| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y de Estudios Avanzados, Campus Guanajuato, Apartado Postal 629, 36500, Irapuato, Guanajuato, Mexico
Correspondence
Alfredo Herrera-Estrella
aherrera{at}ira.cinvestav.mx
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
tvk1 strain were highly hydrophobic, whereas in aerial conidia hydrophobicity was severely reduced. In addition, the tvk1 strain was unable to break the liquid–air interface when the fungus grew in rich medium; however, when it grew in minimal medium the fungus produced large filaments which were much more efficient at breaking the interface than the wild-type. Through cDNA subtractive hybridization between the wild-type and tvk1 grown in submerged culture, five genes encoding hydrophobin-like proteins and two additional genes encoding cell wall proteins were identified. Four hydrophobin-encoding genes (Tv-hfb1, Tv-srh1, tv-cfth1 and Tv-qid3) and a gene encoding a clock-controlled-like protein (Tv-ccg14/TvSm1) were upregulated in tvk1, whereas genes encoding a cell wall protein (tv-qid74) and an additional hydrophobin (tv-hfb3) were absent in the mutant strain. Clear differences in gene expression were shown during conidiation and emergence from the liquid–air interface, suggesting different functions of the corresponding proteins in these two phenomena. The results support a model in which Tvk1 regulates morphology and genes encoding cell wall proteins during development of Trichoderma.
Present address: Max-Planck-Institut für Terrestrische Mikrobiologie, D-35043 Marburg, Germany.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Conidiation of fungi is rarely achieved during submerged culture. However, deletion or the constitutive activity of elements of the cyclic AMP-PKA or MAP kinase pathways results in inappropriate conidiation in submerged cultures in several filamentous fungi (Banno et al., 2005
; Kays et al., 2000; Mendoza-Mendoza et al., 2003; Rocha-Ramírez et al., 2002). Deletion of MAP kinase components results in the production of conidia in shaken cultures, as in the case of deletion of the nrc-1 gene, which encodes a MEK kinase in Neurospora crassa (Kothe & Free, 1998), and of the tvk1 gene in Trichoderma virens, which encodes a MAP kinase (Mendoza-Mendoza et al., 2003).
Conidiation and emergence from liquid–air interfaces represent two processes that are necessary for the survival of fungal species, the first for the propagation of the species and the second during the invasion of new environments. Filamentous fungi normally secrete small and moderately hydrophobic proteins called hydrophobins. These proteins are amphiphiles specific to filamentous fungi (Wösten, 2001), having hydrophilic and hydrophobic regions, and are among the most active surface proteins known (Linder et al., 2005). Hydrophobins have several important roles in fungal physiology, such as adhesion, formation of protective surface coatings and the reduction of the surface tension of water, which allows growth of aerial structures (Wösten, 2001). They mediate escape of hyphae from hydrophilic environments (Wösten et al., 1999) and are involved in formation of structures such as aerial hyphae and fruiting bodies (van Wetter et al., 1996; Wösten et al., 1999). These proteins are also involved in host–fungus interactions, and have been considered as virulence factors in phytopathogenic fungi (Talbot et al., 1996; Wösten et al., 1994a). Two classes of hydrophobins have been reported based on their different solubility and hydropathy profiles. Class II hydrophobin films are significantly less robust and lack the rodlet morphology of class I hydrophobins (Wessels, 1994; Wösten, 2001; Wösten & de Vocht, 2000).
The molecular mechanism by which the expression of hydrophobins is controlled is not clear. However, the expression of mpg1, which encodes a hydrophobin in Magnaporthe grisea, is regulated through the MAP kinase Pmk1 and the cAMP pathway (Soanes et al., 2002). In this paper we describe for the first time the differential regulation of five putative hydrophobin genes through a MAP kinase. The expression of these genes and the morphology of T. virens mediated by Tvk1 was influenced by the environmental conditions.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
tvk1 null mutant (tvk24, Mendoza-Mendoza et al., 2003) strains were maintained on PDA (Potato Dextrose Agar, Difco) medium at 28 °C. Escherichia coli DH5
(Invitrogen) was used for plasmid transformation using pCR2.1 vector (Invitrogen). All chemicals used in this study were purchased from Sigma-Aldrich.
Fungal growth conditions.
To generate the cDNA libraries, spores of the wild-type and the tvk1 null mutant (tvk1) of T. virens (1x106 spores per ml medium) were inoculated at 28 °C using an orbital shaker (170 r.p.m.) in 2 litre flasks containing 500 ml Vogel's minimal medium supplemented with 1.5 % sucrose (VMS) (Mendoza-Mendoza et al., 2003). Samples were collected by filtration, frozen in liquid nitrogen and stored at –70 °C. A small aliquot of each sample was fixed with formaldehyde (1.0 %) and analysed by light microscopy (BX60, Olympus).
To perform the liquid–air interface breaking analysis we applied 50 µl drops of conidia (1x106 ml–1) suspended in PDB (Potato Dextrose Broth, Difco) or VMS media to Parafilm and incubated them at 28 °C. Emergence of hyphae was analysed by using a stereo microscope (Olympus) and the images were captured and modified by using the programs IMAGE-PRO PLUS 4.0 (Media Cybernetics) and PHOTOSHOP (Adobe Systems), respectively.
Gene expression in submerged culture was compared by cultivating 1x106 conidia ml–1 of each strain of T. virens in 25 ml Vogel's medium in shaken flasks at 28 °C. To test the influence of carbon or nitrogen sources on gene expression we incubated the spores for 48 h before transfer to fresh Vogel's medium with or without nitrogen or carbon sources. Samples were then collected at 0, 1, 2 and 6 h after transfer to the induction medium.
Subtraction hybridization and subtractive library construction.
Subtractive libraries were used to prepare a cDNA library specific for T. virens conidiation in submerged culture. From the different time points analysed we chose those that gave us the most representative genes for each differentiation stage: 58, 60 and 64 h post-inoculation. Total RNA was obtained using a RNeasy kit (Qiagen) following the manufacturer's instructions. Subtractive libraries were made using the PCR-Select cDNA subtraction kit (Clontech), according to the manufacturer's instructions. cDNA of the wild-type strain was subtracted from cDNA of the tvk1 null mutant strain; the corresponding clones were designated k plus the clone number (Table 1). In addition cDNA from the tvk1 strain was subtracted from cDNA of the wild-type; these clones were designated wt plus the clone number (Table 2). The cDNAs obtained by subtraction hybridization were ligated into pCRII-Topo vector using the Topo TA cloning kit (Invitrogen) and transformed into E. coli DH5 . The presence of inserts in pCRII Topo was corroborated by PCR using primers AH-con1 (5'-GCCGCCAGTGTGATG-3') and AH-con2 (5'-GCCAGTGTGCTGGAATTC-3'). A total of 587 independent clones were sequenced using an ABI 377 DNA sequencer, and the sequences were compared with public sequence databases using the BLASTX algorithm (NCBI).
|
|
), except when indicated otherwise. To study the hydrophilic–hydrophobic interface between liquid and air environment, 50 µl drops of conidia (1x106 conidia ml–1) were applied to Parafilm. Twenty drops were scraped off the Parafilm and total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Northern blot analyses were performed under high-stringency conditions (Sambrook & Russell, 2001). Fragments contained in the cDNA library were amplified by PCR using primers AH-con1 and AH-con2 and the resulting PCR products were labelled with [32P]dCTP by random priming, using the Readyprime kit (Amersham) according to the manufacturer's specification.
Surface hydrophobicity of aerial and submerged conidia.
Hydrophobicity of conidia was determined by the phase distribution test (Mozes & Rouxhet, 1987), assessing the distribution ratio of cells between water and an organic phase (toluene). Aerial conidia were collected from 7 day old plate cultures, and filtered through crossed layers of cleaning tissues (Kimwipes, Kimberly Clark). Submerged conidia were obtained from a 72 h old cultures in VMS. Aerial and submerged conidia were collected by centrifugation at 10 000 g at room temperature, washed once with water and suspended in Tris buffer (100 mM) at three different pHs (3, 6 and 9), to an OD600 of 0.6. Samples (4 ml) of suspensions were transferred into 25 ml glass bottles and an equivalent volume of toluene was added. After vigorous vortexing for 10 s, the mixture was left to stand for 30 min. The OD600 was measured and the results were expressed as a proportion of the conidia that were excluded from the aqueous phase according to the formula 100x(ODi–ODf)/ODi, where ODi and ODf are the initial and final optical densities of the aqueous phase.
Transmission electron microscopy (TEM).
A PDB liquid culture of 72 h old tvk1 strains was filtered using cleaning tissues (Kimwipes, Kimberly Clark), centrifuged for 10 min at 10 000 g at 4 °C and suspended in sterile distilled water. Suspensions of aerial conidia were prepared by adding sterile distilled water to the PDA plates of 7 day old cultures, agitating the culture surface with a rubber-tipped glass rod and filtering the resulting suspension through crossed layers of Kimwipes). Fungal material was fixed with 3.0 % glutaraldehyde and 1.0 % OsO4 for analysis by TEM as described previously (Puyesky et al., 1999).
Scanning electron microscopy (SEM).
Aerial and submerged conidia were analysed using a JEOL JSM-6700F microscope equipped with a cold-field-emission gun. Conidia from submerged cultures or pieces of PDA agar containing aerial conidia from the wild-type and the tvk1 strains were frozen using a Gatan Alto 2500 cryo-stage chamber. Samples were frozen in slush nitrogen and cryo-transferred under vacuum to the preparation chamber for ice sublimation and gold/palladium sputter coating. Material was transferred from the Gatan preparation chamber to the SEM stage for sample observation.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
tvk1 conidiate abundantly in submerged culture, whereas the wild-type strain does not produce conidia under these conditions. In contrast, on solid medium the mutants yield fewer conidia than the wild-type and show a slight alteration of their characteristic green colour (Mendoza-Mendoza et al., 2003). SEM revealed that cell walls from the aerial spores produced by the wild-type and tvk1 null mutant strains were covered with an extracellular material forming papilla-like structures; these were uniformly distributed in the wild-type strain but showed a heterogeneous arrangement in the tvk1 null mutant (Fig. 1A, B). Contrary to the papilla-like structures shown in aerial conidia, conidia generated in submerged culture showed a smooth phenotype (Fig. 1A). Analysis by TEM showed that cell walls of aerial conidia were composed of an electron-transparent inner layer (W1) and an electron-dense outer layer (W2). In contrast to the W1 layer, which was similar in both strains, W2 was thinner in the tvk1 strain than in the wild-type (Fig. 1C), particularly in the case of submerged culture (Fig. 1C, bottom panel).
|
tvk1 strain was studied in Vogel's minimal medium with sucrose (VMS). Morphological changes became evident 60 h post-inoculation in the tvk1 mutant strain (Fig. 2A–E), whereas the wild-type strain did not show changes even after prolonged incubation (Fig. 2F–H). Pellets of the tvk1 strain were reduced in size (180 µm diameter) compared to the wild-type strain (420 µm diameter). This was accompanied by increasing branching of the mutant, which was followed by the development of a phialide in each newly formed hypha (Fig. 2B, C). During the next 8 h conidia differentiated (Fig. 2C, D), after which they were released into the medium (Fig. 2E). Interestingly, in several cases we observed the release of the conidium and philaide together, indicating that phialides were not strongly attached to the rest of the hyphae (Fig. 2E).
|
tvk1 null mutant strains grown in submerged culture. A total of 567 clones were sequenced, representing 90 different genes. Classification of the matches obtained upon BLAST analysis of the sequences revealed similarities to proteins involved in a range of functions, including protein fate, primary metabolism, oxidative stress, signal transduction, DNA processing, protein biosynthesis, cell wall components and proteins with unknown function (see Tables 1
and 2).
Seven genes identified in the subtractive cDNA libraries encoded putative cell wall proteins. These genes were called Tv-hfb1, Tv-srh1, Tv-qid3, Tv-cfthI, Tv-hfb3, Tv-qid74 and Tv-ccg14/Sm1. Eight cysteine residues were identified in five of the deduced proteins. We clearly identified two consensus patterns of cysteine residues: (1) C-X7–10-CC-X11-C-X15–16-C-X6–9-CC-X10–11-C-X7–8 and (2) C-X6-CC-X9-C-X5-C-X5-CC-X8-C-X16. Both patterns showed the hallmarks of hydrophobins (Wessels, 1994). Because the N-terminus is highly variable in hydrophobin-like proteins, we used only the sequences between the first and eighth cysteine residues. Tv-hfb1, Tv-srh1, Tv-cfth1 and Tv-qid3 belong to the class II hydrophobins, whereas Tv-hfb3 belongs to the class I hydrophobins. Tv-hfb1 showed the highest identity (64 %) to HFBI from Trichoderma reesei, whereas Tv-srh1 was 56 % identical to HFBII from T. reesei and 58 % identical to Srh1 from Hypocrea lixii. Tv-cfth1 showed high similarity with the three domains of the tri-hydrophobin Cfth1 from Claviceps fusiformis (57 %), whereas Tv-qid3 showed high identity (66.0 %) to QID3 from H. lixii. On the other hand, Tv-hfb3 had high identity (87 %) to TasHyd1 from Trichoderma asperellum, 56 % identity to a hypothetical protein from Gibberella zeae (gi|42545447) and 41 % identity to hydrophobin Pbhyd1 from Paracoccidioides brasilensis.
In addition to the genes encoding hydrophobin-like proteins we identified one additional gene encoding a cell wall protein (Tv-qid74) and one which encodes a small secreted protein (Tv-ccg14/Sm1). The latter corresponds to the recently reported gene Sm1 from T. virens (Djonovi
et al., 2006), which encodes a protein that displayed strong identity to a probable SnodProt1 precursor from N. crassa (50 % identity), which has been reported also as a clock-controlled gene (ccg-14) in N. crassa (Zhu et al., 2001). A comparative analysis of Tv-snodprot1 and its homologues in other fungi showed high identity among them (Djonovi et al., 2006). Tv-qid74 had a high identity to the repetitive domains of Qid74, a cell wall protein reported in H. lixii (Rey et al., 1998).
Genes encoding cell wall proteins are differentially expressed during the conidiation process
Expression of the genes encoding cell wall proteins mentioned above was investigated and compared between the wild-type and tvk1 null mutant strains. Total RNA from mycelium of wild-type and tvk1 strains grown in liquid shaken cultures in VMS medium was used for this analysis (Fig. 3). Tv-hfb1, Tv-srh1, Tv-cfth1 and Tv-ccg14/Sm1 were expressed in both strains; however, higher expression was found in the tvk1 strain (Fig. 3). The expression pattern of these genes was similar, most of them showing maximum expression 52 h post-inoculation, a few hours before noticeable morphological changes occurred, and thereafter their transcript levels started to decrease. Interestingly, Tv-qid74 and Tv-hfb3 were expressed at higher levels in the wild-type strain than in the tvk1 null mutant, in which their expression level was scarcely detectable (Fig. 3). Tv-qid74 reached its highest level of expression 60 h post-inoculation; after this its expression decreased sharply (Fig. 3). On the other hand, Tv-hfb3 showed the highest expression level between 60 and 64 h (Fig. 3). The highest level of expression of Tv-qid74 and Tv-hfb3 coincided with the start of the production of phialides and conidia (Fig. 2b–c).
|
tvk1 strains were inoculated in VMS and incubated for 24 h at 28 °C. The mycelium was then transferred to Vogel's minimal medium (VMM) without carbon (VM–C) or nitrogen sources (VM–N). In these experiments the tvk1 null mutant always showed higher expression levels of Tv-hfb1, Tv-srh1, Tv-cfth1 and Tv-ccg14/Sm1 than the wild-type strain. Unexpectedly, no differences were observed between the samples obtained from VMM and VM–C; however, a discrete increase in Tv-hfb1, Tv-srh1, Tv-cfth1 and Tv-ccg14/Sm1 expression in VM–N with respect the VMM was observed (data not shown).
Conidia produced in submerged culture show higher hydrophobicity than those generated on solid media
The hydrophobic character of the reproductive structures generated in liquid and on solid media was determined according to the phase distribution method (Mozes & Rouxhet, 1987). The hydrophobicity indices obtained indicated that aerial conidia produced by the wild-type strain were highly hydrophobic whereas those obtained from the tvk1 mutant had notably reduced hydrophobicity (Table 3). Surprisingly, conidia of the tvk1 mutant generated in submerged cultures were the most hydrophobic and this characteristic was prevalent at the different pH values analysed. In contrast, hydrophobicity indices from aerial conidia decreased at high pH.
|
tvk1 straintvk1 and wild-type strains to escape from a hydrophilic to a hydrophobic environment was evaluated. Wild-type and tvk1 spores were grown in PDB or VMS medium placed on Parafilm. Depending on the medium used, two interesting differences, opposite to each other, in the tvk1 strain were observed. (1) In VMS medium, the tvk1 strain formed the most prominent and strong filaments; these filaments were not strongly attached to the hydrophobic surface, since they easily came off when the drop was collected with a micropipette tip. Together with this prominent filament formation, a collapse in the middle of the drop was observed (Fig. 4a). (2) In contrast, in PDB the mutant was unable to break the liquid–air interface (Fig. 4a). This characteristic was not due to growth alterations in the tvk1 strain, which appeared to form normal filaments in the drops.
|
). Tv-srh1 mRNA levels decreased with time and those of Tv-qid3 increased in both strains. However, the highest expression level of both genes was observed in the tvk1 strain. In the wild-type strain Tv-hfb3 was not expressed until 40 h post-inoculation, when it reached its maximum level, with a clear reduction after this point. Tv-qid74 was expressed at all times examined; it increased slightly by 40 h after drop deposition and then started to decrease. The pattern of expression of Tv-cfth1 in the wild-type strain was similar to that of Tv-qid74, except that it reached an apparent maximum by 35 h, with a significant reduction at 40 h and increased again by 45 h. In contrast, in the tvk1 mutant the expression of Tv-cfth1 reached a maximum by 35 h, with a pronounced drop at 45 h. Interestingly, Tv-hfb3 and Tv-qid74 were expressed exclusively in the wild-type strain. In contrast, Tv-ccg14/Sm1 showed increasing levels of expression over time and was detected only in the tvk1 mutant. Tv-hfb1 was expressed weakly in the tvk1 strain in VMS and was completely absent in PDB (Fig. 4b and data not shown). A good correlation between hydrophobin gene expression and hyphal emergence under the conditions tested was shown (Fig. 4). Taken together these data suggest a complex regulation of hydrophobin gene expression and cell wall components during changes in environment. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
tvk1 strains of T. virens showed morphological differences in the first cell wall layer (W2). The W2 layer has been considered to be spore specific and it could be related to pigmentation (Muñoz et al., 1995). Because tvk1 aerial conidia are pale green, in contrast to the intense dark green colour from the wild-type strain, the idea of thickness and green colour being related would be supported by our observations. However, this correlation is not valid for submerged conidia, which contain a very thin outer layer (W2) and have an intense green colour. The developmental programme of T. virens was completed in 72 h, when the conidia were released from the conidiophore, probably through enzymic activities. Tv-nag1, a gene found among those expressed only in tvk1, encodes an N-acetyl-β-D-glucosaminidase and its homologue in Trichoderma atroviride is secreted and partially bound to the cell wall (Brunner et al., 2003). Another interesting finding was that of a Gβ-subunit-encoding gene identified in the subtractive library of the genes absent in tvk1. Gβ subunit proteins are considered negative regulators of conidiation in submerged culture in Aspergillus nidulans and N. crassa (Rosen et al., 1999; Yang et al., 2002).
Five hydrophobin-like proteins have been reported in Trichoderma, and four belong to class II (Linder et al., 2005; Viterbo & Chet, 2006). We identified two additional hydrophobin-like proteins in T. virens. Tv-hfb3 encodes a novel protein related to class I hydrophobins. Although Tv-hfb3 almost contains the ‘classical’ cysteine distribution, it has an unusual spacing between the fourth and fifth cysteine. A similar difference was also observed in Hyd3, a hydrophobin from Fusarium verticillioides, but in this case the discrepancy was located between the third and fourth cysteine (Fuchs et al., 2004). Tv-cfth1 encodes the second novel hydrophobin-like protein in T. virens. The open reading frame deduced from the genomic clone of the gene showed high identity to each domain of the tri-hydrophobin Cfth1 from C. fusiformis (de Vries et al., 1999), and allowed us to identify eight cysteines in the deduced protein (data not shown). However, one of the cysteines is located in an unusual position, between the fourth and fifth cysteine found in typical hydrophobins. Another class II hydrophobin with an additional cysteine in an equivalent location is HFBIII from T. reesei. Furthermore, when the structure of Tv-cfth1 was compared to the structure of hydrophobin HFBII (PDB code 1R2M), the cysteine was found on the protein surface and in the region of the -helix in HFBII. Therefore, Tv-cfth1 is very likely to adopt the typical structure of hydrophobins. On the other hand, Tv-qid74 is highly homologous to Qid74, an extracellular protein which has been detected in several species of Trichoderma; however, the physiological role of this protein is still unknown (Rey et al., 1998).
Our results indicate that Tvk1 is involved in the negative regulation of the genes encoding type II hydrophobins, Tv-hfb1, Tv-srh1 and Tv-cfth1, in submerged culture. Furthermore, the high expression of Tv-hfb1, Tv-srh1 and Tv-cfth1 during conidiation in submerged culture in tvk1 suggests a role for the corresponding proteins in this developmental programme. The expression of Tv-hfb3 and Tv-qid74 was abolished in the absence of Tvk1, suggesting a positive role for Tvk1 in the expression of these genes. The fact that neither gene was expressed during conidiation in submerged culture indicates that they are dispensable for this process. Viterbo & Chet (2006) showed that Tashyd1, the homologue of Tv-hfb3, is needed for hydrophobicity of young hyphae and during plant root colonization. The class I hydrophobin Mpg1 of Magnaporthe grisea is required for appresoria function (Talbot et al., 1996) and regulated by the homologue of tvk1 (Soanes et al., 2002). These data suggest conserved signalling in the regulation of this class of hydrophobins through Pmk1 homologues. Conidia of tvk1 generated in submerged cultures were more hydrophobic than those generated on plates. Although these results were the opposite of those reported for other systems (Muñoz et al., 1995; Pascual et al., 2000), they clearly correlated with the high levels of expression of the hydrophobin-encoding genes in submerged culture.
Hydrophobins seem to serve a dual role by first lowering the water surface tension, which allows the fungus to penetrate physical barriers, and then forming a protective coating on aerial structures and spores (Wösten et al., 1994b, 1999). In T. reesei HFBII is involved in conidiation and HFBI is involved in breaking hydrophilic–hydrophobic interfaces (Askolin et al., 2005). Hyperconidiation and the high level of expression of Tv-srh1 in submerged cultures could be explained if we consider that Tv-srh1 is also needed for conidiation as in T. reesei (Askolin et al., 2005). Nevertheless, expression of hfb2 from T. reesei in both conidiation and normal vegetative growth suggests that the protein has other functions in addition to its presumed role in protecting the spores (Nakari-Setala et al., 1997). Deletion of hfb2 in T. reesei promoted intense conidiation when lactose medium was used as the carbon source, but when glucose was used the mutant produced fewer conidia than the wild-type (Askolin et al., 2005; Bailey et al., 2002). These phenotypes resemble those of tvk1, which shows reduced conidiation on solid medium and hyperconidiates in submerged culture (Mendoza-Mendoza et al., 2003).
In contrast to HFBII, which is secreted, most HFBI is attached to the cell walls in T. reesei (Askolin et al., 2001). Deletion of hfb1 affects sporulation, probably due to the reduction in aerial hyphae formation. The role of HFBI in hyphal development and during the breaking of the liquid–air interface by filaments was shown in glucose-containing medium (Askolin et al., 2005). Our results indicate that during breakage of the liquid–air interface Tv-hfb1 was expressed at very low levels, whereas the expression of Tv-srh1 and Tv-qid3 was very high, and they may perform the function of Tv-hfb1 in this process. The differences in expression and behaviour of the fungi in the same situation depend on the environment, as reflected by the ability of tvk1 to break the hydrophilic–hydrophobic interface in VMS and its inability to do so in PDB. However, the breaking of the interface in PDB in the wild-type could be explained by the participation of other hydrophobins expressed in this medium. Tv-qid3 expression was reduced in submerged cultures of the tvk1 strain until it became undetectable. In contrast, it was highly expressed during breakage of the hydrophilic–hydrophobic interface in the tvk1 strain, whereas the expression in the wild-type was lower. These data suggest a role of Tv-qid3 in filamentous growth in hydrophobic environments but not in liquid media or during submerged conidiation.
All things considered, the MAP kinase Tvk1 appears to play a major role in the control of the production of conidia through its action as a molecular switch for the expression of a set of genes involved in the corresponding developmental programme.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: H. A. B. Wösten
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Askolin, S., Penttilä, M., Wösten, H. A. B. & Nakari-Setala, T. (2005). The Trichoderma reesei hydrophobin genes hfb1 and hfb2 have diverse functions in fungal development. FEMS Microbiol Lett 253, 281–288.[CrossRef][Medline]
Bailey, M. J., Askolin, S., Horhammer, N., Tenkanen, M., Linder, M., Penttilä, M. & Nakari-Setala, T. (2002). Process technological effects of deletion and amplification of hydrophobins I and II in transformants of Trichoderma reesei. Appl Microbiol Biotechnol 58, 721–727.[CrossRef][Medline]
Banno, S., Ochiai, N., Noguchi, R., Kimura, M., Yamaguchi, I., Kanzaki, S., Murayama, T. & Fujimura, M. (2005). A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa. Genes Genet Syst 80, 25–34.[CrossRef][Medline]
Brunner, K., Peterbauer, C. K., Mach, R. L., Lorito, M., Zeilinger, S. & Kubicek, C. P. (2003). The Nag1 N-acetylglucosaminidase of Trichoderma atroviride is essential for chitinase induction by chitin and of major relevance to biocontrol. Curr Genet 43, 289–295.[CrossRef][Medline]
de Vries, O. M., Moore, S., Arntz, C., Wessels, J. G. & Tudzynski, P. (1999). Identification and characterization of a tri-partite hydrophobin from Claviceps fusiformis. A novel type of class II hydrophobin. Eur J Biochem 262, 377–385.[Medline]
Djonovi, S., Pozo, M. J., Dangott, L. J., Howell, C. R. & Kenerley, C. M. (2006). Sm1, a proteinaceous elicitor secreted by the biocontrol fungus Trichoderma virens induces plant defense responses and systemic resistance. Mol Plant Microbe Interact 19, 838–853.[Medline]
Fuchs, U., Czymmek, K. & Sweigard, J. A. (2004). Five hydrophobin genes in Fusarium verticillioides include two required for microconidial chain formation. Fungal Genet Biol 41, 852–864.[CrossRef][Medline]
Herrera-Estrella, A. & Chet, I. (2003). The biological control agent Trichoderma: from fundamentals to applications. In Handbook of Fungal Biotechnology, pp. 147–156. Edited by D. Arora. New York: Marcel Dekker.
Kays, A. M., Rowley, P. S., Baasiri, R. A. & Borkovich, K. A. (2000). Regulation of conidiation and adenylyl cyclase levels by the G protein GNA-3 in Neurospora crassa. Mol Cell Biol 20, 7693–7705.
Kothe, G. O. & Free, S. J. (1998). The isolation and characterization of nrc-1 and nrc-2, two genes encoding protein kinases that control growth and development in Neurospora crassa. Genetics 149, 117–130.
Linder, M. B., Silvia, G. R., Nakari-Setälä, T. & Penttilä, M. E. (2005). Hydrophobins: the protein-amphiphiles of filamentous fungi. FEMS Microbiol Rev 29, 877–896.[CrossRef][Medline]
Mendoza-Mendoza, A., Pozo, M. J., Grzegorski, D., Martínez, P., García, J. M., Olmedo-Monfil, V., Cortés, C., Kenerley, C. & Herrera-Estrella, A. (2003). Enhanced biocontrol activity of Trichoderma through inactivation of a mitogen-activated protein kinase. Proc Natl Acad Sci U S A 100, 15965–15970.
Mozes, N. & Rouxhet, P. G. (1987). Methods for measuring hydrophobicity of microorganisms. J Microbiol Methods 6, 99–112.[CrossRef]
Muñoz, G., Agosin, E., Cotoras, M., San Martin, R. & Volpe, M. D. (1995). Comparison of aerial and submerged spore properties for Trichoderma harzianum. FEMS Microbiol Lett 125, 63–69.
Nakari-Setala, T., Aro, N., Ilmen, M., Muñoz, G., Kalkkinen, N. & Penttilä, M. (1997). Differential expression of the vegetative and spore-bound hydrophobins of Trichoderma reesei – cloning and characterization of the hfb2 gene. Eur J Biochem 248, 415–423.[Medline]
Pascual, S., De Cal, A., Magan, N. & Melgarejo, P. (2000). Surface hydrophobicity, viability and efficacy in biological control of Penicillium oxalicum spores produced in aerial and submerged culture. J Appl Microbiol 89, 847–853.[CrossRef][Medline]
Puyesky, M., Benhamou, N., Noyola, P., Bauw, G., Ziv, T., van Montagu, M., Herrera-Estrella, A. & Horwitz, B. A. (1999). Developmental regulation of cmp1, a gene encoding a multidomain conidiospore surface protein of Trichoderma. Fungal Genet Biol 27, 88–99.[CrossRef][Medline]
Rey, M., Ohno, S., Pintor-Toro, J. A., Llobell, A. & Benitez, T. (1998). Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus. Proc Natl Acad Sci U S A 95, 6212–6216.
Rocha-Ramírez, V., Omero, C., Chet, I., Horwitz, B. A. & Herrera-Estrella, A. (2002). Trichoderma atroviride G-protein subunit gene tga1 is involved in mycoparasitic, coiling and conidiation. Eukaryot Cell 1, 594–605.
Rosen, S., Yu, J. H. & Adams, T. H. (1999). The Aspergillus nidulans sfaD gene encodes a G protein β subunit that is required for normal growth and repression of sporulation. EMBO J 18, 5592–5600.[CrossRef][Medline]
Sambrook, J. & Russell, D. (2001). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Soanes, D. M., Kershaw, M. J., Cooley, R. N. & Talbot, N. J. (2002). Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea. Mol Plant Microbe Interact 15, 1253–1267.[Medline]
Talbot, N. J., Kershaw, M. J., Wakley, G. E., de Vries, O. M., Wessels, J. & Hamer, J. E. (1996). MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related development of Magnaporthe grisea. Plant Cell 8, 985–999.[Abstract]
van Wetter, M. A., Schuren, F. H. J., Schuurs, T. A. & Wessels, J. G. H. (1996). Targeted mutation of the SC3 hydrophobin gene of Schizophyllum commune affects formation of aerial hyphae. FEMS Microbiol Lett 140, 265–269.
Viterbo, A. & Chet, I. (2006). TasHyd1, a new hydrophobin gene from the biocontrol agent Trichoderma asperellum, is involved in plant root colonization. Mol Plant Pathol 7, 249–258.[CrossRef]
Wessels, J. G. H. (1994). Developmental regulation of fungal cell wall formation. Annu Rev Phytopathol 32, 413–437.
Wösten, H. A. B. (2001). Hydrophobins: multipurpose proteins. Annu Rev Microbiol 55, 625–646.[CrossRef][Medline]
Wösten, H. A. B. & de Vocht, M. L. (2000). Hydrophobins, the fungal coat unravelled. Biochim Biophys Acta 1469, 79–86.[Medline]
Wösten, H. A. B., Schuren, F. H. J. & Wessels, J. G. H. (1994a). Interfacial self-assembly of a hydrophobin into an amphipathic protein membrane mediates fungal attachment to hydrophobic surfaces. EMBO J 13, 5848–5854.[Medline]
Wösten, H. A. B., Asgeirsdottir, S. A., Krook, J. H., Drenth, J. H. & Wessels, J. G. (1994b). The fungal hydrophobin Sc3p self-assembles at the surface of aerial hyphae as a protein membrane constituting the hydrophobic rodlet layer. Eur J Cell Biol 63, 122–129.[Medline]
Wösten, H. A. B., van Wetter, M. A., Lugones, L. G., van der Mei, H. C., Busscher, H. J. & Wessels, J. G. (1999). How a fungus escapes the water to grow into the air. Curr Biol 9, 85–88.[CrossRef][Medline]
Yang, Q., Poole, S. I. & Borkovich, K. A. (2002). A G-protein beta subunit required for sexual and vegetative development and maintenance of normal G alpha protein levels in Neurospora crassa. Eukaryot Cell 1, 378–390.
Zhu, H., Nowrousian, M., Kupfer, D., Colot, H. V., Berrocal-Tito, G., Lai, H., Bell-Pedersen, D., Roe, B. A., Loros, J. J. & Dunlap, J. C. (2001). Analysis of expressed sequence tags from two starvations, time-of-day-specific libraries of Neurospora crassa reveals novel clock controlled genes. Genetics 157, 1057–1065.
Received 21 December 2006;
revised 16 February 2007;
accepted 20 February 2007.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
