ECFG 9 Parallel Session 3 Post-translational Regulation and Signaling


Apical dominance in fungal hyphae: the primacy of the tip

Camile Semighini, Aleksandra Virag, Steven Harris

University of Nebraska, Lincoln, NE, United States

The fungal mycelium consists of individual hyphae that radiate outwards from the colony center. Each hypha grows solely by tip extension, and generates new hyphae via the formation of lateral branches. To achieve this characteristic pattern of mycelial organization, individual hyphae must exhibit apical dominance, whereby the growing tip is dominant and appears to suppress the formation of other tips (i.e., lateral branches) in its general vicinity. At a physiological level, apical dominance presumably reflects the exclusive targeting of cellular resources (i.e., vesicles laden with precursors required for cell surface expansion and cell wall deposition) to the hyphal tip at the expense of potential branching sites. Accordingly, branch sites would only become active once they are a sufficient distance from the growing tip. Here, we present results that suggest possible mechanisms underlying apical dominance in the model fungus Aspergillus nidulans. In particular, we show that reactive oxygen species (ROS) generated by NADPH oxidase (Nox) accumulate at the hyphal tip. The absence of ROS, or its inappropriate localization, leads to aberrant patterns of branching. In contrast, excessive accumulation of ROS severely disrupts polarity establishment. Our results suggest that a tip-associated Nox complex (which includes Cdc42, Rac1, NoxR, and BemA) produces ROS that serves a spatial marker to define a single dominant axis of hyphal extension. Additional observations also implicate an ARF GTPase signaling module in the regulation of apical dominance.




Cell wall dynamics in Aspergillus niger

Robbert Damveld, Mark Arentshorst, Benjamin Nitsche, Vera Meyer, Cees van den Hondel, Arthur Ram

Institute of Biology Leiden, Leiden, Netherlands

The Aspergillus cell wall is a complex structure composed of a network of polysaccharides (alpha-glucan, beta-glucan, chitin and galactomannan) and cell wall proteins and is an essential factor in maintaining the shape and the integrity of the fungal cell. To ensure the integrity of the cell wall, fungi have evolved highly dynamic reinforcement and remodeling mechanisms to escape from cell wall threatening conditions. We have used a combination of genetic and genomic approaches to understand in more detail cell wall dynamics in the filamentous fungus Aspergillus niger. Treatment of A. niger with cell wall disturbing compounds, such as caspofungin (an inhibitor of beta-1,3-glucan synthesis) or Calcofluor White (interferes with chitin and glucan assembly) induces remodeling of the fungal cell wall by specific induction of genes that encode proteins involved in alpha-glucan synthesis, chitin synthesis and certain cell wall proteins. The cell wall stress response was also observed by interfering with polar growth. Overexpression of a dominant active form of the small GTPase RacA (RacAG12V), has a dramatic effect on fungal morphology and results in loss of polarized cell growth. Whereas the wild-type strain and the strain overexpressing the wild-type form of RacA form long hyphae, cells expressing RacAG12V grow isotropically, resulting in round cells. Transcriptomic studies using Affymetrix microarrays have been performed to identify genes that are differentially expressed during polarized or isotropical cell growth and again the agsA gene (encoding a putative alpha-glucan synthase) was found to be differentially expressed. We have thus used the agsA promoter region to develop a GFP-based reporter system to identify cell wall stress inducing compounds. In addition, the agsA promoter region was also used to design a new strategy for isolating cell wall mutants with a constitutively activated cell wall stress response pathway. We show that a mutation that affects galactomannan biosynthesis in A. niger strongly induces agsA expression and that galactomannan biosynthesis is an attractive target for the development of new antifungals.





Protein degradation and fungal development

Gerhard Braus, Özgür Bayram, Kerstin Helmstaedt, Elke Schwier, Oliver Valerius

Georg August Univ, Göttingen, Germany

The homothallic filamentous ascomycete A. nidulans is able to form fruitbodies (cleistothecia) either by mating of two strains or by selfing in the absence of a partner. The three-dimensional A. nidulans cleistothecium is the most complicated structure this fungus is able to form. This includes an energy and material consuming process where parts of hyphae have to be dissolved and locally rearranged. Fungal development is coordinated with the control of a broad secondary metabolism and external stimuli as light, nutrition or airation (Braus et al., 2002). We have recently shown that filamentous fungi possess an eight subunit COP9 signalosome CSN which is reminiscent to the corresponding plant and vertebrate complex. CSN is required in the model A. nidulans for accurate fungal development, because deletion of the gene for subunits of CSN result in a block in sexual development. Furthermore csn mutants are blind towards light, whereas the wildtype prefers asexual development in the presence of light and sexual development in darkness, respectively. Thirdly, csn mutants are impaired in the control of secondary metabolism (Busch et al., 2003, Busch et al., 2007). The COP9 signalosome is involved in the control of protein degradation including SCF E3 ubiquitin ligase activities. The ubiquitinylation activity of SCF can be modulated by neddylation, a reversible conjugation of the ubiquitin-related protein NEDD8/Rub1 on the cullin subunit. Among the ubiquitin-like protein family, NEDD8 is most homologous to ubiquitin. The intrinsic COP9 signalosome deneddylase activity seems to be critical for the coordination of fungal development, secondary metabolism and light. The A. nidulans genome contains approximately 70 genes for putative F-boxes which are the adaptors of SCFs and are putative targets of CSN action. Twenty of these genes are specifically expressed during development. Deletion of one of these genes has already been shown to result in impaired fungal development (Krappmann et al., 2006). I will discuss the importance of a coordinated control of protein degradation in the interplay between light and its receptors, proteins involved in signal transduction and regulators of fungal development and secondary metabolism using the fungus A. nidulans as model.




Ubiquitylation without degradation of a WC-1-like activator mediates repression of carotenogenesis by a fungal RING-finger protein

Fátima Silva, Eusebio Navarro, Ascensión Peñaranda, Santiago Torres-Martínez, Victoriano Garre

Universidad de Murcia, Murcia, Spain

Ubiquitylation of proteins plays a major role in regulation of cellular processes mainly by proteasome-dependent degradation, although it has become increasingly clear that it is also involved in other processes different to targeting to the proteasome. Light is a major environmental cue that controls the behavior of living organisms, mainly though regulation of gene expression. In the fungus Mucor circinelloides, blue light regulates the synthesis of carotenes. Two regulatory genes involved in the production of carotenes, crgA and mcwc-1c, have been previously described in Mucor. The crgA gene encodes a RING-finger protein that represses carotenogenesis in the dark, whereas mcwc-1c is required for light induction of carotenogenic transcript and carotene accumulation. Other two white collar-1 homologous genes have been identified in Mucor: mcwc-1a, which is involved in phototropism, and mcwc-1b, of unknown function. The MCWC-1 deduced protein sequences show similarity with the well characterised WC-1 photoreceptor of Neurospora crassa, which also works as a transcription factor. We have generated double knockout mutants for crgA and every mcwc-1 gene, and the analysis of these mutants demonstrated the existence of two regulatory independent pathways controlling carotenogenesis. One of them is involved in light regulation and requires mcwc-1c, whereas the other is light-independent and mediated by crgA and mcwc-1b. Deletion or loss-of-function mutation of mcwc-1b suppressed the effect provoked by the crgA mutation, indicating that the repression of carotenogenesis by crgA is dependent on mcwc-1b, which works as a carotenogenesis activator. Northern blot analyses revealed that crgA does not control mcwc-1b function at transcriptional level since mcwc-1b mRNA accumulation was similar in the wild-type strain and a null crgA mutant. Protein analysis showed that the wild-type strain presented the MCWC-1b protein in an inactive and stable ubiquitylated state. The fact of crgA mutant lacked ubiquitylated MCWC-1b indicates that CrgA is involved in the ubiquitylation without degradation of MCWC-1b. Thus, our data reveal that carotenogenesis in a filamentous fungus is regulated by the proteolysis-independent ubiquitylation of an activator, suggesting that this mechanism of transcriptional regulation could be more widespread than previously thought.





The IME2-related protein kinase ImeB inhibits sexual development in Aspergillus nidulans

Fatih Sari1, Özgür Bayram1, Gerhard H. Braus1, Stefan Irniger2

1Institute of Microbiology and Genetics, Georg-August University, Göttingen, Germany, 2Institute of Microbiology, Leibniz-University, Hannover, Germany

The IME2 gene of the yeast Saccharomyces cerevisiae encodes a protein kinase essential for meiosis and sporulation. Ime2 related proteins have been identified in fungi and mammals. A survey of the Aspergillus nidulans genome data base revealed a putative IME2 homolog, termed imeB. The ImeB protein has 40% identity to Ime2 in the N‐terminal region. We found that ImeB mRNA is upregulated during both sexual and asexual development and that an ImeB-GFP fusion protein is localised to the nucleus. To elucidate the role of imeB in A. nidulans, we constructed an imeB deletion strain. Deletion mutants show a slower growth, but have an increased number of sexual fruiting bodies, cleistothecia, on plates. This indicates that ImeB acts as a negative regulator of sexual development. Indeed, imeB mutants produce vast amounts of Hülle cells in submerged cultures, whereas wild type strains develop only vegetative hyphae. These Hülle cells are physiologically active. Transcript levels of the developmental regulators stuA, nsdD and veA are upregulated in imeB mutant strains. Overexpression of imeB also results in slower growth and in a fluffy phenotype lacking any conidia or cleistothecia when grown in dark. ImeB belongs to the family of mitogen activated protein (MAP) kinases characterised by a TXY motif in the activation loop. A site‐directed mutagenesis of the TXY motif, TTY in ImeB, showed that a mutation of any single amino acid of the motif leads to a phenotype reminiscent of imeB deletion strains. Thus, every single amino acid this of motif is essential for ImeB function.

Our findings suggest that ImeB is a member of the MAP kinase family and has a role in transducing environmental signals that presumably stimulate A. nidulans cells to block sexual development. We will present our current work focussing on investigations to unravel other players of this MAP kinase module




Direct involvement of the proteasome in PacC processing

América Hervás-Aguilar1, José M. Rodríguez1, Joan Tilburn2, Herbert N.Jr Arst2, Miguel A. Peñalva1

1Centro de Investigaciones Biológicas, CSIC, Madrid, Spain, 2Imperial College London, London, United Kingdom

The eukaryotic 26S proteasome complex is a multichambered protease involved in proteolytic degradation. However, transcription factors as NF-k B p105 and Ci/Gli3 are processed (i.e. partially proteolyzed to yield a functional form) by the proteasome. Both proteins possess the minimum signal necessary to undergo partial degradation by the proteasome: a simple sequence preceding, in the direction of proteasome movement, a tightly folded domain (a Rel homology domain and a zinc finger region respectively). The A. nidulans zinc finger transcription factor PacC, distantly related to Ci, is activated by two proteolytic steps in response to ambient pH alkalinization. The first, carried out by the "signalling protease", removes the C-terminal processing-inhibitory domain from full length PacC72, yielding a truncated product, PacC53. PacC53 is accessible to the second "processing protease" which, in a way that agrees with the proteasome’s modus operandi, renders PacC27. In a direct approach, we used the proteasome inhibitor MG132 which, though inefficiently taken up by fungi, impairs PacC processing, shifting cleavage site specificity. In ex vivo experiments, substitution of catalytic Lys108 by Arg or Ala on Pre2p (the S. cerevisiae proteasome b 5 subunit) impairs PacC processing, yielding alternative PacC27 products. This effect is specific for Pre2p, one of the three proteasome proteolytic subunits. Thus, we characterised the orthologue of S. cerevisiae PRE2 in A. nidulans and named it preB. We constructed, using gene replacements, two missense mutations in preB: preB1K101A and preB2K101R. While preB1K101A is lethal, viable preB2K101R impairs growth and chymotryptic activity (like pre2K108R in yeast), which correlates with an increase of 2-3 times in the levels of the NimE mitotic cyclin (a prototypic proteasome substrate). The active site mutation preB2K101R consistently shifts position of the scissile bonds when PacC is processed in A. nidulans, resembling S. cerevisiae pre2K108R, and appears to result in PacC instability. Besides, when a PacC processing efficiency determinant is deleted, preB2K101R compensates the impairment in PacC27 formation. We conclude that PacC must be a direct substrate of the proteasome: PacC is processed by the proteasome (the "processing protease") to yield PacC27.





An RNA binding protein from Magnaporthe oryzae and its role in pathogenicity.

Sara L. Tucker, Marina Franceschetti, Concepcion Gomez-Mena, Ane Sesma

John Innes Centre, Norwich, Norfolk, United Kingdom

It was recently demonstrated that Magnaporthe can infect the roots of cereal plants, as well as their aerial parts. However, using previously characterised M. oryzae mutant strains it became clear that the method of root infection, and therefore the genes involved, differ from those required for leaf infection. To elucidate the differential regulation of root infection we generated an M. oryzae library of insertional mutants in strain Guy-11. This library was screened to identify strains impaired in their ability to infect rice roots. The mutant, RBP35, is considerably affected in its ability to cause disease on roots while there is a small reduction in virulence on the aerial parts of plants.

RBP35 has undergone insertional inactivation of a gene encoding a predicted RNA binding protein. Such proteins are characterised based on the presence of one or more RNA recognition motifs (RRMs). The RRM is a nucleic acid binding domain that can act together and/or in association with other domains which then contribute to protein cooperativity and/or affinity for ssRNA. RBP35 also contains several RGG motifs which are involved in protein-protein interactions and can bind directly to RNA. We have generated an RBP35:GFP fusion construct to determine the subcellular location of RBP35. The RGG motifs have been altered to determine their role in the RNA-binding function and any effect on the interaction with other RNAs/proteins. The full length cDNA has been isolated and analysed. Currently we are fusing RBP35 to Myc- and FLAG-tags and optimising methods for protein purification of RBP35 along with its associated cellular components, the so called ribonucleoprotein complex.



Investigation of post-translational processing during nitrogen metabolism in Aspergillus nidulans

Magdalena Mos, Igor Morozov, Meriel G. Jones, Mark X. Caddick

University of Liverpool, Liverpool, Merseyside, United Kingdom

From transcriptomics and proteomic studies into nitrogen metabolism and its regulation in A. nidulans we identified a wide variety of genes and proteins showing differential expression. The function of many of these proteins is unknown but an initial bioinformatic analysis suggested that several might have interesting and important functions in post-translational regulation. Four proteins were chosen for further analysis, a 14:3:3 protein, two F-box proteins and a putative scaffold protein for the SCF ubiquitin ligase complex. The respective genes were deleted with the objective of characterising their role with specific reference to nitrogen metabolism and its regulation. All four deleted strains are viable. Northern analysis is currently being used to investigate the effect of each deletion on areA and niaD transcription and Western analysis to investigate effects on levels of AreA protein. These experiments have been designed to monitor the dynamics of nitrogen metabolite repression and derepression. Initial results suggest that these proteins may have a role in nitrogen regulation suggesting that post-translational processes are involved.



Msb2, a putative membrane mucin functioning in signalling and pathogenesis of Fusarium oxysporum

Elena Perez-Nadales, Antonio Di Pietro

University of Cordoba, Cordoba, Spain

The soilborne vascular wilt fungus Fusarium oxysporum infects a wide variety of plant species by directly penetrating roots, invading the cortex and colonizing the vascular tissue. The mitogen activated protein kinase (MAPK) Fmk1 is essential for plant infection. The signalling components upstream of the Fmk1 cascade are currently unknown. We have identified a gene from F. oxysporum whose predicted product shows homology with Msb2, a mucin functioning at the head of the filamentation MAPK cascade in yeast. To test whether F. oxysporum Msb2 is involved in signalling through the Fmk1 pathway, we produced targeted knockout mutants both in the wild type and the D fmk1 background. The ∆msb2 strains were still able to grow invasively on fruit tissue and across cellophane sheets, but are less virulent on tomato plants than the wild type strain. Similar to D fmk1 strains, D msb2 mutants also showed reduced secretion of pectinolytic enzymes. In contrast to D fmk1 mutants, growth of D msb2 strains was specifically affected by the cell wall-targeting compounds Congo Red (CR) and Calcofluor White (CFW). Interestingly, a D fmk1D msb2 double mutant had dramatically impaired growth in the presence of CR and CFW, suggesting that Fmk1 and Msb2 interact genetically to regulate cell integrity in F. oxysporum. Western analysis with phospho-ERK antibody revealed similarities in the phosphorylation pattern between D fmk1 and D msb2 strains. These results suggest that F. oxysporum Msb2 interacts with the Fmk1 cascade to regulate a number of virulence-related functions as well as response to cell wall stress.