichment for proteins predicted to be involved in plant cell wall degradation, but also many genes encoding proteins of unknown function. As a complement to expression data, the secretome associated with N. crassa growth on Miscanthus and cellulose was determined using a shotgun proteomics approach. Over 50 proteins were identified, including 10 of the 23 predicted N. crassa cellulases. Strains containing deletions in genes encoding 16 proteins detected in both the microarray and mass spectrometry experiments were analyzed for phenotypic changes during growth on crystalline cellulose and for cellulase activity. While growth of some of the deletion strains on cellulose was severely diminished, other deletion strains produced higher levels of extracellular proteins that showed increased cellulase activity. These results show that the powerful tools available in N. crassa allow for a comprehensive system level understanding of plant cell wall degradation mechanisms used by a ubiquitous filamentous fungus.cellulase ͉ secretome ͉ transcriptome

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Fungal degradation of plant biomass may provide insights for improving cellulosic biofuel production. We show that the model cellulolytic fungus Neurospora crassa relies on a high-affinity cellodextrin transport system for rapid growth on cellulose. Reconstitution of the N. crassa cellodextrin transport system in Saccharomyces cerevisiae promotes efficient growth of this yeast on cellodextrins. In simultaneous saccharification and fermentation experiments, the engineered yeast strains more rapidly convert cellulose to ethanol when compared with yeast lacking this system.

The microRNA miR393 has been shown to play a role in plant development and in the stress response by targeting mRNAs that code for the auxin receptors in Arabidopsis. In this study, we verified that two rice auxin receptor gene homologs (OsTIR1 and OsAFB2) could be targeted by OsmiR393 (Os for Oryza sativa). Two new phenotypes (increased tillers and early flowering) and two previously observed phenotypes (reduced tolerance to salt and drought and hyposensitivity to auxin) were observed in the OsmiR393-overexpressing rice plants. The OsmiR393-overexpressing rice demonstrated hyposensitivity to synthetic auxin-analog treatments. These data indicated that the phenotypes of OsmiR393-overexpressing rice may be caused through hyposensitivity to the auxin signal by reduced expression of two auxin receptor genes (OsTIR1 and OsAFB2). The expression of an auxin transporter (OsAUX1) and a tillering inhibitor (OsTB1) were downregulated by overexpression of OsmiR393, which suggested that a gene chain from OsmiR393 to rice tillering may be from OsTIR1 and OsAFB2 to OsAUX1, which affected the transportation of auxin, then to OsTB1, which finally controlled tillering. The positive phenotypes (increased tillers and early flowering) and negative phenotypes (reduced tolerance to salt and hyposensitivity to auxin) of OsmiR393-overexpressing rice present a dilemma for molecular breeding.