Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Sciences Building 036, College Park, MD 20742, USA
Correspondence
Donald L. Nuss
nuss{at}umbi.umd.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
*These authors contributed equally to this paper.
Present address: Department of Biology, New Mexico State University, Las Cruces, NM 88003, USA.
Present address: Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health Bldg 50, Bethesda, MD 20892-2715, USA.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cryphonectria parasitica, the chestnut blight fungus, is the plant pathogen responsible for the destruction of the native populations of American chestnut throughout the natural range of this tree, beginning in the early 20th century (Merkel, 1906). A relationship between G-protein-signalling pathways and virulence of C. parasitica has been suggested by the deletion of the genes encoding two G
subunits (cpg-1 and cpg-2). Absence of CPG-1 results in an avirulent strain that is also defective in asexual sporulation and pigment production and is reduced in growth, while a deficiency in CPG-2 leads to only minor alterations in phenotype from wild-type and little change in virulence (Gao & Nuss, 1996
). A G
subunit, CPGB-1, has also been identified in this organism (Kasahara & Nuss, 1997
) and the
cpgb-1 strain is similar to
cpg-1 in that it is minimally virulent with no pigmentation and greatly reduced sporulation. However, in contrast to the phenotype of the G
knockout, the lack of CPGB-1 results in increased biomass production (Kasahara & Nuss, 1997
). Although precise functions have not yet been determined for the G-protein subunits identified in C. parasitica, the disruption of signalling caused by the absence of CPG-1 or CPGB-1 mimics some of the phenotypic changes induced by infection with members of the virulence-attenuating virus family Hypoviridae that occur in natural populations of C. parasitica. Specifically, the prototypic hypovirus CHV1-EP713 has been shown to reduce growth (in common with
cpg-1) as well as asexual sporulation, pigmentation and virulence (in common with
cpg-1 and
cpgb-1 (reviewed by Dawe & Nuss, 2001
).
Chen et al. (1996) demonstrated that a potentially large number of changes in transcript accumulation occurred in response to hypovirus infection of C. parasitica by using mRNA differential display technology. During this study the authors also noted a connection between G-protein-mediated cAMP levels and aspects of the hypovirulent phenotype. Further analysis of the effect of hypovirus infection has been facilitated by the development of a robust spotted cDNA microarray platform (Allen et al., 2003
) based on approximately 2200 unique ORFs previously identified by expressed sequence tags (Dawe et al., 2003
). Allen et al. (2003)
examined the transcriptional changes of this population of genes in the presence of hypovirus compared to the expression levels in wild-type (uninfected) mycelium, concluding that significant and persistent reprogramming of the C. parasitica transcriptome occurred in the presence of CHV1-EP713. A comparison of the effects on transcript accumulation of CHV1-EP713 and CHV1-Euro7, a naturally occurring hypovirus that is in the same family but produces less severe symptoms on fungal phenotype, has demonstrated the utility of comparing multiple datasets from this microarray system (Allen & Nuss, 2004
).
Therefore, as part of a continuing effort to understand the role of G-protein-signalling in the growth, reproduction and virulence of this plant pathogen, we have expanded our use of the microarray platform to examine the effect of the absence of CPG-1 or CPGB-1 on global transcriptional profiles. We note striking similarities and overlap between those genes whose transcripts are altered in abundance when either G-protein component is lacking. In an attempt to better understand the relationship between G-protein-mediated signalling and hypovirus infection, we have used antibodies to the G and G
subunits to quantify a reduction in the accumulation of both proteins in the presence of hypovirus. Subsequently, we also compared the above microarray data to those obtained by examining hypovirus-infected mycelium and noted additional significant overlap between the datasets. The observed transcriptional profiles point to the modulation of G-protein-signalling pathways as a significant contributor to the phenotype of hypovirus-infected mycelium. Implications for the modulation of specific genes are discussed in the context of fungal development and virulence.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Extract preparations and Western blotting.
Total protein extracts for Western blotting were prepared using the method of Parsley et al. (2003). Dilution immunoblot quantification of relative G-protein subunit levels was performed as described previously using antisera raised against purified CPG-1 and CPGB-1 that specifically recognizes these proteins (Parsley et al., 2003
). Total RNA extracts were prepared as described by Allen et al. (2003)
.
Microarray slide printing.
A second-generation spotted cDNA chip was constructed from the same C. parasitica EST library printing plates used to construct the first-generation chips profiled in Allen et al. (2003). The EST library represented approximately 2200 independent transcripts as determined by single pass sequences and contig analysis (Dawe et al., 2003
). For the second print-run, GAPS II-coated slides from Corning (coated with
-amino-propyl-silane) were used. Purified PCR products were singly arrayed with a mean spot diameter of 100 µm and a spot spacing of 300 µm. Following printing, the spotted cDNA was cross-linked, washed and blocked as described by Allen et al. (2003)
.
Fluorescent probe generation, hybridization and scanning.
Fluorescence-labelled cDNA probes were prepared from total RNA (25 µg per probe) by direct incorporation of Cy3- or Cy5-dUTP using the CyScribe First-Strand cDNA Labelling Kit (Amersham Pharmacia) primed with oligo(dT) according to the manufacturer's instructions. Unincorporated nucleotides were removed using a Microcon-30 spin column and processed according to Allen et al. (2003). Pre-hybridization, hybridization and post-hybridization wash steps were as suggested by the manufacturer of GAPS II slides (Corning). Each hybridized chip was scanned in both Cy3 and Cy5 channels with an Affymetrix 418 Scanner as described by Allen et al. (2003)
.
Microarray data management, processing and analysis.
Scanned images were interpreted with Spotfinder (version 1.0; http://www.tigr.org). Raw data files are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org. Further data processing and analyses were as described by Allen et al. (2003). Briefly, normalization of raw fluorescent signal was achieved by utilizing algorithms present in MIDAS software (http://www.tigr.org/software). Dye-bias effects were corrected utilizing LOWESS, a local linear regression algorithm, while differences between repeated hybridizations were managed by standard deviation regularization, i.e. by rescaling each spot's cy3 and cy5 measurement using the standard deviation of cy3 and cy5 values as calculated across all experiments. Differentially expressed genes were identified as spots with log2 ratios equal to or greater than ±2 standard deviations from the mean log2 ratio of each dataset in a minimum of three of four hybridizations. To ensure that independent lists of differentially expressed genes reflected biological differences rather than technical differences associated with different print runs, the hybridizations described by Allen et al. (2003)
between hypovirus-free C. parasitica strain (EP155) and C. parasitica infected with the prototypic hypovirus CHV1-EP713 were repeated. Therefore, all profiling results reported here are from hybridizations performed on the same set of second generation chips. Where indicated, mean fold change data were derived from log2 data, such that fold change=
). Comparisons between lists and curation of differentially expressed clone data were performed as described by Allen & Nuss (2004)
. Minor refinement of the non-redundant lists was achieved by re-examining any clones whose BLASTX hit E-value was reported by Dawe et al. (2003)
as greater than 1x1010. Any such clones were verified for redundancy within the library population using BLASTN results obtained by comparing the library to itself, thus eliminating clones that had different, but weak, BLASTX hits yet were in fact derived from the same mRNA sequence. Complete redundant data tables are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org
Real-time RT-PCR validation of microarray data.
Probes and oligonucleotides for the particular clones to be tested were obtained from Integrated DNA Technologies (Coralville). Each clone to be verified was tested using RNA from two separate preparations, in triplicate on each occasion, using an Applied Biosystems GeneAMP 5700 sequence detection system and an Applied Biosystems TaqMan reverse transcription kit, according to the manufacturer's instructions and as described by Allen et al. (2003). Validation failure was determined to be those instances where microarray and real-time RT-PCR values indicated opposite effects on transcription of the tested transcript in the same strain with respect to wild-type values
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Microarray analysis revealed changes deemed to be significant in 416 clones, representing 216 different transcripts (116 downregulated, 100 upregulated) when the cpg-1 strain was compared to the isogenic wild-type strain EP155 (supplementary data table A). When the
cpgb-1 strain was similarly compared to EP155, 300 clones, representing 163 transcripts (69 downregulated, 94 upregulated) were altered in abundance (supplementary data table B). Comparison of the two non-redundant lists of altered transcripts showed considerable overlap (Fig. 1a
). One hundred transcripts were found to be changed in both strains, representing 46·3 % of the total for
cpg-1 and 61·3 % of the total for
cpgb-1. Further analysis of these 100 transcripts demonstrated even closer similarity, since all of these changes were coordinately regulated in both strains (i.e. a clone upregulated in the absence of CPG-1 was also upregulated in the absence of CPGB-1; Fig. 1b
). As a general trend, the magnitude of change observed for a given transcript in the
cpg-1 mutant was greater than that observed in the
cpgb-1 strain.
|
|
|
Class ABV comprised 45 transcripts, representing almost one-third (33·1 %) of the total genes altered by hypovirus infection, that were also found to be altered in both of the G-protein mutants (Table 1). Analysis of this class using hierarchical clustering (Fig. 3c
) illustrated that 43 transcripts (96 %) were coordinately changed across all three samples. The two clones that contradicted this behaviour were upregulated in both G-protein mutants, but downregulated in the presence of CHV1-EP713. Additionally, the magnitude (expressed as the mean fold change, where fold change=
) of transcriptional changes undergone by these 43 genes were generally lower in the hypovirus-infected strain than in either the
cpg-1 or
cpgb-1 mutant as indicated by the brightness of colour on the cluster diagram (Fig. 3c
) and the values in Table 1
.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A comparison of the data obtained from the two G-protein subunit deletion mutants demonstrated significant similarity, despite their superficially divergent phenotypes. The overlap of 100 similarly altered genes, 46·3 % of the total for cpg-1 and 61·3 % of the total for
cpgb-1, is likely to represent those targets that are the most sensitive to disruptions of the G-protein pathway. Also, this observation highlights the difficulties of interpreting the functional significance of phenotype based on single-gene knockouts of G-protein subunits or components of G-protein-signalling pathways. It is particularly striking that all of these transcripts are not just altered in abundance in both scenarios, but are also changed in the same direction. A likely explanation for the considerable similarity in the transcriptional profiles of the two G-protein knockout strains can be attributed to a previously observed two- to fourfold reduction in the levels of CPG-1 in the absence of CPGB-1 and vice versa (Parsley et al., 2003
). Interestingly, these protein reductions were specific to CPG-1 and CPGB-1; the deletion of a second G
subunit, CPG-2 (Choi et al., 1995
) had no effect on the accumulation of CPGB-1, suggesting that CPGB-1 and CPG-1 may represent cognate functional partners, a hypothesis that our data further support. Alternatively, the profile similarities may reflect a degree of crosstalk between the CPG-1 and CPGB-1-mediated cascades, or a combination of immediate functional interaction and downstream crosstalk. As a general theme, we also noted that the magnitude of transcript changes was larger in the absence of CPG-1 rather than CPGB-1. A specific explanation for this observation is not apparent at this time. However, there could be a correlation between this phenomenon and the severity of the phenotype of the
cpg-1 strain since it is significantly more impaired in growth and development than
cpgb-1.
Potentially important signalling proteins that may represent candidates responsible for significant contributions to the G-protein mutant phenotypes include a phosducin-like-protein BDM2 (AEST-19-D-04) and the PAS/LOV family member VVD (AEST-27-H-09). Both showed considerable reduction in transcript level in both G-protein deletion strains in comparison to EP155 (supplementary data Tables A and B). BDM2, a homologue of PLP2p from Saccharomyces cerevisiae (Flanary et al., 2000) represents a second phosducin-like protein that is also similar to BDM-1 (23 % identical, 36 % similar), a negative regulator of the G
-signalling pathway (Kasahara et al., 2000
) whose gene is closely linked to the cpgb-1 gene in the C. parasitica genome (Dawe et al., 2003
). Deletion of bdm-1 resulted in a phenotype identical to that of
cpgb-1 (Kasahara et al., 2000
) and a large reduction in the amount of CPGB-1 protein compared to wild-type (A. L. Dawe & D. L. Nuss, unpublished observations). Proteins similar to BDM-1 and PLP2 have been identified in Dictyostelium discoideum as well as S. cerevisiae, and in both cases the BDM-1 homologue appears to be involved in G
-signalling while deletion of the bdm2/plp2 gene results in a lethal phenotype (Blaauw et al., 2003
; Flanary et al., 2000
). It is presently unclear how the function of BDM2/PLP2 might fit into the overall G-protein-signalling scheme of C. parasitica, but the reduction of the bdm2 transcript in the absence of either CPG-1 or CPGB-1 may indicate a connection that would benefit from further study.
In Neurospora crassa, the VIVID (VVD) protein serves as a blue-light receptor for photo-adaptation (Schwerdtfeger & Linden, 2003) and contributes to mechanisms that control the regulation of circadian rhythms (Heintzen et al., 2001
). Both G-protein mutants are deficient in asexual sporulation and pigmentation (Gao & Nuss, 1996
; Kasahara & Nuss, 1997
), two characteristics that, in wild-type mycelium, are enhanced by high light intensity. Therefore the observation of a reduction in the level of vvd transcript provides for a potential mechanism by which sporulation and pigmentation are affected in the G-protein mutants. Intriguingly, although these same characteristics are also altered in the hypovirus-infected mycelium, the vvd transcript was not detected as changed by either microarray or real-time RT-PCR. This presents the possibility that sporulation and pigmentation are affected by a G-protein-independent mechanism in the presence of CHV1-EP713.
Previous studies had implicated the G-protein-signalling pathway as a possible target of hypovirus-mediated alteration that could contribute to the pleiotropic effects observed in infected mycelia (Choi et al., 1995; Gao & Nuss, 1996
). In our experiments, we have used antiserum raised against full-length CPG-1 in a method that has been previously determined to provide accurate assessment of relative G-protein subunit accumulation in C. parasitica (Parsley et al., 2003
). Our results permitted a measurement of the amount of this subunit in the presence of CHV1-EP713, determined to be 25 % of that present in uninfected EP155 mycelium. Additionally, we detected a similar reduction in the relative quantity of CPGB-1 protein. This represents the first indication of hypovirus-mediated alteration of the levels of a G
subunit and is further evidence of a considerable modulation of G-protein-signalling pathways in general by the hypovirus CHV1-EP713. Given the fact that in both cases we observed the transcript levels to be slightly elevated, it is likely that these changes are the result of increased turnover of the proteins in question. Whether this is an effect of the reduction of one subunit, which then results in the corresponding reduction of the putative functional partner (as described by Parsley et al., 2003
) or represents hypovirus-mediated attenuation of both G-protein components, is presently unclear.
Combined observations from the three hybridization conditions analysed in this study illuminated a considerable overlap in the expression profile changes that occurred in the absence of CPG-1 or CPGB-1, or in the presence of CHV1-EP713. Of the 100 genes commonly changed between cpg-1 and
cpgb-1 (class AB), almost half (45 %) were also changed in the presence of hypovirus (class ABV). Of particular note, 43 of these 45 genes were coordinately changed in all three scenarios, suggesting a considerable influence of CHV1-EP713 upon G-protein-signalling. CHV1-EP713 appears to affect CPG-1- and CPGB-1-mediated pathways to an equal extent since classes AV (17 genes) and BV (11 genes) are of similar size, and both are much smaller than ABV. This is consistent with the observation that CHV1-EP713 infection reduces the accumulation of both subunits and reflects a general modulation of G-protein-controlled pathways. Also apparent was the generally larger magnitude of the changes observed in either the
cpg-1 or
cpgb-1 strains compared to CHV1-EP713-infected EP155. This presumably reflects the reduction in abundance of these subunits at the protein level in the presence of hypovirus, with the effects on genes targeted by the G-protein-signalling pathway becoming more pronounced in the complete absence of those proteins.
Previous analysis of the changes wrought by hypovirus infection (Allen et al., 2003) had speculated on the possible involvement of stress response-related proteins, including homologues of heat-shock protein 70 (HSP70) and glutathione S-transferase (GST) on fungal phenotype and virulence. HSP70 (AEST-38-C-04) was downregulated in the presence of CHV1-EP713, while GST (AEST-38-H-09) was upregulated, and both transcripts behave identically in each of the G-protein knockout strains. These results suggest that: (i) either HSP70- and GST-like transcripts may be controlled by G-protein-responsive pathways; or (ii) the deletion of a key regulating component such as a G
or G
subunit results in a stress response that has some similarities to the presence and replication in the cytoplasm of the 12·7 kb RNA genome of the hypovirus CHV1-EP713.
Allen et al. (2003) also noted the specific alteration of homologues of three transcription factors in the presence of CHV1-EP713: Pro1 (regulation of fruiting body development and asexual sporulation in Sordaria macrospora; Masloff et al., 2002
), HoxX (regulation of hydrogenase biosynthesis in Bradyrhizobium japonicum; Durmowicz & Maier, 1997
) and Mst12 (regulation of growth of infectious hyphae in Magnaporthe grisea; Park et al., 2002
). Most interestingly, only Mst12 shows similar regulation in the absence of CPG-1 and CPGB-1. Mst12 was identified based on its similarity to the Ste12 gene from S. cerevisiae, a transcription factor that is activated in response to stimulation by mating pheromones through a G-protein-coupled receptor, and leads to the induction of various genes required for mating. In this respect, it is interesting to note that both CHV1-EP713-infected EP155 and the
cpg-1 strain exhibit female infertility (Anagnostakis, 1984
; Gao & Nuss, 1996
). Thus, the C. parasitica Mst12 homologue represents an intriguing candidate that is likely to play a role in modulating the expression of genes important for mating and virulence in response to a G-protein-initiated cascade.
Protease secretion is an important component of pathogenic fungal behaviour (reviewed by Monod & Borg-von, 2002). Specifically, production of secreted aspartyl proteinases was shown to be crucial for virulence, tissue invasion, adhesion and interference with host defence systems by the human pathogen Candida albicans (Agabian et al., 1994
; Hoegl et al., 1996
; Hube et al., 1994
; Ruchel et al., 1992
). Forty-nine proteases were identified during the analysis of C. parasitica EST sequences by Dawe et al. (2003)
that were used to prepare the spotted cDNA microarray employed in this study. Eleven of these 49 transcripts (22 %) were observed to be changed in abundance in at least one of the tested strains. However, this population of 49 proteases includes 13 predicted acid-proteases of which six (46 %: EapA, -B and -C plus AEST-28-E-10, -29-E-12 and -35-B-04) were changed. Intriguingly, all six are changed in both G-protein-deleted mutants (all are downregulated), but only two are similarly altered in the presence of hypovirus. Previous studies have demonstrated that null mutants of EapA and EapC produced altered extracellular proteolytic profiles, with EapA contributing 97 % of the proteolytic activity detected in C. parasitica culture medium (Jara et al., 1996
; Razanamparany et al., 1992
). However, information concerning the virulence of these strains is presently unavailable. These data provide evidence for modulation of protease production by G-protein-signalling pathways and are consistent with the reported reduction in extracellular protease production by mutants of Botrytis cinerea lacking the G
subunit BCG-1 (Gronover et al., 2001
). Our information did not permit a comprehensive analysis of potentially secreted proteases since only partial cDNA sequences were available (Dawe et al., 2003
). However, we conclude that downregulation of genes encoding secreted acid proteases may contribute to the reduction in virulence observed in the
cpg-1 and
cpgb-1 strains of C. parasitica. Given the importance of EapA to external proteolytic activity and considering that this transcript is also downregulated by hypovirus infection, the above conclusion might be valid for CHV1-EP713-infected mycelium as well.
In this study, we have presented data that illustrate the considerable crosstalk and overlap between downstream targets of both G- and G
-signalling in the filamentous fungus C. parasitica. Furthermore, we have shown that global transcriptional changes brought about by the presence of the hypovirus CHV1-EP713 bear a striking similarity to those noted for strains deficient in G-protein-signalling. Coupled with our observation of a reduced accumulation of G-protein components CPG-1 and CPGB-1, these data support the notion that interference of G-protein-mediated signal transduction pathways provides for a major contribution to the phenotype of hypovirus-infected mycelium. Furthermore, our studies have illuminated future directions that will provide for more detailed analysis of pathways that impact fungal virulence and also a better understanding of hostvirus interactions at a molecular level.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allen, T. & Nuss, D. (2004). Specific and common alterations in host gene transcript accumulation following infection of the chestnut blight fungus by mild and severe hypoviruses. J Virol 78, 41454155.
Allen, T. D., Dawe, A. L. & Nuss, D. L. (2003). Use of cDNA microarrays to monitor transcriptional responses of the chestnut blight fungus Cryphonetria parasitica to infection by virulence-attenuating hypoviruses. Eukaryot Cell 2, 12531265.
Anagnostakis, S. L. (1984). The mycelial biology of Endothia parasitica. I. Nuclear and cytoplasmic genes that determine morphology and virulence. In The Ecology and Physiology of the Fungal Mycelium, pp. 353366. Edited by D. H. Jennings & A. D. M. Rayner. Cambridge: Cambridge University Press.
Blaauw, M., Knol, J. C., Kortholt, A., Roelofs, J., Ruchira Postma, M., Visser, A. J. & van Haastert, P. J. (2003). Phosducin-like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins. EMBO J 22, 50475057.
Bölker, M. (1998). Sex and crime: heterotrimeric G proteins in fungal mating and pathogenesis. Fungal Genet Biol 25, 143156.[CrossRef][Medline]
Chen, B., Gao, S., Choi, G. H. & Nuss, D. L. (1996). Extensive alteration of fungal gene transcript accumulation and elevation of G-protein-regulated cAMP levels by a virulence-attenuating hypovirus. Proc Natl Acad Sci U S A 93, 79968000.
Choi, G. H. & Nuss, D. L. (1992). Hypovirulence of chestnut blight fungus conferred by an infectious viral cDNA. Science 257, 800803.[Medline]
Choi, G. H., Chen, B. & Nuss, D. L. (1995). Virus-mediated or transgenic suppression of a G-protein alpha subunit and attenuation of fungal virulence. Proc Natl Acad Sci U S A 92, 305309.[Abstract]
Cole, S. W., Galic, Z. & Zack, J. A. (2003). Controlling false-negative errors in microarray differential expression analysis: a PRIM approach. Bioinformatics 19, 18081816.
Dawe, A. L. & Nuss, D. L. (2001). Hypoviruses and chestnut blight: exploiting viruses to understand and modulate fungal pathogenesis. Annu Rev Genet 35, 129.[CrossRef][Medline]
Dawe, A. L., McMains, V. C., Panglao, M., Kasahara, S., Chen, B. & Nuss, D. L. (2003). An ordered collection of expressed sequences from Cryphonectria parasitica and evidence of genomic microsynteny with Neurospora crassa and Magnaporthe grisea. Microbiology 149, 23732384.[CrossRef][Medline]
Durmowicz, M. C. & Maier, R. J. (1997). Roles of HoxX and HoxA in biosynthesis of hydrogenase in Bradyrhizobium japonicum. J Bacteriol 179, 36763682.[Abstract]
Flanary, P. L., DiBello, P. R., Estrada, P. & Dohlman, H. G. (2000). Functional analysis of Plp1 and Plp2, two homologues of phosducin in yeast. J Biol Chem 275, 1846218469.
Gao, S. & Nuss, D. L. (1996). Distinct roles for two G protein alpha subunits in fungal virulence, morphology, and reproduction revealed by targeted gene disruption. Proc Natl Acad Sci U S A 93, 1412214127.
Gronover, C. S., Kasulke, D., Tudzynski, P. & Tudzynski, B. (2001). The role of G-protein alpha subunits in the infection process of the gray mold fungus Botrytis cinerea. Mol PlantMicrobe Interact 14, 12931302.[Medline]
Hamm, H. E. (1998). The many faces of G protein signaling. J Biol Chem 273, 669672.
Heintzen, C., Loros, J. J. & Dunlap, J. C. (2001). The PAS protein VIVID defines a clock-associated feedback loop that represses light input, modulates gating, and regulates clock resetting. Cell 104, 453464.[Medline]
Hoegl, L., Ollert, M. & Korting, H. C. (1996). The role of Candida albicans secreted aspartic proteinase in the development of candidoses. J Mol Med 74, 135142.[Medline]
Hube, B., Monod, M., Schofield, D. A., Brown, A. J. & Gow, N. A. (1994). Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol Microbiol 14, 8799.[Medline]
Jara, P., Gilbert, S., Delmas, P., Guillemot, J. C., Kaghad, M., Ferrara, P. & Loison, G. (1996). Cloning and characterization of the eapB and eapC genes of Cryphonectria parasitica encoding two new acid proteinases, and disruption of eapC. Mol Gen Genet 250, 97105.[CrossRef][Medline]
Kasahara, S. & Nuss, D. L. (1997). Targeted disruption of a fungal G-protein beta subunit gene results in increased vegetative growth but reduced virulence. Mol PlantMicrobe Interact 10, 984993.[Medline]
Kasahara, S., Wang, P. & Nuss, D. L. (2000). Identification of bdm-1, a gene involved in G protein beta-subunit function and alpha-subunit accumulation. Proc Natl Acad Sci U S A 97, 412417.
Lengeler, K. B., Davidson, R. C., D'Souza, C., Harashima, T., Shen, W. C., Wang, P., Pan, X., Waugh, M. & Heitman, J. (2000). Signal transduction cascades regulating fungal development and virulence. Microbiol Mol Biol Rev 64, 746785.
Masloff, S., Jacobsen, S., Poggeler, S. & Kuck, U. (2002). Functional analysis of the C6 zinc finger gene pro1 involved in fungal sexual development. Fungal Genet Biol 36, 107116.[CrossRef][Medline]
Merkel, H. W. (1906). A deadly fungus on the American chestnut. NY Zool Soc Annu Rep 10, 97103.
Monod, M. & Borg-von, Z. M. (2002). Secreted aspartic proteases as virulence factors of Candida species. Biol Chem 383, 10871093.[Medline]
Park, G., Xue, C., Zheng, L., Lam, S. & Xu, J. R. (2002). MST12 regulates infectious growth but not appressorium formation in the rice blast fungus Magnaporthe grisea. Mol PlantMicrobe Interact 15, 183192.[Medline]
Parsley, T. B., Segers, G. C., Nuss, D. L. & Dawe, A. L. (2003). Analysis of altered G-protein subunit accumulation in Cryphonectria parasitica reveals a third Galpha homologue. Curr Genet 43, 2433.[Medline]
Razanamparany, V., Jara, P., Legoux, R., Delmas, P., Msayeh, F., Kaghad, M. & Loison, G. (1992). Cloning and mutation of the gene encoding endothiapepsin from Cryphonectria parasitica. Curr Genet 21, 455461.[Medline]
Ruchel, R., de Bernardis, F., Ray, T. L., Sullivan, P. A. & Cole, G. T. (1992). Candida acid proteinases. J Med Vet Mycol 30 (Suppl. 1), 123132.[Medline]
Schwerdtfeger, C. & Linden, H. (2003). VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation. EMBO J 22, 48464855.
Received 18 May 2004;
revised 3 September 2004;
accepted 7 September 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |