Structural insights into the dual activities of the two-barrel RNA polymerase QDE-1.

Neurospora crassa protein QDE-1, a member of the two-barrel polymerase superfamily, possesses both DNA- and RNA-dependent RNA polymerase (DdRP and RdRP) activities. The dual activities are essential for the production of double-stranded RNAs (dsRNAs), the precursors of small interfering RNAs (siRNAs) in N. crassa. Here, we report five complex structures of N-terminal truncated QDE-1 (QDE-1ΔN), representing four different reaction states: DNA/RNA-templated elongation, the de novo initiation of RNA synthesis, the first step of nucleotide condensation during de novo initiation and initial NTP loading. The template strand is aligned by a bridge-helix and double-psi beta-barrels 2 (DPBB2), the RNA product is held by DPBB1 and the slab domain. The DNA template unpairs with the RNA product at position -7, but the RNA template remains paired. The NTP analog coordinates with cations and is precisely positioned at the addition site by a rigid trigger loop and a proline-containing loop in the active center. The unique C-terminal tail from the QDE-1 dimer partner inserts into the substrate-binding cleft and plays regulatory roles in RNA synthesis. Collectively, this work elucidates the conserved mechanisms for DNA/RNA-dependent dual activities by QDE-1 and other two-barrel polymerase superfamily members.

An integrative NMR-SAXS approach for structural determination of large RNAs defines the substrate-free state of a trans-cleaving Neurospora Varkud Satellite ribozyme.

The divide-and-conquer strategy is commonly used for protein structure determination, but its applications to high-resolution structure determination of RNAs have been limited. Here, we introduce an integrative approach based on the divide-and-conquer strategy that was undertaken to determine the solution structure of an RNA model system, the Neurospora VS ribozyme. NMR and SAXS studies were conducted on a minimal trans VS ribozyme as well as several isolated subdomains. A multi-step procedure was used for structure determination that first involved pairing refined NMR structures with SAXS data to obtain structural subensembles of the various subdomains. These subdomain structures were then assembled to build a large set of structural models of the ribozyme, which was subsequently filtered using SAXS data. The resulting NMR-SAXS structural ensemble shares several similarities with the reported crystal structures of the VS ribozyme. However, a local structural difference is observed that affects the global fold by shifting the relative orientation of the two three-way junctions. Thus, this finding highlights a global conformational change associated with substrate binding in the VS ribozyme that is likely critical for its enzymatic activity. Structural studies of other large RNAs should benefit from similar integrative approaches that allow conformational sampling of assembled fragments.

NLR surveillance of essential SEC-9 SNARE proteins induces programmed cell death upon allorecognition in filamentous fungi.

In plants and metazoans, intracellular receptors that belong to the NOD-like receptor (NLR) family are major contributors to innate immunity. Filamentous fungal genomes contain large repertoires of genes encoding for proteins with similar architecture to plant and animal NLRs with mostly unknown function. Here, we identify and molecularly characterize patatin-like phospholipase-1 (PLP-1), an NLR-like protein containing an N-terminal patatin-like phospholipase domain, a nucleotide-binding domain (NBD), and a C-terminal tetratricopeptide repeat (TPR) domain. PLP-1 guards the essential SNARE protein SEC-9; genetic differences at plp-1 and sec-9 function to trigger allorecognition and cell death in two distantly related fungal species, Neurospora crassa and Podospora anserina Analyses of Neurospora population samples revealed that plp-1 and sec-9 alleles are highly polymorphic, segregate into discrete haplotypes, and show transspecies polymorphism. Upon fusion between cells bearing incompatible sec-9 and plp-1 alleles, allorecognition and cell death are induced, which are dependent upon physical interaction between SEC-9 and PLP-1. The central NBD and patatin-like phospholipase activity of PLP-1 are essential for allorecognition and cell death, while the TPR domain and the polymorphic SNARE domain of SEC-9 function in conferring allelic specificity. Our data indicate that fungal NLR-like proteins function similar to NLR immune receptors in plants and animals, showing that NLRs are major contributors to innate immunity in plants and animals and for allorecognition in fungi.

Light-activated protein interaction with high spatial subcellular confinement.

Methods to acutely manipulate protein interactions at the subcellular level are powerful tools in cell biology. Several blue-light-dependent optical dimerization tools have been developed. In these systems one protein component of the dimer (the bait) is directed to a specific subcellular location, while the other component (the prey) is fused to the protein of interest. Upon illumination, binding of the prey to the bait results in its subcellular redistribution. Here, we compared and quantified the extent of light-dependent dimer occurrence in small, subcellular volumes controlled by three such tools: Cry2/CIB1, iLID, and Magnets. We show that both the location of the photoreceptor protein(s) in the dimer pair and its (their) switch-off kinetics determine the subcellular volume where dimer formation occurs and the amount of protein recruited in the illuminated volume. Efficient spatial confinement of dimer to the area of illumination is achieved when the photosensitive component of the dimerization pair is tethered to the membrane of intracellular compartments and when on and off kinetics are extremely fast, as achieved with iLID or Magnets. Magnets and the iLID variants with the fastest switch-off kinetics induce and maintain protein dimerization in the smallest volume, although this comes at the expense of the total amount of dimer. These findings highlight the distinct features of different optical dimerization systems and will be useful guides in the choice of tools for specific applications.

The Fungal Iron Chelator Desferricoprogen Inhibits Atherosclerotic Plaque Formation.

Hemoglobin, heme and iron are implicated in the progression of atherosclerosis. Therefore, we investigated whether the hydrophobic fungal iron chelator siderophore, desferricoprogen (DFC) inhibits atherosclerosis. DFC reduced atherosclerotic plaque formation in ApoE-/- mice on an atherogenic diet. It lowered the plasma level of oxidized LDL (oxLDL) and inhibited lipid peroxidation in aortic roots. The elevated collagen/elastin content and enhanced expression of adhesion molecule VCAM-1 were decreased. DFC diminished oxidation of Low-density Lipoprotein (LDL) and plaque lipids catalyzed by heme or hemoglobin. Formation of foam cells, uptake of oxLDL by macrophages, upregulation of CD36 and increased expression of TNF-α were reduced by DFC in macrophages. TNF-triggered endothelial cell activation (vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecules (ICAMs), E-selectin) and increased adhesion of monocytes to endothelium were attenuated. The increased endothelial permeability and intracellular gap formation provoked by TNF-α was also prevented by DFC. DFC acted as a cytoprotectant in endothelial cells and macrophages challenged with a lethal dose of oxLDL and lowered the expression of stress-responsive heme oxygenase-1 as sublethal dose was employed. Saturation of desferrisiderophore with iron led to the loss of the beneficial effects. We demonstrated that DFC accumulated within the atheromas of the aorta in ApoE-/- mice. DFC represents a novel therapeutic approach to control the progression of atherosclerosis.

Neurospora crassa family GH72 glucanosyltransferases function to crosslink cell wall glycoprotein N-linked galactomannan to cell wall lichenin.

The formation of a glucan/chitin/glycoprotein cell wall matrix is vital for fungal survival, growth, and morphogenesis. The cell wall proteins are important cell wall components and function in adhesion, signal transduction, and as cell wall structural elements. In this report we demonstrate that Neurospora crassa GH72 glucan transferases function to crosslink cell wall glycoproteins into the cell wall. With an in vitro assay, we show that the glucan transferases are able to attach lichenin, a cell wall glucan with a repeating ß-1,4-glucose-ß-1,4-glucose-ß-1,3-glucose structure, to cell wall glycoproteins. We propose that the pathway for attachment of lichenin to the glycoprotein has four steps. First, N-linked oligosaccharides present on the glycoproteins are modified by the addition of a galactomannan. As part of our report we have characterized the structure of the galactomannan, which consists of an α-1,6-mannose backbone with galactofuranose side chains. In the second step, the galactomannan is processed by members of the GH76 α-1,6-mannanases. In the third step, the glucan transferases cleave the lichenin and create substrate-enzyme intermediates. In the final step, the transferases transfer the lichenin to the processed galactomannan. We demonstrate that the N. crassa glucan transferases have demonstrate specificity for the processed galactomannan and for lichenin. The energy from the cleaved glycosidic bond in lichenin is retained in the substrate-enzyme intermediate and used to create a new glycosidic bond between the lichenin and the processed galactomannan. The pathway effectively crosslinks glycoproteins into the fungal cell wall.

Non-optimal codon usage affects expression, structure and function of clock protein FRQ.

Codon-usage bias has been observed in almost all genomes and is thought to result from selection for efficient and accurate translation of highly expressed genes. Codon usage is also implicated in the control of transcription, splicing and RNA structure. Many genes exhibit little codon-usage bias, which is thought to reflect a lack of selection for messenger RNA translation. Alternatively, however, non-optimal codon usage may be of biological importance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. Here we show that, unlike most genes in Neurospora, frq exhibits non-optimal codon usage across its entire open reading frame. Optimization of frq codon usage abolishes both overt and molecular circadian rhythms. Codon optimization not only increases FRQ levels but, unexpectedly, also results in conformational changes in FRQ protein, altered FRQ phosphorylation profile and stability, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function. Our study provides an example of how non-optimal codon usage functions to regulate protein expression and to achieve optimal protein structure and function.

Poly-Saturated Dolichols from Filamentous Fungi Modulate Activity of Dolichol-Dependent Glycosyltransferase and Physical Properties of Membranes.

 Mono-saturated polyprenols (dolichols) have been found in almost all Eukaryotic cells, however, dolichols containing additional saturated bonds at the ω-end, have been identified in A. fumigatus and A. niger. Here we confirm using an LC-ESI-QTOF-MS analysis, that poly-saturated dolichols are abundant in other filamentous fungi, Trichoderma reesei, A. nidulans and Neurospora crassa, while the yeast Saccharomyces cerevisiae only contains the typical mono-saturated dolichols. We also show, using differential scanning calorimetry (DSC) and fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) that the structure of dolichols modulates the properties of membranes and affects the functioning of dolichyl diphosphate mannose synthase (DPMS). The activity of this enzyme from T. reesei and S. cerevisiae was strongly affected by the structure of dolichols. Additionally, the structure of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) model membranes was more strongly disturbed by the poly-saturated dolichols from Trichoderma than by the mono-saturated dolichols from yeast. By comparing the lipidome of filamentous fungi with that from S. cerevisiae, we revealed significant differences in the PC/PE ratio and fatty acids composition. Filamentous fungi differ from S. cerevisiae in the lipid composition of their membranes and the structure of dolichols. The structure of dolichols profoundly affects the functioning of dolichol-dependent enzyme, DPMS.

The DEAD-Box Protein CYT-19 Uses Arginine Residues in Its C-Tail To Tether RNA Substrates.

DEAD-box proteins are nonprocessive RNA helicases that play diverse roles in cellular processes. The Neurospora crassa DEAD-box protein CYT-19 promotes mitochondrial group I intron splicing and functions as a general RNA chaperone. CYT-19 includes a disordered, arginine-rich "C-tail" that binds RNA, positioning the helicase core to capture and unwind nearby RNA helices. Here we probed the C-tail further by varying the number and positions of arginines within it. We found that removing sets of as few as four of the 11 arginines reduced RNA unwinding activity (kcat/KM) to a degree equivalent to that seen upon removal of the C-tail, suggesting that a minimum or "threshold" number of arginines is required. In addition, a mutant with 16 arginines displayed RNA unwinding activity greater than that of wild-type CYT-19. The C-tail modifications impacted unwinding only of RNA helices within constructs that included an adjacent helix or structured RNA element that would allow C-tail binding, indicating that the helicase core remained active in the mutants. In addition, changes in RNA unwinding efficiency of the mutants were mirrored by changes in functional RNA affinity, as determined from the RNA concentration dependence of ATPase activity, suggesting that the C-tail functions primarily to increase RNA affinity. Interestingly, the salt concentration dependence of RNA unwinding activity is unaffected by C-tail composition, suggesting that the C-tail uses primarily hydrogen bonding, not electrostatic interactions, to bind double-stranded RNA. Our results provide insights into how an unstructured C-tail contributes to DEAD-box protein activity and suggest parallels with other families of RNA- and DNA-binding proteins.

Heat Shock Protein 90 kDa (Hsp90) Has a Second Functional Interaction Site with the Mitochondrial Import Receptor Tom70.

To accomplish its crucial role, mitochondria require proteins that are produced in the cytosol, delivered by cytosolic Hsp90, and translocated to its interior by the translocase outer membrane (TOM) complex. Hsp90 is a dimeric molecular chaperone and its function is modulated by its interaction with a large variety of co-chaperones expressed within the cell. An important family of co-chaperones is characterized by the presence of one TPR (tetratricopeptide repeat) domain, which binds to the C-terminal MEEVD motif of Hsp90. These include Tom70, an important component of the TOM complex. Despite a wealth of studies conducted on the relevance of Tom70·Hsp90 complex formation, there is a dearth of information regarding the exact molecular mode of interaction. To help fill this void, we have employed a combined experimental strategy consisting of cross-linking/mass spectrometry to investigate binding of the C-terminal Hsp90 domain to the cytosolic domain of Tom70. This approach has identified a novel region of contact between C-Hsp90 and Tom70, a finding that is confirmed by probing the corresponding peptides derived from cross-linking experiments via isothermal titration calorimetry and mitochondrial import assays. The data generated in this study are combined to input constraints for a molecular model of the Hsp90/Tom70 interaction, which has been validated by small angle x-ray scattering, hydrogen/deuterium exchange, and mass spectrometry. The resultant model suggests that only one of the MEEVD motifs within dimeric Hsp90 contacts Tom70. Collectively, our findings provide significant insight on the mechanisms by which preproteins interact with Hsp90 and are translocated via Tom70 to the mitochondria.

The putative cellodextrin transporter-like protein CLP1 is involved in cellulase induction in Neurospora crassa.

Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3ßG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3ßG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators.

Tubulin tail sequences and post-translational modifications regulate closure of mitochondrial voltage-dependent anion channel (VDAC).

It was previously shown that tubulin dimer interaction with the mitochondrial outer membrane protein voltage-dependent anion channel (VDAC) blocks traffic through the channel and reduces oxidative metabolism and that this requires the unstructured anionic C-terminal tail peptides found on both α- and ß-tubulin subunits. It was unclear whether the α- and ß-tubulin tails contribute equally to VDAC blockade and what effects might be due to sequence variations in these tail peptides or to tubulin post-translational modifications, which mostly occur on the tails. The nature of the contribution of the tubulin body beyond acting as an anchor for the tails had not been clarified either. Here we present peptide-protein chimeras to address these questions. These constructs allow us to easily combine a tail peptide with different proteins or combine different tail peptides with a particular protein. The results show that a single tail grafted to an inert protein is sufficient to produce channel closure similar to that observed with tubulin. We show that the ß-tail is more than an order of magnitude more potent than the α-tail and that the lower α-tail activity is largely due to the presence of a terminal tyrosine. Detyrosination activates the α-tail, and activation is reversed by the removal of the glutamic acid penultimate to the tyrosine. Nitration of tyrosine reverses the tyrosine inhibition of binding and even induces prolonged VDAC closures. Our results demonstrate that small changes in sequence or post-translational modification of the unstructured tails of tubulin result in substantial changes in VDAC closure.

O-Glycan analysis of cellobiohydrolase I from Neurospora crassa.

We describe here the composition of the O-linked glycans on the Neurospora crassa cellobiohydrolase I (CBHI), which accounts for approximately 40% of the protein secreted by cells growing in the presence of cellulose. CBHI is O-glycosylated with six types of linear, and three types of branched, O-glycans containing approximately equal amounts of mannose and galactose. In addition to the classic fungal O-glycans with reducing end mannoses, we also identified reducing end galactoses which suggest the existence of a protein-O-galactosyltransferase in N. crassa Because of the excellent genetic resources available for N. crassa, the knowledge of the CBHI O-glycans may enable the future evaluation of the role of O-glycosylation on cellulase function and the development of directed O-glycan/cellulase engineering.

Linear Discriminant Analysis Identifies Mitochondrially Localized Proteins in Neurospora crassa.

Besides their role as powerhouses, mitochondria play a pivotal role in the spatial organization of numerous enzymatic functions. They are connected to the ER, and many pathways are organized through the mitochondrial membranes. Thus, the precise definition of mitochondrial proteomes remains a challenging task. Here, we have established a proteomic strategy to accurately determine the mitochondrial localization of proteins from the fungal model organism Neurospora crassa. This strategy relies on both highly pure mitochondria as well as the quantitative monitoring of mitochondrial components along their consecutive enrichment. Pure intact mitochondria were obtained by a multistep approach combining differential and density Percoll (ultra) centrifugations. When compared with three other intermediate enrichment stages, peptide sequencing and quantitative profiling of pure mitochondrial fractions revealed prototypic regulatory profiles of per se mitochondrial components. These regulatory profiles constitute a distinct cluster defining the mitochondrial compartment and support linear discriminant analyses, which rationalized the annotation process. In total, this approach experimentally validated the mitochondrial localization of 512 proteins including 57 proteins that had not been reported for N. crassa before.

Role of Saccharomyces cerevisiae Trk1 in stabilization of intracellular potassium content upon changes in external potassium levels.

Saccharomyces cerevisiae cells are able to grow at very different potassium concentrations adapting its intracellular cation levels to changes in the external milieu. Potassium homeostasis in wild type cells resuspended in media with low potassium is an example of non-perfect adaptation since the same intracellular concentration is not approached irrespective of the extracellular levels of the cation. By using yeasts lacking the Trk1,2 system or expressing different versions of the mutated main plasma membrane potassium transporter (Trk1), we show that Trk1 is not essential for adaptation to potassium changes but the dynamics of potassium loss is very different in the wild type and in trk1,2 mutant or in yeasts expressing Trk1 versions with highly impaired transport characteristics. We also show that the pattern here described can be also fulfilled by heterologous expression of NcHAK1, a potassium transporter not belonging to the TRK family. Hyperpolarization and cationic drugs sensitivity in mutants with defective transport capacity provide additional support to the hypothesis of connections between the activity of the Trk system and the plasma membrane H(+) ATPase (Pma1) in the adaptive process.

Deciphering the uniqueness of Mucoromycotina cell walls by combining biochemical and phylogenomic approaches.

Most fungi from the Mucoromycotina lineage occur in ecosystems as saprobes, although some species are phytopathogens or may induce human mycosis. Mucoromycotina represent early diverging models that are most valuable for understanding fungal evolution. Here we reveal the uniqueness of the cell wall structure of the Mucoromycotina Rhizopus oryzae and Phycomyces blakesleeanus compared with the better characterized cell wall of the ascomycete Neurospora crassa. We have analysed the corresponding polysaccharide biosynthetic and modifying pathways, and highlight their evolutionary features and higher complexity in terms of gene copy numbers compared with species from other lineages. This work uncovers the presence in Mucoromycotina of abundant fucose-based polysaccharides similar to algal fucoidans. These unexpected polymers are associated with unusually low amounts of glucans and a higher proportion of chitin compared with N. crassa. The specific structural features are supported by the identification of genes potentially involved in the corresponding metabolic pathways. Phylogenomic analyses of genes encoding carbohydrate synthases, polysaccharide modifying enzymes and enzymes involved in nucleotide-sugar formation provide evidence for duplication events during evolution of cell wall metabolism in fungi. Altogether, the data highlight the specificity of Mucoromycotina cell walls and pave the way for a finer understanding of their metabolism.

Transient transformation of Podosphaera xanthii by electroporation of conidia.

BACKGROUND: Powdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. Powdery mildew fungi are obligate biotrophic parasites unable to grow on culture media, a fact that has significantly limited their genetic manipulation. In this work, we report a protocol based on the electroporation of fungal conidia, for the transient transformation of Podosphaera fusca (synonym Podosphaera xanthii), the main causal agent of cucurbit powdery mildew. RESULTS: To introduce DNA into P. xanthii conidia, we applied two square-wave pulses of 1.7 kV for 1 ms with an interval of 5 s. We tested these conditions with several plasmids bearing as selective markers hygromycin B resistance (hph), carbendazim resistance (TUB2) or GFP (gfp) under control of endogenous regulatory elements from Aspergillus nidulans, Neurospora crassa or P. xanthii to drive their expression. An in planta selection procedure using the MBC fungicide carbendazim permitted the propagation of transformants onto zucchini cotyledons and avoided the phytotoxicity associated with hygromycin B. CONCLUSION: This is the first report on the transformation of P. xanthii and the transformation of powdery mildew fungi using electroporation. Although the transformants are transient, this represents a feasible method for the genetic manipulation of this important group of plant pathogens.

A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation.

Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.

Fungal type III polyketide synthases.

This article covers the literature on fungal type III polyketide synthases (PKSs) published from 2005 to 2014. Since the first discovery of fungal type III PKS genes in Aspergillus oryzae, reported in 2005, putative genes for type III PKSs have been discovered in fungal genomes. Compared with type I PKSs, type III PKSs are much less abundant in fungi. However, type III PKSs could have some critical roles in fungi. This article summarizes the studies on fungal type III PKS functional analysis, including Neurospora crassa ORAS, Aspergillus niger AnPKS, Botrytis cinerea BPKS and Aspergillus oryzae CsyA and CsyB. It is mostly in vitro analysis using their recombinant enzymes that has revealed their starter and product specificities. Of these, CsyB was found to be a new kind of type III PKS that catalyses the coupling of two ß-keto fatty acyl CoAs. Homology modelling reported in this article supports the importance of the capacity of the acyl binding tunnel and active site cavity in fungal type III PKSs.

The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation.

Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ)-based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium vs sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.