Center for Biosystems Research, University of Maryland Biotechnology Institute, 5115 Plant Sciences Building, College Park, MD 20742, USA
Correspondence
Donald L. Nuss
nuss{at}umbi.umd.edu
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ABSTRACT |
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The GenBank accession numbers for the sequences determined in this work are CB686454CB690670.
Tables of clones classified according to molecular function and biological process are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org.
Present address: Children's National Medical Center, Center for Genetic Medicine, 111 Michigan Avenue NW, Washington DC 20010, USA.
Present address: Department of Molecular Biology, Keio University School of Medicine, 144 : 8 Ogura, Saiwai, Kawasaki, Kanagawa 212-0054, Japan.
Present address: Biotechnology Research Center, Guangxi University, 13 Xuiling Road, Nanning, Guangxi 530005, PR China.
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INTRODUCTION |
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The focus of our studies, the chestnut blight fungus Cryphonectria parasitica, was accidentally introduced into the United States in the early part of the twentieth century (Merkel, 1906) and has been responsible for the destruction of the native population of the American chestnut throughout almost the entire natural range of this hardwood species. C. parasitica is a Sordariomycete, a classification of ascomycetes that includes many phytopathogenic fungi, including the genera Ophiostoma (containing the species responsible for Dutch elm disease) and Magnaporthe. Further interest in C. parasitica stems from the observation that natural populations of the fungus can contain virulence-attenuating dsRNA elements, or hypoviruses (Anagnostakis & Day, 1979
). The application of molecular biology techniques enabled the development of rigorously tested hypovirus and C. parasitica reverse genetics systems (reviewed by Dawe & Nuss, 2001
). Subsequent studies led to the isolation and characterization of a number of genes important for virulence, including components of G-protein signalling pathways (Choi et al., 1995
; Kasahara & Nuss, 1997
; Kasahara et al., 2000
; Parsley et al., 2003
). Consistent with the prediction that hypoviruses alter host phenotype by modulating cellular signal transduction pathways, differential mRNA display (Chen et al., 1996
) indicated that infection with hypovirus CHV1-EP713 results in a large change in the expression profile. While informative, differential display analyses do not readily reveal which host genes are differentially expressed. Cloning and investigation of the C. parasitica gene termed 13-1 has allowed the construction of a promoter-based reporter system with which to further analyse viral determinants that influence cellular signal transduction pathways (Parsley et al., 2002
). However, such studies are also limited due to the use of a single-gene readout to monitor pathway activity, which may not reflect larger, global, changes occurring through the whole transcriptome.
As part of our continuing efforts to understand the interactions of hypoviruses and their host, C. parasitica, and the factors that affect fungal virulence, we have undertaken a sequencing project to generate a collection of expressed sequences. At the time of writing, only about 40 C. parasitica genes were represented in the NCBI databases. In the absence of a large-scale sequencing effort, we describe below the identification of approximately 2200 new genes using a moderate-throughput approach. By comparison of the recovered sequences to the publicly available information from NCBI, we demonstrate that these ESTs relate to a wide variety of genes with known roles in other organisms and thus provide for preliminary functional assignment of 1255 genes. Further, we explore a small region of genomic microsynteny that appears conserved across three genera and may have functional relevance to G-protein signalling. We anticipate that this large increase in available sequence data for C. parasitica will prove of considerable utility to the phytopathogenic research community while also enabling future studies that will take advantage of new microarray technologies to examine large-scale transcriptional alterations under a variety of conditions.
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METHODS |
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RNA extraction and library construction.
Single-stranded RNA was prepared from solid-medium-grown EP155 and virus-infected cultures according to Chen et al. (1996). Poly(A)+ mRNA was isolated with oligo-d(T) cellulose (Gibco-BRL). Double-stranded cDNA was synthesized and SalI adapters added, then equal amounts of the cDNA from both EP155 and virus-infected EP155 were mixed prior to cloning using the SuperScript Lambda system (Gibco-BRL/Invitrogen) according to the manufacturer's instructions. The in vitro packaging of the resulting phage constructs was accomplished with the Gigapack III procedure (Stratagene) and Escherichia coli strain Y1090 (ZL) for phage propagation and subsequent storage. Finally, the prepared phage were used to infect E. coli DH10B(Zip) (Gibco-BRL/Invitrogen), which permitted plasmid excision resulting in colonies containing cDNA constructs representing both mRNA populations. Individual colonies were transferred into 96-well microtitre plates for cataloguing and storage. Preliminary insert size verification was performed on 20 randomly selected colonies by miniprep and restriction digestion with NotI/SalI. For subsequent experiments not included in this paper, all the clones that provided good sequence data were PCR amplified using T7 and SP6 primers, permitting a larger sampling of insert size data. An additional 120 randomly selected clones were sized in this manner.
cDNA sequence analyses.
Plasmids were prepared from cultures of stored colonies using the Qiaprep 8 system (Qiagen) with a vacuum manifold. Each preparation was checked for yield by electrophoresis, and then submitted to the DNA Sequencing Core Facility at the Center for Biosystems Research for analysis on ABI 3100 or 377 (Applied Biosystems) machines. Single sequence reads in one direction from the 5' end of the cloned fragment were obtained using the M13 reverse primer. Results files were scanned using the SeqMan unit of DNAStar running on a Macintosh G4 computer for the presence of known vector sequence and quality of data so that erroneous results (such as those plasmids with no insert, or multiple templates per reaction) could be removed. Short sequences (less than 100 bp) were also eliminated at this time. Following cleanup, the sequences were assembled into contigs using SeqMan and minimum match percentage of 80 % across at least 50 bp. The resulting contigs combined the similar sequences of duplicate clones or different clones of the same original cDNA product, thus providing an approximation of the number of individual sequences represented in the dataset. Analysis of the total sequence data was then accomplished in two stages, both performed on an IBM PC running the Red Hat Linux operating system and components of the BLAST program suite (Altschul et al., 1997). In the first stage, a library of DNA sequences was prepared from the known C. parasitica items in the NCBI database combined with unpublished sequences from our laboratory. These sequences were then compared, using the BLASTN algorithm, to all of the unknown individual EST sequences (not contigs) so that clones representing known C. parasitica genes could be identified. The second stage used the EST sequences in a BLASTX analysis against the entire non-redundant protein database as available from NCBI. Tables that comprised lists of the clones and their matches, together with NCBI information and alignments for each hit, were generated from the BLAST output using BioPerl (http://www.bioperl.org) software tools. Gene ontology (GO) information was manually added using the Gene Ontology Consortium's Amigo browser (http://www.godatabase.org/cgi-bin/go.cgi). The entire collection of redundant sequences has been submitted to the NCBI (accession numbers for dbEST, 1747447417478690, corresponding to GenBank numbers CB686454CB690670) and are also publicly searchable at the COGEME phytopathogen database (cogeme.ex.ac.uk).
Genomic sequence analyses.
An EcoRIEcoRI fragment approximately 15 kb in length isolated during characterization of the C. parasitica G-protein -subunit gene (cpgb-1; Kasahara & Nuss, 1997
) was subcloned into pBluescript II SK+ (Stratagene). Following restriction mapping, this insert was divided into smaller sections and subcloned into pBluescript II SK+ or pUC18 to facilitate sequencing, with specific primers being designed to read sequence where required. Sequencing was performed at the CBR core facility and at Keio University, Tokyo, Japan. Assembly of the sequence information with reads in both directions was accomplished using the SeqMan utility of DNAStar. The single contig obtained was determined to accurately represent this region of the C. parasitica genome by comparing the location of the restriction sites predicted with those determined experimentally above. All genomic information for Neurospora crassa and Magnaporthe grisea was obtained from the publicly available databases at the Whitehead Institute/MIT Center for Genome Research (www-genome.wi.mit.edu). Data for Saccharomyces cerevisiae and Schizosaccharomyces pombe were obtained from publicly available information at the Saccharomyces genome resource (genome-www.stanford.edu/Saccharomyces/) and the Sanger Institute (http://www.sanger.ac.uk/Projects/S_pombe/), respectively.
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RESULTS AND DISCUSSION |
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Identification of known C. parasitica sequences
A BLASTN analysis was performed using a specific collection of DNA sequences consisting of the publicly available C. parasitica information, as well as unpublished data from the Nuss laboratory, in comparison to our entire EST sequence data. In this manner we determined that 598 clones represented 13 known C. parasitica genes as detailed in Table 1, 12 from NCBI submitted data and one from unpublished information. Two cDNA sequences were present in far greater abundance than any others and represented almost 6 % of the total sequences each. These corresponded to ORF B of hypovirus CHV1-EP713 and cryparin, a secreted hydrophobin that has previously been shown to be highly expressed (Zhang et al., 1994
). Of the 40 sequences from NCBI that were included in this analysis, the fact that 12 were contained within our collection correlates with the projection of approximately 25 % coverage above.
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Given the dynamic hostpathogen interface, the importance of pathways that transmit signals from the cell surface of the pathogen to the nucleus cannot be overstated. Our laboratory has previously identified a number of signal transduction components, including the G-proteins CPG-1, CPG-2 (Choi et al., 1995), CPG-3 (Parsley et al., 2003
) and CPGB-1 (Kasahara & Nuss, 1997
) as well as other proteins required for their function such as BDM-1 (Kasahara et al., 2000
) and a regulator of G
, RGS (G. C. Segers & D. L. Nuss, unpublished). Identifying specific sequences of interest in our collection, we have also noted a number of other genes that appear to be involved in perception and transfer of signals. These include additional internal transducers: examples of the small GTPase RHO family, a RAB family member, Drab11, and a RAN-binding protein (Sbp1p) probably involved in cell cycle control. Other genes of predicted importance for growth include DFG5, a glycosylphosphatidylinositol (GPI)-anchored protein essential for cell wall biogenesis in S. cerevisiae (Kitagaki et al., 2002
) and two septins. Similarly, cellular structural components were found (making up 11·6 % of the structural protein category, Fig. 2a
), including tubulin and actin as well as components that regulate their organization and biogenesis such as Dam1p, Cct7p and the Arp2/3 complex. Chen et al. (1996)
noted the alteration of cAMP levels in response to hypovirus infection; therefore the identification of a RAS-interacting protein (RIP3) that, in Dictyostelium discoideum, is required for chemotaxis and cAMP-mediated signal transduction (Lee et al., 1999
) is potentially relevant. Most interestingly, we have also identified an orthologue of the Ras guanine nucleotide exchange factor (RasGEF) AleA, a likely functional partner of RIP3. Together, these two components may form part of a C. parasitica RAS-regulated pathway as has been postulated for D. discoideum (Lee et al., 1999
).
During growth of C. parasitica on solid medium, rings of developmentally distinct regions become apparent, areas that are primarily the sites of asexual sporulation and pigmentation. The frequency of the occurrence of these bands corresponds to the light cycle in which the culture is being grown and thus provides evidence of the light-responsive nature of these processes. In N. crassa, substantial advances have been made in studying the mechanism of light response and maintenance of circadian rhythms upon which similar developmental steps including conidiation are dependent (reviewed by Dunlap, 1996). One key component of the process is the PAS protein VVD (Heintzen et al., 2001
). We have identified an orthologue of this gene as well as a clock-controlled gene whose expression in N. crassa is dependent on the clock cycle (Bell-Pedersen et al., 1996
) and a cDNA similar to the nop1 gene of N. crassa that encodes a putative rhodopsin orthologue (Bieszke et al., 1999
). In an additional effort unrelated to the C. parasitica EST library, we have also recovered a partial sequence of the transcriptional regulator FRQ from C. parasitica by degenerate PCR (A. L. Dawe & D. L. Nuss, unpublished observations). Further study of the mechanisms of light response and circadian rhythm maintenance may provide insights into the importance of these developmental regulatory pathways for fungal plant pathogenicity.
Our studies are also predicated in part upon efforts to enhance the naturally occurring virulence attenuation characteristics of the virus family Hypoviridae as a method of biological control (reviewed by Dawe & Nuss, 2001). Effective dissemination of the hypovirus through a native population requires the passing of the dsRNA elements from infected to non-infected fungus by means of anastomosis (hyphal fusion: Anagnostakis & Day, 1979
). This process is dependent upon the vegetative incompatibility (vic) loci of each strain, and virus transmission can be greatly inhibited by heteroallelism between pairs (Cortesi et al., 2001
). At the time of writing, public information concerning the sequence of the vic genes was unavailable, but we have isolated five new C. parasitica sequences in this study that appear to be related to this important process. Genetic analysis of the fungus P. anserina has identified at least 10 loci (het-c, -d and -e; mod-a, -d and -e; idi-1, -2 and -3; pspA), as being involved in the vegetative incompatibility reaction (reviewed by Glass & Kaneko, 2003
) and their characterization has revealed proteins probably involved in signal transduction and membrane structural modifications. The HET-C protein has recently been shown to increase glycosphingolipid transfer rates (Mattjus et al., 2003
) and also is involved in growth and sporulation (Saupe et al., 1994
). Our analysis yielded three EST clones corresponding to het-c. Complete sequencing of these inserts from both 5' and 3' ends has enabled us to construct a complete predicted amino acid sequence of the C. parasitica HET-C orthologue. When compared to the sequence for het-c of P. anserina and the predicted protein of N. crassa locus NCU07947.1, significant identity throughout the polypeptides is clear (Fig. 3
). Each protein is 5363 % identical to the other two, suggesting considerable conservation of incompatibility components between these fungi. This is further supported by the presence of C. parasitica ESTs corresponding to the vegetative incompatibility proteins HET-D, a WD-40 domain protein of unknown function from P. anserina (Espagne et al., 2002
) and HET-6 of N. crassa, whose function is also presently unspecified. The mod genes of P. anserina are believed to code for signalling components that mediate the incompatibility response that can lead to programmed cell death. MOD-A and MOD-E were represented in the C. parasitica library and a sixth C. parasitica gene, the G
subunit CPG-2 identified by Choi et al. (1995)
, may also be relevant since it is closely related to the MOD-D protein (Loubradou et al., 1999
). In contrast to the CPG-1
subunit, which caused extensive phenotypic changes when absent, deletion of cpg-2 caused only minor changes from wild-type and therefore was not required for pigmentation, sporulation or virulence (Choi et al., 1995
). Further analysis of the clones identified in this study may, in conjunction with characterized loci in C. parasitica and other organisms, provide additional insights into this phenomenon and its relevance to the engineering of hypoviruses for enhanced potential as biological control agents.
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The synteny appears absent in the genomes of S. cerevisiae and Sch. pombe, since the two genes of interest are at least 100 kb and 40 kb apart, respectively (data not shown). Furthermore, of the two phosducin-like proteins Plp1p (gi number 6320389; most similar to BDM-1) and Plp2p (gi number 6324856) in S. cerevisiae (Flanary et al., 2000), PLP2 is located closer to the
-subunit STE4. We have also noted an EST clone (26C10, tentatively termed bdm-2) from C. parasitica that appears to correspond to PLP2 (E=9x10-34), a gene that gave a lethal phenotype when knocked out in S. cerevisiae (Flanary et al., 2000
). Examining the genome data available for N. crassa and M. grisea we have determined that sequences that resemble PLP2/bdm-2 are present in both of these fungi also (NCU00617.1 and MG02871.1, respectively) but are located on different linkage groups and supercontigs. Therefore, while both Plp1p and Plp2p were characterized as phosducin-like proteins from S. cerevisiae, it appears as if the functional relationship of the Plp2p family to G
subunits may be quite different from that which has been hypothesized for BDM-1 (Kasahara et al., 2000
) and Plp1p (Flanary et al., 2000
).
Of additional note, several N. crassa EST clones were found to map to a region approximately 700 bp downstream of the G subunit, but comparison with the entire cDNA fragment of gnb-1 (Yang et al., 2002
) indicated that they represented an untranslated region of this ORF (data not shown). There are no other ESTs or predicted ORFs that map to this region of the N. crassa genome. Similarly, we have not isolated any C. parasitica ESTs that correspond to the equivalent region in this organism. Therefore, it appears as if the two genes are maintained in this arrangement across genera without any intervening genes. Additional comparison of the intervening regions of N. crassa and C. parasitica failed to indicate any conserved regions of sequence identity or similarity (data not shown). This suggests that the physical proximity of the two genes is the most important feature, perhaps reflecting a requirement for co-segregation of these loci.
Conclusions
We have catalogued the largest collection of expressed genes thus far produced for C. parasitica, resulting in approximately 1·3 million bp of previously unknown sequence. We have demonstrated that the sequences represented a wide variety of molecular functions involved in many biological processes. The acquired data pointed to considerable similarity of expressed genes between related species and suggested that this may extend to conserved genomic arrangements. With the available database created in this study, used in conjunction with microarray technology, we now have the opportunity to explore on a large scale the transcriptional changes that occur during events such as hypovirus infection. This will undoubtedly lead to enhanced understanding of hypovirus-mediated modulation of host gene expression as well as provide opportunities for future studies of genes that are likely to be critical for fungal plant pathogenesis.
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ACKNOWLEDGEMENTS |
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Received 28 March 2003;
revised 12 June 2003;
accepted 13 June 2003.