Project description:We have studied the physiological response of the fungus Aspergillus niger when exposed to wheat straw as a model lignocellulosic substrate. Using RNA-sequencing we showed that, 24 hours after exposure to straw, gene expression of known plant cell wall degrading enzymes represents a huge investment for the cells (about 20 % of the total mRNA). Our results also uncovered new esterases and surface interacting proteins that might form part of the fungal degradative arsenal. We also show that antisense transcripts are abundant and that their expression can be regulated by conditions. Triplicate samples of A. niger N402 taken at each of three timepoints: After 48 h growth in minimal media with Glucose as sole carbon source, After transfer to Wheat Straw media for 24 h and 5 h after after the exogenous addition of glucose to the wheat starw media.
Project description:Pathogenicity of the phytopathogenic enterobacterium Erwinia chrysanthemi, the causative agent of soft rot disease in many plants, is a complex process involving several factors whose production is subject to temporal regulation during infection. After penetrating into its host plant, E. chrysanthemi resides latently in the plant intercellular spaces without provoking any symptoms, and disease occurs only when the environmental conditions are favourable for massive bacterial multiplication and production of plant cell wall degrading enzymes. PecS is a transcriptional regulator of the MarR family that represses production of plant cell wall degrading enzymes. Here, we used microarray analysis to define the PecS regulon and demonstrated that PecS exerts wide-ranging effects on gene expression in E. chrysanthemi. However, the major effects of PecS are largely confined to specific genes that could be linked to pathogenicity and to a group of genes concerned with evading host defences. Among the identified targets are the genes encoding plant cell wall degrading enzymes, secretion systems, the genes involved in flagella synthesis, in biosurfactant synthesis, in oxidative stress response as well as genes encoding toxin-like virulence factors such as NipE and Hemolysin-coregulated proteins. Electromobility shift assays and DNAse I footprinting demonstrated that PecS directly interacts with the regulatory regions of five new targets, ahpC, rhlA, nipE, virK, avrL, that define three different functional classes of genes: oxidative stress response genes (ahpC), biosurfactant synthesis gene (rhlA), genes encoding exported proteins related to other plant associated bacteria proteins (nipE, virK, avrL). Based on this work, we propose a pivotal role of PecS in the switch from a saprophytic to a parasitic lifestyle.
Project description:The basidiomycete white-rot fungus Obba rivulosa, a close relative of Gelatoporia (Ceriporiopsis) subvermispora, is an efficient degrader of softwood. The dikaryotic O. rivulosa strain T241i (FBCC949) has been shown to selectively remove lignin from spruce wood prior to depolymerization of plant cell wall polysaccharides, thus possessing potential in biotechnological applications such as pretreatment of wood in pulp and paper industry. In this work, we studied the time-course of the conversion of spruce by the genome-sequenced monokaryotic O. rivulosa strain 3A-2, which is derived from the dikaryon T241i, to get insights to transcriptome level changes during prolonged solid state cultivation. During 8-week cultivation, O. rivulosa expressed a constitutive set of genes encoding putative plant cell wall degrading enzymes. High level of expression of the genes targeted towards all plant cell wall polymers was detected at 2-week time point, after which majority of the genes showed reduced expression. This implicated non-selective degradation of lignin by the O. rivulosa monokaryon. These results suggest high variation between mono- and dikaryotic strains of the white-rot fungi with respect to their abilities to convert plant cell wall polymers. Overall design: The aim of the study is to study nutritional demands during a prolonged cultivation on wood by looking at the transcriptome at different time points, with special interes at CAZymes and central carbon metabolic genes. Obba rivulosa 3 A-2 growing on its natural substrate, spruce wood sticks was studied after 2, 4 and 8 weeks of solid state cultivation in order to follow wood depolymerization in more natural like conditions.
Project description:The genome of the lignocellulose-degrading, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus encodes genes comprising clusters of glycoside hydrolases, ABC transporters and metabolic enzymes that are transcriptionally responsive to carbohydrates. Transcriptomic and biosolubilization analyses were used to determine if C. saccharolyticus could be deployed as a probe to assess the characteristics of plant biomass feedstocks and efficacy of pre-treatment methods, as these both relate to deconstruction strategies for biofuels production. Based on the response of C. saccharolyticus to plant cell wall polysaccharides, genomic loci were identified that reflected the availability of cellulose, glucomannan, pectin and xylan in biomass to microbial degradation. Furthermore, these loci were useful in assessing how various plant biomass feedstocks (genetically and chemically modified Populus sp., unpretreated Populus sp., and chemically modified switchgrass) were amenable C. saccharolyticus solubilization. Overall design: Three independant 4-slide loop designs were used with a total of ten treatments. Biological repeats were harvested for each treatment. Loop #1 consists of plant model polysaccharides: crystalline cellulose (Avicel), mannan (konjac glucomannan), xylan (birchwood) and pectin. Loop #2 consists of comically pretreated and genetically modified plant biomass: Populus trichopcarpa (unpretreated), dilute acid pretreated switchgrass, dilute pretreated Populus trichocarpa x deltoides and genetically modified, lignin-reduced P. trichocarpa (as4CL1). Loop #3 consists of genetically modified P. trichocarpa: P. trichocarpa (unpretreated), and two variants of cellulose-reduced P. trichocarpa (7-1 and 7-2). Each condition was labeled with cy3 and cy5 dyes.
Project description:The induction of genes in response to exposure of T. reesei to wheat straw was explored using genome-wide RNA-seq and compared to published RNA-seq data and model of how A. niger senses and responds to the lignocellulose. After 24 h of exposure to straw, transcript levels of known and predicted lignocellulose-degrading enzymes increased to around 8% of total cellular mRNA in T. reesei, which was much less when compared to A. niger. The bulk of enzymes used to deconstruct wheat straw is similar in both fungi. Other, non-plant cell wall-degrading enzymes which may aid in lignocellulose degradation were also uncovered in T. reesei and similar to those described in A. niger. Antisense transcripts were also shown to be present in T. reesei and their expession can be regulated by the respective growth condition. Triplicate samples of T. reesei cultivated in each of the three following conditions were taken: 1) After 48 h growth in glucose-based minimal media; 2) After transfer of mycelia from glucose-based media into media containing wheat straw as a sole carbon source and 3) 5 h after addition of glucose to straw cultures.
Project description:Infection-related development of phytopathogenic fungi is initiated by sensing and responding to plant surface cues. This response results in the formation of specialized infection structures, so-called appressoria. To unravel the program inducing appressoria in the biotrophic smut fungus Ustilago maydis, we exposed cells to a hydrophobic surface and the cutin monomer 16-hydroxy hexadecanoic acid. Genome-wide transcriptional profiling under these appressorium-inducing in vitro conditions revealed dramatic transcriptional changes in almost 20% of the genes. Comparisons with the U. maydis sho1 msb2 double mutant, lacking two putative sensors for plant surface cues, revealed that these plasma membrane receptors regulate a small subset of the surface cue-induced genes. These genes comprised mainly secreted proteins including plant cell wall degrading enzymes that facilitate plant penetration and secreted effectors that are essential virulence factors, with functions after penetration. Our data also demonstrate specific effects on two transcription factors that redirect the transcriptional regulatory network towards appressorium formation and plant penetration. Thus, plant surface cues prime U. maydis for biotrophic development. Solopathogenic Ustilago maydis strain AM1 and its derivate AM1Δsho1Δmsb2 were grown to mid-log phase in YEPSL medium and resuspended in 2% YEPSL plus/minus 16-hydroxy hexadecanoic acid (HDA). The cell suspensions were sprayed on hydrophobic surface (Parafilm) and incubated for 12 h. As control, cells were sprayed on hydrophilic glass surface and incubated for 2 h. After RNA extraction Affymetrix microarrays were performed.
Project description:The zygomycete fungi-like Rhizomucor miehei have been extensively exploited for the production of various enzymes. As a thermophilic fungus, R. miehei is capable of growing at temperatures that approach the upper limits for all eukaryotes. In order to study the thermophilic mechanism, the transcriptional profiles of R. miehei CAU432 grown at two different temperatures (at 30°C and 50°C) were investigated by RNA-seq analysis. Approximately 35 million high-quality reads were generated from each library, and 62% reads were uniquely mapped to the genome. A high percentage of reads (67.1%) were mapped to predicted protein-coding genes, while 3.96% reads were distributed in splice junctions, 3.49% reads in antisense transcripts, 2.27% in introns, and 21.9% in other genomic regions. The frequency of reads which mapped to different genes ranged from one to over 300,000. More than 90% of predicted genes (9,680, 93% at 30°C; 9,618, 93.6% at 50°C) were detected with at least one read, while 128 genes and 190 genes were uniquely expressed at 50°C and 30°C, respectively. The results show that 2,117 genes were differently expressed (P<0.001) by the fungus with more than two-fold changes. These genes include 849 up-regulated and 1,268 down-regulated genes in mycelia grown at 50°C. These significantly differently expressed genes include many genes, putatively involved in thermophilic process, such as HSP, chaperones and proteasome. Overall design: 2 samples examined: mycelium growing at 30°C and 50°C
Project description:Digestive fluids isolated from the foregut (crop) of adult specimens of T. domestica were analysed by shotgun proteomics in order to identify the putative carbohydrate-active enzymes involved in plant cell wall digestion.
Project description:Enzymatic degradation of plant biomass requires a complex mixture of many different enzymes. Like most fungi, thermophilic Myceliophthora species therefore have a large set of enzymes targeting different linkages in plant polysaccharides. The majority of these enzymes have not been functionally characterized and their role in plant biomass degradation is unknown. This study describes a strategy using sexual crossing and screening with the thermophilic fungus Myceliophthora heterothallica to identify specific enzymes associated with improved sugar beet pulp saccharification.Two genetically diverse M. heterothallica strains CBS 203.75 and CBS 663.74 were used to generate progenies with improved growth on sugar beet pulp. One progeny, named SBP.F1.2.11, had a different genetic pattern from the parental strains, and had improved saccharification activity after growth on 3% sugar beet pulp. Exo-proteome analysis of progeny and parental strains after 7 days growth on sugar beet pulp showed that only 17 of the 133 secreted CAZy enzymes were more abundant in progeny SBP.F1.2.11. Particularly one enzyme belonging to the carbohydrate esterase family 5 (CE5) was more present in SBP.F1.2.11. This CE5-CBM1 enzyme, named as Axe1, was phylogenetically related to acetyl xylan esterases.