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Suppressive subtractive hybridization and differential screening identified genes differentially expressed in yeast and mycelial forms of Ophiostoma piceae
FEMS Microbiology Letters 238 (2004) 175–181 www.fems-microbiology.org
Suppressive subtractive hybridization and differential screening identified genes differentially expressed in yeast and mycelial forms of Ophiostoma piceae Nisha Dogra *, Colette Breuil Department of Wood Science, 2424 Main Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z4 Received 30 December 2003; received in revised form 29 March 2004; accepted 15 July 2004 First published online 28 July 2004
Abstract Ophiostoma piceae is a sap-staining fungus that colonizes and discolours wood. It has been established that the mycelium form but not the yeast form becomes pigmented in vitro. Suppressive subtractive hybridization PCR was used to isolate transcripts specifically upregulated in either the yeast or mycelial forms of O. piceae. Subtracted cDNAs were cloned and transformed into Escherichia coli. The yeast and mycelium specific clones were then screened by differential screening to reduce the possibility of isolating false positive clones and the differential expression of the two sets of cDNAs was confirmed by reverse Northern hybridization. Numerous genes appear to be specifically expressed in either the yeast or mycelial forms. Sequence analysis identified several cDNAs similar to known genes. However, a few cDNAs showed no similarity to sequences in the public databases. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Ophiostoma piceae; Sapstain; Yeast; Mycelium; SSH; Differential screening
1. Introduction Ophiostomatoid fungi are a complex group of pathogenic and saprophytic species responsible for serious and economically important tree diseases in North America. Ophiostoma piceae, one of the most common sap-staining fungi, grows mostly on coniferous trees [1]. When O. piceae colonizes wood, it produces melanized hyphae and induces discolouration of the wood known as blue to gray-black stain. The dark pigment has been identified as melanin and is classified as a secondary metabolite [2]. Our previous studies have shown that several features displayed by O. piceae and other * Corresponding author at: Department of Medical Genetics, Vancouver General Hospital, 2660 Oak Street, Vancouver, BC, Canada V6H 3Z6. Tel.: +604 875 4111x61572; fax: +604 875 4497. E-mail address: [email protected] (N. Dogra).
sap-stain fungi grown on wood, could be reproduced and manipulated in vitro. For example, several extracellular enzymes (lipase, proteases, etc.) are secreted by sap-staining fungi grown in defined artificial media [3–5]. Using yeast-like cells of O. piceae, an efficient transformation system has been developed for gene disruption studies [6]. In culture media, yeast-like forms can be obtained when the organism is grown in liquid culture medium under shaking conditions, while the mycelial form can be grown in solid or liquid medium under static conditions. Melanin pigmentation is present in the mycelial form of O. piceae, whereas the yeast form is devoid of pigmentation. Genomic data in Ophiostomatoid fungi is very limited as for fungi in general. The identification of differentially expressed genes required for yeast and mycelium specificity is of prime importance to understand the genetics of O. piceae. Suppressive subtractive hybridiza-
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.07.033
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tion (SSH) is a method based on suppressive PCR that allows creation of subtracted cDNA libraries for the identification of genes differentially expressed in response to an experimental stimulus. It requires relatively few steps to identify cDNAs corresponding to differentially expressed genes, being especially useful when comparing two populations. The availability of differential cDNA libraries will allow us to investigate various aspects of O. piceae metabolism. For example, expressed sequence tag (EST) arrays can be probed with cDNA libraries from yeast, mycelial-like culture and a glycosylation-deficient mutant. Similarly, EST O. piceae arrays could be hybridized with libraries from cultures that displayed either intense or weak pigmentation. Important genes identified from these subtracted cDNA libraries will allow us to carry out detailed functional analysis of these genes and the proteins they encode. This would be the first large-scale comprehensive search for genes in O. piceae.
cations: Buffer OBB and oligotex suspension were added to the total RNA and incubated at 70 C for 3 min. The sample was placed at room temperature for 30 min instead of 10 min for hybridization between the oligo(dT)30 of the oligotex particle and the poly(A) tail of the mRNA. The second modification was the incorporation of an ethanol precipitation step for the isolation of mRNA after eluting the poly(A) mRNA sample by adding hot (70 C) buffer OEB. From yeast and mycelial cells, 4 lg each of mRNA was extracted and used to generate cDNA by using reverse transcriptase (avian myeloblastosis virus; 20 U) and 1 ll of 10 lM cDNA synthesis primer, buffer conditions as prescribed by the PCR-select subtractive library kit (CLONTECH Laboratories, Palo Alto, CA). A complementary strand for this cDNA was synthesized by using DNA polymerase 1 and DNA ligase, followed by treatment with RNaseH. 2.4. Suppressive subtractive hybridization
2. Materials and methods 2.1. Growth conditions Yeast-like culture of O. piceae strain was grown to mid log phase in an agitated flask (250 rpm) containing complete medium [7] at 23 C for 48 h. For the recovery of the filamentous growth phase of O. piceae, an aliquot of the above culture was plated onto complete medium with 1.5% agar. Incubation was carried out at room temperature for 48 h after overlaying with cellophane (Bio-Rad Laboratories; Missisauga, ON). The resulting mycelial mat from the plates was scraped, washed with normal saline and filtered on four layers of sterile cheese cloth to remove any remaining spores prior to RNA extraction. Washed mycelial hyphae from the cheese cloth were collected and processed immediately for RNA isolation. Yeast-like cells were recovered by growing in liquid complete medium at 23 C for 48 h on an orbital shaker at 250 rpm. The yeast-like cell suspension was passed through four layers of sterile cheese cloth to remove any contaminating mycelia and was immediately processed for RNA isolation. 2.2. RNA extraction Yeast and mycelium samples of O. piceae were separately pulverized under liquid nitrogen and the RNA was extracted by using Trizol reagent (Gibco BRL, USA) according to the manufacturerÕs instructions. 2.3. Isolation of mRNA and cDNA synthesis Polyadenylated RNA was isolated using the Qiagen (Qiagen Inc., ON) mRNA protocol with minor modifi-
SSH was performed on yeast- (Y) and mycelium- (M) like cells of O. piceae by using the PCR-select SSH kit of Clontech (CLONTECH) according to the manufacturerÕs instructions. In brief, cDNAs from Y and M cells were subjected to blunt-ended digestion by restriction enzyme Rsa1. For comparison of two populations and for screening differentially expressed clones, forward and reverse subtractions were performed. In the forward subtraction, Y cDNA served as the tester and M cDNA served as the driver and in reverse subtraction, Y cDNA served as the driver and M cDNA served as the tester. In forward subtraction, Y cDNAs were split into two samples (25 ng each) to which different PCR-primer adaptor sequences were ligated (adaptor 1 for one pool of cDNA and adaptor 2R for other). Third ligation of both adaptors to the tester cDNAs (unsubtracted tester control) was performed and used as a negative control for subtraction. In the first hybridization, 750 ng of mycelium cDNA (driver) was added to each sample of Y testers (T-1 and T-2) and incubated at 68 C for 8 h. Hybridization kinetics led to equalization and enrichment of differentially expressed sequences. In the second hybridization, two primary hybridization samples were mixed together and 500 ng of fresh denatured M cDNA (driver) was added to the mixed sample and incubated at 68 C overnight and subsequently diluted with 200 ll of dilution buffer. The resulting cDNA molecules were then subjected to two rounds of PCR to amplify and enrich the desired differentially expressed sequences by using primers (PCR primer1, nested PCR primer1 and nested PCR primer 2R) of the PCR-select subtractive library kit, (CLONTECH) in a Peltier-effect cycling PCR machine (MJ Research Inc.). In the first round of PCR amplification, PCR primer1 (corresponding to adaptor 1 and 2R
N. Dogra, C. Breuil / FEMS Microbiology Letters 238 (2004) 175–181
sequences) was used for exponential amplification of differentially expressed sequences. In the second round of PCR amplification, nested PCR primer1 (corresponding to adaptor 1 sequence) and nested PCR primer 2R (corresponding to adaptor 2R sequence) were used to reduce background and to enrich for differentially expressed sequences. In the first PCR amplification, 1 ll of the hybridization reaction was used for PCR amplification in a 24 ll reaction volume by using Advantage cDNA PCR kit (CLONTECH Laboratories, Palo Alto, CA). A PCR of 27 cycles was performed under the following parameters: an initial step at 72 C for 5 min to extend the adaptors and fill in overhangs, denaturing at 94 C for 30 s, annealing at 66 C for 30 s and extension at 72 C for 1.5 min. To further reduce the background, the resulting PCR product was further diluted (3 ll in 27 ll) and a second round of amplification was performed by using nested PCR primer1 and 2R (15 cycles for Y and 12 cycles for M). Denaturation was at 94 C for 30 s, annealing at 68 C for 30 s and extension at 72 C for 1.5 min with a final extension incubated at 72 C for 10 min.
2.5. Subtracted cDNA library construction The PCR products of forward subtraction as well as reverse subtraction were then cloned separately using a T/A cloning kit (Invitrogen, Carlsbad, CA). Plasmids were transformed into competent cells of Escherichia coli and grown on LB-agar medium under X-gal and ampicillin selection. Individual white colonies that showed the presence of inserted DNA (by b-galactosidase expression) were randomly picked and further analysed.
2.6. cDNA insert analysis by PCR The two subtracted cDNA libraries produced were enriched for genes specific to Y or M cells of O. piceae in E. coli. Randomly picked white colonies were grown in 96-well plates with 100 ll LB broth and ampicillin (50 lg ml 1) at 37 C. cDNA inserts in individual E. coli colonies were analysed by nested PCR using (MJ Research multiple 96 plate). One ll of overnight culture was used in a 19 ll reaction volume with nested primer1 and 2R. PCR was performed under the following parameters: 94 C for 30 s, 23 cycles of 95 C for 30 s and 68 C for 3 min. PCR product (8 ll) was checked on 2% agarose gel and the rest was used for differential screening.
2.7. Screening of subtracted cDNA clones Differential screening of the arrayed subtracted cDNA clones was performed by using the PCR-select differential screening kit (CLONTECH). Initial screening for positive clones was performed by reverse Northern hybridizations. Equal amounts of amplified cDNA were denatured with 0.6 N NaOH before they were manually spotted (2 ll each) onto two identical nylon membranes (Bio-Rad). To minimize false positive results, negative and positive controls were used. The neg-
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ative control contained cDNA with nested primers supplied with the PCR-select differential screening kit (CLONTECH). The subtracted cDNA corresponding to each probe was used as positive control. The cDNA was immobilised by baking at 80 C for 2 h under a vacuum before hybridization with two different cDNA probes. These two probes are: (1) yeast subtracted cDNA and (2) mycelium subtracted cDNA. Adaptors were removed from subtracted cDNAs before using them as probes. The inserts from subtracted cDNA clones that were arrayed on the membranes also contain the adaptor sequences. Despite their small size, these adaptor sequences cause a very high background during hybridization. Therefore, before using them as probe, adaptors from subtracted cDNAs were removed by digestion with Rsa1, Eag1 and Sma1. 90 ng of adaptor free subtracted cDNAs was labelled by random priming using 50 lCi each of [a-32P]dCTP (3000 Ci/ mmol; Perkin–Elmer Life Sciences, Inc, Boston, MA). Probes were purified from unincorporated label using a CHROMA SPIN-100 (# K1302-1) column and the same amount (107 counts per minute) was added to each membrane. Blots were prehybridized for 2 h at 72 C in 5 ml of CLONTECHÕs ExpressHyb Hybridization Solution (# 8015-1) combined with blocking solution (boiled for 5 min and chilled on ice) containing unpurified nested primer1 and 2R, cDNA synthesis primers and their complementary oligonucleotides. Hybridization was performed using CLONTECHÕs ExpressHyb Hybridization Solution at 72 C overnight with continuous agitation. After washing membranes with low-stringency (2 · SSC and 0.5% SDS) and high-stringency (0.2 · SSC and 0.5% SDS) at 68 C, they were exposed to X-ray film (Kodak Biomax MR film) for varying lengths of time (e.g., 2 h, 4 h, 6 h and overnight).
2.8. cDNA sequencing and sequence analysis The positive clones that passed the differential screening were further analysed by sequencing using M13 Primers. Sequencing reactions were carried out in a Perkin–Elmer DNA thermal cycler using an ABI PRISM dye terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA). Sequences were compared with the non-redundant database using the blastx and blastn programs at the DNA analysis website maintained by NCBI and at the Whitehead Institute site (http://www-genome.wi.mit.edu/annotation/fungi/ neurospora/) to search the Neurospora crassa database for similar sequences. All these sequences have been submitted to EST database of NCBI GenBank. Their dbEST_Ids and GenBank_Accn. nos. are listed in Tables 1 and 2.
3. Results
3.1. Generation and identification of differen-
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Table 1 Yeast and mycelium specific cDNA sequences of Ophiostoma piceae submitted to NCBI GenBank User_Id
DbEST_Id
GenBank_Accn. no.
Yeast OPY5 OPY6 OPY8 OPY19 OPY22 OPY24 OPY32 OPY34 OPY40 OPY41 OPY46 OPY50 OPY63 OPY66 OPY68 OPY71 OPY72 OPY91 OPY92 OPY94 OPY122
15281405 15281406 15281407 15281408 15281409 15281410 15281411 15281412 15281413 15281414 15281415 15281416 15281418 15281419 15281420 15281421 15281422 15281423 15281424 15281425 15281426
CA453145 CA453146 CA453147 CA453148 CA453149 CA453150 CA453151 CA453152 CA453153 CA453154 CA453155 CA453156 CA453158 CA453159 CA453160 CA453161 CA453162 CA453163 CA453164 CA453165 CA453166
Mycelium OPM5 OPM9 OPM82 OPM16 OPM23 OPM46 OPM79 OPM95 OPM98 OPM20 OPM24 OPM29
17070174 17070175 17070176 17068120 17068121 17068122 17068123 17068124 17068125 15281427 15281428 15281429
CB333772 CB333773 CB333774 CB331849 CB331850 CB331851 CB331852 CB331853 CB331854 CA453167 CA453168 CA453169
tially expressed cDNA fragments Subtractive hybridization of Y–M enriched 358 cDNA clones. These clones were candidates for tran-
scripts specific to or up-regulated in Y at 48 h. Subtractive hybridization of M–Y enriched 285 cDNA clones. These clones were candidates for transcripts specific to or upregulated in M at 48 h. Positive clones were screened from candidates by a differential screening procedure. This screening of the arrayed subtracted cDNA clones (358 Y specific and 285 M specific) was performed by nested PCR by using nested PCR primer 1 and nested PCR primer 2R. As a result, only those clones were amplified which had cDNA inserts carrying adaptor 1 and adaptor 2R on both ends. Fig. 1 shows an example of insert screening analysis. Amplified cDNA inserts ranged in size from 0.12 to 1.2 kb as analysed by agarose gel electrophoresis. In total, 260 of 358 Yspecific cDNA clones and 151 of 285 M-specific cDNA clones passed this screening test and were used for further analysis. The rest of the clones were either false positives or had an insert size less than 0.12 kb. 3.2. Confirmation of differential screening Equal amounts of amplified cDNA were arrayed on a nylon membrane for duplicate screening. Two identical membranes were hybridized with two different probes. These were: (a) Y subtracted probe which is a purified secondary PCR product of the forward subtraction; (b) M subtracted probe which is a purified secondary PCR product of the reverse subtraction. The hybridization results of two subtracted probes were compared. Fig. 2 is a representative reverse Northern blot showing results of the differential screening analysis. The negative control (Fig. 2, boxed) did not hybridize to either blot, demonstrating the specificity of hybridization. Positive controls in both the blots showed strong signals (Fig. 2, shown with arrows). A wide range of intensities of signals were observed from very strong to faint in Panel A of Fig. 2. Faint signals are presumed to represent rarely transcribed differentially expressed genes. Clones that
Table 2 Clones specific to or upregulated in yeast (OPY) and mycelium (OPM) form of Ophiostoma piceae Clones
Length (bp)
Database Accession No.
Gene product
Organism
E-value
OPY5 OPY32 OPY41 OPY34 OPY58 OPY68 OPY66 OPY94 OPY72 OPY122 OPM23 OPM46 OPM79 OPM82 OPM98
269 334 209 476 762 351 506 151 507 784 342 310 150 145 125
CAB44984 D22735 – NP_705622 AF271619 P00411 X62722 AW722674 S78197 AC72265 – CA305759 – AW714904 –
Glucose repressible gene Protein (GRG-1) Hypothetical nox3 protein Novel ORF 307 Complete cds Cox2 nuclear PhO2 gene for specific p-nitrophenylphosphatase Morning cDNA library Probable maturase protein Cytochrome C oxidase subunit COI i2 protein Novel EST rice Xa7/avrxa7 subt. library Novel Evening cDNA library Novel
Podospora anserina Emericella nidulans – Schizosaccharomyces pombe Aspergillus tubingensis Yarrowia lipolytica Schizosaccharomyces pombe Neurospora crassa Schizosaccharomyces pombe Agocybe aegrita – Oryza sativa – Neurospora crassa –
3e 4e – 1e 5e 4e 9e 7e 1e 2e – 7e – 2e –
05 39 34 06 30 13 26 49 63 06 07
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Fig. 1. Insert screening analysis. Randomly picked white colonies from yeast and mycelium-specific subtracted clones of Ophiostoma piceae were subjected to nested primer PCR and analysed on a 2.0% agarose gel. Lane 10 is /X174 DNA/Hae III digest size markers; Lanes 1–9 and 11–17 are different band fragments of cDNA insert.
Fig. 2. Comparison of differential screening approach of yeast and mycelium-specific subtracted cDNAs of Ophiostoma piceae. Equal amounts of amplified yeast cDNAs were arrayed onto two sets of membranes and hybridized with two different probes. Panel A: Blots hybridized with forward (yeast) subtracted cDNA probe. Panel B: Blots hybridized with reverse (mycelium) subtracted cDNA probe. Differentially expressed yeast transcripts were shown as clones with dark signals in Panel A. On each membrane, negative hybridization control (boxed); positive hybridization control (shown with arrow).
hybridized only to the forward-subtracted probe were strong candidates for differential expression. As was shown in Panel A of Fig. 2 almost 70% of the clones are strong candidates for differential expression. Clones that fail to hybridize may represent extremely rare transcripts and therefore lie below the sensitivity limit (Fig. 2; Panel A; D2, D10, E6). These clones typically correspond to low abundance transcripts which were enriched during the subtraction. It was found that 192 out of 260 Y specific and 108 out of 151 M specific clones passed this screening test and were strong candidates for differential expression. Identified clones were arranged as O. piceae Y-specific clones (OPYs) and O. piceae M-specific clones (OPMs), respectively, and sequenced.
3.3. Sequence analysis of differentially expressed clones A random selection of 35 OPYs and 15 OPMs were sequenced. All ESTs sequenced from the libraries of
subtracted cDNAs had sequences of adaptor 1 and adaptor 2R on both ends indicating that contaminating background due to non-specific amplification of the tester was very low. The results of sequence matching with known genes are summarized in Table 2. Sequence analysis revealed that 22 out of 35 OPYs sequences matched known genes, whereas the remainder were novel sequences. Sequence analysis of OPMs revealed that 12 out of 15 sequences showed similarity with known genes. While the remainder could be novel sequences. Out of the 15 OPM sequences, 7 gave significant matches with the sequences in the N. crassa genome, With NCBI blastn search OPY94 showed similarity with the morning specific library of N. crassa and OPM82 showed similarity with evening specific library of N. crassa [8]. Yeast specific clone OPY5 showed similarity with glucose repressible gene protein (GRG-1) of Podospora anadian which is very important in ophiostomatoid fungi. Mycelium specific transcript OPM46
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showed similarity to rice xA7/avrX a7 subtracted library ESTs of Oryza sativa (Accession No. CA305759). Since each cDNA clone obtained by subtraction was digested with restriction enzyme before subtractive hybridization [9] they covered a partial region of full-length cDNA.
4. Discussion In terms of growth environment of O. piceae, Y differs from M in many respects. Many genes which are upregulated or downregulated in Y or M could be involved in such differences. Identification of the genes responsible for these phenotypic differences requires methods which rapidly and efficiently compare the transcripts expressed in these two cell forms. An equalizing cDNA subtraction method, termed SSH, provides the technical basis for such a comparison. The availability of differential cDNA libraries allowed us to investigate various aspects of O. piceae metabolism. Isolation of differentially expressed Y and M specific genes would serve as an initial step towards understanding the basic mechanism of sapstain. Through the analysis of reverse Northern blot results, it has been found that mostly differentially expressed cDNAs were enriched during SSH procedure as they were monitored by nested primer PCR at the initial screening step. Furthermore, for high throughput analysis, it is reasonable to limit differential screening by using forward and reverse subtracted cDNA probes, in this case, confirming that differential expression of the candidate clones is probably unnecessary [10–12]. In this study, subtracted libraries generated 30% of new sequences in Y and 20% in M (Table 2) which is consistent with the average percentage of new sequences in SSH subtracted libraries reported by others [11,13,14]. Taken together about 70% of OPYs and OPMs encoded aa sequences similar to functionally defined proteins (Table 2). The remainder had no predictable function, based on their sequences. Since each cDNA clone obtained by subtraction was digested with restriction enzyme before subtractive hybridization [9], they generally covered a partial region of full-length cDNA. Isolation of full-length cDNA and genomic DNA is necessary for functional and expression analysis. These subtracted cDNA fragments will be available as probes for this purpose. In our present study, to obtain longer cDNA clones, we will use an O. piceae EST database as a source for screening of known genes. Among cDNAs isolated by the subtraction, we identified independent clones that encode predicted proteins significantly similar to very important proteins. For example, clone OPY5 has shown similarity with a glucose repressible gene protein (GRG-1) of Podospora anserina [15]. Although, understanding the mechanism of glucose repression in yeast has proved to be a difficult and challenging problem, a multitude of genes in different path-
ways are repressed by glucose at the level of transcription [16]. In S. cerevisiae, the initial signal for glucose repression could be linked to an increased glycolytic flux [17]. Interruption of glycosylation in Ophiostomatoid fungi may thus be an interesting avenue for control. Genes controlling protein glycosylation affect the fungal cell wall carbohydrate composition which may in turn affect a large number of pathogenicity determinants [18]. It is interesting to note that some of the yeast specific transcripts showed similarity with morning specific library of N. crassa and that mycelium specific transcripts showed similarity with evening specific library of N. crassa. Some of the Y and M specific transcripts gave significant matches with known genes or sequences like putative homing endonuclease which are generally associated with unusual DNA splicing and incorporation events [19]. Here, in this study we have shown that SSH used in conjunction with high throughput differential screening allows rapid and easy identification of differentially expressed genes. The system introduced in this paper virtually excludes the possibility of isolating false positive clones. To the best of our knowledge, this is the first report describing an efficient methodology for isolating differentially expressed genes in Ophiostomatoid fungi, opening promising avenues of research into their presumably growth specific functions with regard to sapstain and development of efficient and environmentally acceptable control methods.
Acknowledgements We thank Jennifer Wong for her assistance with this work and Dr. Devki Nandan for reviewing this manuscript. This work was supported by a grant of the Natural Science and Engineering Research Council.
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Suppressive subtractive hybridization and differential screening identified genes differentially expressed in yeast and mycelial forms of Ophiostoma piceae Nisha Dogra *, Colette Breuil Department of Wood Science, 2424 Main Mall, University of British Columbia, Vancouver, BC, Canada V6T 1Z4 Received 30 December 2003; received in revised form 29 March 2004; accepted 15 July 2004 First published online 28 July 2004
Abstract Ophiostoma piceae is a sap-staining fungus that colonizes and discolours wood. It has been established that the mycelium form but not the yeast form becomes pigmented in vitro. Suppressive subtractive hybridization PCR was used to isolate transcripts specifically upregulated in either the yeast or mycelial forms of O. piceae. Subtracted cDNAs were cloned and transformed into Escherichia coli. The yeast and mycelium specific clones were then screened by differential screening to reduce the possibility of isolating false positive clones and the differential expression of the two sets of cDNAs was confirmed by reverse Northern hybridization. Numerous genes appear to be specifically expressed in either the yeast or mycelial forms. Sequence analysis identified several cDNAs similar to known genes. However, a few cDNAs showed no similarity to sequences in the public databases. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Ophiostoma piceae; Sapstain; Yeast; Mycelium; SSH; Differential screening
1. Introduction Ophiostomatoid fungi are a complex group of pathogenic and saprophytic species responsible for serious and economically important tree diseases in North America. Ophiostoma piceae, one of the most common sap-staining fungi, grows mostly on coniferous trees [1]. When O. piceae colonizes wood, it produces melanized hyphae and induces discolouration of the wood known as blue to gray-black stain. The dark pigment has been identified as melanin and is classified as a secondary metabolite [2]. Our previous studies have shown that several features displayed by O. piceae and other * Corresponding author at: Department of Medical Genetics, Vancouver General Hospital, 2660 Oak Street, Vancouver, BC, Canada V6H 3Z6. Tel.: +604 875 4111x61572; fax: +604 875 4497. E-mail address: [email protected] (N. Dogra).
sap-stain fungi grown on wood, could be reproduced and manipulated in vitro. For example, several extracellular enzymes (lipase, proteases, etc.) are secreted by sap-staining fungi grown in defined artificial media [3–5]. Using yeast-like cells of O. piceae, an efficient transformation system has been developed for gene disruption studies [6]. In culture media, yeast-like forms can be obtained when the organism is grown in liquid culture medium under shaking conditions, while the mycelial form can be grown in solid or liquid medium under static conditions. Melanin pigmentation is present in the mycelial form of O. piceae, whereas the yeast form is devoid of pigmentation. Genomic data in Ophiostomatoid fungi is very limited as for fungi in general. The identification of differentially expressed genes required for yeast and mycelium specificity is of prime importance to understand the genetics of O. piceae. Suppressive subtractive hybridiza-
0378-1097/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.07.033
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tion (SSH) is a method based on suppressive PCR that allows creation of subtracted cDNA libraries for the identification of genes differentially expressed in response to an experimental stimulus. It requires relatively few steps to identify cDNAs corresponding to differentially expressed genes, being especially useful when comparing two populations. The availability of differential cDNA libraries will allow us to investigate various aspects of O. piceae metabolism. For example, expressed sequence tag (EST) arrays can be probed with cDNA libraries from yeast, mycelial-like culture and a glycosylation-deficient mutant. Similarly, EST O. piceae arrays could be hybridized with libraries from cultures that displayed either intense or weak pigmentation. Important genes identified from these subtracted cDNA libraries will allow us to carry out detailed functional analysis of these genes and the proteins they encode. This would be the first large-scale comprehensive search for genes in O. piceae.
cations: Buffer OBB and oligotex suspension were added to the total RNA and incubated at 70 C for 3 min. The sample was placed at room temperature for 30 min instead of 10 min for hybridization between the oligo(dT)30 of the oligotex particle and the poly(A) tail of the mRNA. The second modification was the incorporation of an ethanol precipitation step for the isolation of mRNA after eluting the poly(A) mRNA sample by adding hot (70 C) buffer OEB. From yeast and mycelial cells, 4 lg each of mRNA was extracted and used to generate cDNA by using reverse transcriptase (avian myeloblastosis virus; 20 U) and 1 ll of 10 lM cDNA synthesis primer, buffer conditions as prescribed by the PCR-select subtractive library kit (CLONTECH Laboratories, Palo Alto, CA). A complementary strand for this cDNA was synthesized by using DNA polymerase 1 and DNA ligase, followed by treatment with RNaseH. 2.4. Suppressive subtractive hybridization
2. Materials and methods 2.1. Growth conditions Yeast-like culture of O. piceae strain was grown to mid log phase in an agitated flask (250 rpm) containing complete medium [7] at 23 C for 48 h. For the recovery of the filamentous growth phase of O. piceae, an aliquot of the above culture was plated onto complete medium with 1.5% agar. Incubation was carried out at room temperature for 48 h after overlaying with cellophane (Bio-Rad Laboratories; Missisauga, ON). The resulting mycelial mat from the plates was scraped, washed with normal saline and filtered on four layers of sterile cheese cloth to remove any remaining spores prior to RNA extraction. Washed mycelial hyphae from the cheese cloth were collected and processed immediately for RNA isolation. Yeast-like cells were recovered by growing in liquid complete medium at 23 C for 48 h on an orbital shaker at 250 rpm. The yeast-like cell suspension was passed through four layers of sterile cheese cloth to remove any contaminating mycelia and was immediately processed for RNA isolation. 2.2. RNA extraction Yeast and mycelium samples of O. piceae were separately pulverized under liquid nitrogen and the RNA was extracted by using Trizol reagent (Gibco BRL, USA) according to the manufacturerÕs instructions. 2.3. Isolation of mRNA and cDNA synthesis Polyadenylated RNA was isolated using the Qiagen (Qiagen Inc., ON) mRNA protocol with minor modifi-
SSH was performed on yeast- (Y) and mycelium- (M) like cells of O. piceae by using the PCR-select SSH kit of Clontech (CLONTECH) according to the manufacturerÕs instructions. In brief, cDNAs from Y and M cells were subjected to blunt-ended digestion by restriction enzyme Rsa1. For comparison of two populations and for screening differentially expressed clones, forward and reverse subtractions were performed. In the forward subtraction, Y cDNA served as the tester and M cDNA served as the driver and in reverse subtraction, Y cDNA served as the driver and M cDNA served as the tester. In forward subtraction, Y cDNAs were split into two samples (25 ng each) to which different PCR-primer adaptor sequences were ligated (adaptor 1 for one pool of cDNA and adaptor 2R for other). Third ligation of both adaptors to the tester cDNAs (unsubtracted tester control) was performed and used as a negative control for subtraction. In the first hybridization, 750 ng of mycelium cDNA (driver) was added to each sample of Y testers (T-1 and T-2) and incubated at 68 C for 8 h. Hybridization kinetics led to equalization and enrichment of differentially expressed sequences. In the second hybridization, two primary hybridization samples were mixed together and 500 ng of fresh denatured M cDNA (driver) was added to the mixed sample and incubated at 68 C overnight and subsequently diluted with 200 ll of dilution buffer. The resulting cDNA molecules were then subjected to two rounds of PCR to amplify and enrich the desired differentially expressed sequences by using primers (PCR primer1, nested PCR primer1 and nested PCR primer 2R) of the PCR-select subtractive library kit, (CLONTECH) in a Peltier-effect cycling PCR machine (MJ Research Inc.). In the first round of PCR amplification, PCR primer1 (corresponding to adaptor 1 and 2R
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sequences) was used for exponential amplification of differentially expressed sequences. In the second round of PCR amplification, nested PCR primer1 (corresponding to adaptor 1 sequence) and nested PCR primer 2R (corresponding to adaptor 2R sequence) were used to reduce background and to enrich for differentially expressed sequences. In the first PCR amplification, 1 ll of the hybridization reaction was used for PCR amplification in a 24 ll reaction volume by using Advantage cDNA PCR kit (CLONTECH Laboratories, Palo Alto, CA). A PCR of 27 cycles was performed under the following parameters: an initial step at 72 C for 5 min to extend the adaptors and fill in overhangs, denaturing at 94 C for 30 s, annealing at 66 C for 30 s and extension at 72 C for 1.5 min. To further reduce the background, the resulting PCR product was further diluted (3 ll in 27 ll) and a second round of amplification was performed by using nested PCR primer1 and 2R (15 cycles for Y and 12 cycles for M). Denaturation was at 94 C for 30 s, annealing at 68 C for 30 s and extension at 72 C for 1.5 min with a final extension incubated at 72 C for 10 min.
2.5. Subtracted cDNA library construction The PCR products of forward subtraction as well as reverse subtraction were then cloned separately using a T/A cloning kit (Invitrogen, Carlsbad, CA). Plasmids were transformed into competent cells of Escherichia coli and grown on LB-agar medium under X-gal and ampicillin selection. Individual white colonies that showed the presence of inserted DNA (by b-galactosidase expression) were randomly picked and further analysed.
2.6. cDNA insert analysis by PCR The two subtracted cDNA libraries produced were enriched for genes specific to Y or M cells of O. piceae in E. coli. Randomly picked white colonies were grown in 96-well plates with 100 ll LB broth and ampicillin (50 lg ml 1) at 37 C. cDNA inserts in individual E. coli colonies were analysed by nested PCR using (MJ Research multiple 96 plate). One ll of overnight culture was used in a 19 ll reaction volume with nested primer1 and 2R. PCR was performed under the following parameters: 94 C for 30 s, 23 cycles of 95 C for 30 s and 68 C for 3 min. PCR product (8 ll) was checked on 2% agarose gel and the rest was used for differential screening.
2.7. Screening of subtracted cDNA clones Differential screening of the arrayed subtracted cDNA clones was performed by using the PCR-select differential screening kit (CLONTECH). Initial screening for positive clones was performed by reverse Northern hybridizations. Equal amounts of amplified cDNA were denatured with 0.6 N NaOH before they were manually spotted (2 ll each) onto two identical nylon membranes (Bio-Rad). To minimize false positive results, negative and positive controls were used. The neg-
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ative control contained cDNA with nested primers supplied with the PCR-select differential screening kit (CLONTECH). The subtracted cDNA corresponding to each probe was used as positive control. The cDNA was immobilised by baking at 80 C for 2 h under a vacuum before hybridization with two different cDNA probes. These two probes are: (1) yeast subtracted cDNA and (2) mycelium subtracted cDNA. Adaptors were removed from subtracted cDNAs before using them as probes. The inserts from subtracted cDNA clones that were arrayed on the membranes also contain the adaptor sequences. Despite their small size, these adaptor sequences cause a very high background during hybridization. Therefore, before using them as probe, adaptors from subtracted cDNAs were removed by digestion with Rsa1, Eag1 and Sma1. 90 ng of adaptor free subtracted cDNAs was labelled by random priming using 50 lCi each of [a-32P]dCTP (3000 Ci/ mmol; Perkin–Elmer Life Sciences, Inc, Boston, MA). Probes were purified from unincorporated label using a CHROMA SPIN-100 (# K1302-1) column and the same amount (107 counts per minute) was added to each membrane. Blots were prehybridized for 2 h at 72 C in 5 ml of CLONTECHÕs ExpressHyb Hybridization Solution (# 8015-1) combined with blocking solution (boiled for 5 min and chilled on ice) containing unpurified nested primer1 and 2R, cDNA synthesis primers and their complementary oligonucleotides. Hybridization was performed using CLONTECHÕs ExpressHyb Hybridization Solution at 72 C overnight with continuous agitation. After washing membranes with low-stringency (2 · SSC and 0.5% SDS) and high-stringency (0.2 · SSC and 0.5% SDS) at 68 C, they were exposed to X-ray film (Kodak Biomax MR film) for varying lengths of time (e.g., 2 h, 4 h, 6 h and overnight).
2.8. cDNA sequencing and sequence analysis The positive clones that passed the differential screening were further analysed by sequencing using M13 Primers. Sequencing reactions were carried out in a Perkin–Elmer DNA thermal cycler using an ABI PRISM dye terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA). Sequences were compared with the non-redundant database using the blastx and blastn programs at the DNA analysis website maintained by NCBI and at the Whitehead Institute site (http://www-genome.wi.mit.edu/annotation/fungi/ neurospora/) to search the Neurospora crassa database for similar sequences. All these sequences have been submitted to EST database of NCBI GenBank. Their dbEST_Ids and GenBank_Accn. nos. are listed in Tables 1 and 2.
3. Results
3.1. Generation and identification of differen-
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Table 1 Yeast and mycelium specific cDNA sequences of Ophiostoma piceae submitted to NCBI GenBank User_Id
DbEST_Id
GenBank_Accn. no.
Yeast OPY5 OPY6 OPY8 OPY19 OPY22 OPY24 OPY32 OPY34 OPY40 OPY41 OPY46 OPY50 OPY63 OPY66 OPY68 OPY71 OPY72 OPY91 OPY92 OPY94 OPY122
15281405 15281406 15281407 15281408 15281409 15281410 15281411 15281412 15281413 15281414 15281415 15281416 15281418 15281419 15281420 15281421 15281422 15281423 15281424 15281425 15281426
CA453145 CA453146 CA453147 CA453148 CA453149 CA453150 CA453151 CA453152 CA453153 CA453154 CA453155 CA453156 CA453158 CA453159 CA453160 CA453161 CA453162 CA453163 CA453164 CA453165 CA453166
Mycelium OPM5 OPM9 OPM82 OPM16 OPM23 OPM46 OPM79 OPM95 OPM98 OPM20 OPM24 OPM29
17070174 17070175 17070176 17068120 17068121 17068122 17068123 17068124 17068125 15281427 15281428 15281429
CB333772 CB333773 CB333774 CB331849 CB331850 CB331851 CB331852 CB331853 CB331854 CA453167 CA453168 CA453169
tially expressed cDNA fragments Subtractive hybridization of Y–M enriched 358 cDNA clones. These clones were candidates for tran-
scripts specific to or up-regulated in Y at 48 h. Subtractive hybridization of M–Y enriched 285 cDNA clones. These clones were candidates for transcripts specific to or upregulated in M at 48 h. Positive clones were screened from candidates by a differential screening procedure. This screening of the arrayed subtracted cDNA clones (358 Y specific and 285 M specific) was performed by nested PCR by using nested PCR primer 1 and nested PCR primer 2R. As a result, only those clones were amplified which had cDNA inserts carrying adaptor 1 and adaptor 2R on both ends. Fig. 1 shows an example of insert screening analysis. Amplified cDNA inserts ranged in size from 0.12 to 1.2 kb as analysed by agarose gel electrophoresis. In total, 260 of 358 Yspecific cDNA clones and 151 of 285 M-specific cDNA clones passed this screening test and were used for further analysis. The rest of the clones were either false positives or had an insert size less than 0.12 kb. 3.2. Confirmation of differential screening Equal amounts of amplified cDNA were arrayed on a nylon membrane for duplicate screening. Two identical membranes were hybridized with two different probes. These were: (a) Y subtracted probe which is a purified secondary PCR product of the forward subtraction; (b) M subtracted probe which is a purified secondary PCR product of the reverse subtraction. The hybridization results of two subtracted probes were compared. Fig. 2 is a representative reverse Northern blot showing results of the differential screening analysis. The negative control (Fig. 2, boxed) did not hybridize to either blot, demonstrating the specificity of hybridization. Positive controls in both the blots showed strong signals (Fig. 2, shown with arrows). A wide range of intensities of signals were observed from very strong to faint in Panel A of Fig. 2. Faint signals are presumed to represent rarely transcribed differentially expressed genes. Clones that
Table 2 Clones specific to or upregulated in yeast (OPY) and mycelium (OPM) form of Ophiostoma piceae Clones
Length (bp)
Database Accession No.
Gene product
Organism
E-value
OPY5 OPY32 OPY41 OPY34 OPY58 OPY68 OPY66 OPY94 OPY72 OPY122 OPM23 OPM46 OPM79 OPM82 OPM98
269 334 209 476 762 351 506 151 507 784 342 310 150 145 125
CAB44984 D22735 – NP_705622 AF271619 P00411 X62722 AW722674 S78197 AC72265 – CA305759 – AW714904 –
Glucose repressible gene Protein (GRG-1) Hypothetical nox3 protein Novel ORF 307 Complete cds Cox2 nuclear PhO2 gene for specific p-nitrophenylphosphatase Morning cDNA library Probable maturase protein Cytochrome C oxidase subunit COI i2 protein Novel EST rice Xa7/avrxa7 subt. library Novel Evening cDNA library Novel
Podospora anserina Emericella nidulans – Schizosaccharomyces pombe Aspergillus tubingensis Yarrowia lipolytica Schizosaccharomyces pombe Neurospora crassa Schizosaccharomyces pombe Agocybe aegrita – Oryza sativa – Neurospora crassa –
3e 4e – 1e 5e 4e 9e 7e 1e 2e – 7e – 2e –
05 39 34 06 30 13 26 49 63 06 07
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Fig. 1. Insert screening analysis. Randomly picked white colonies from yeast and mycelium-specific subtracted clones of Ophiostoma piceae were subjected to nested primer PCR and analysed on a 2.0% agarose gel. Lane 10 is /X174 DNA/Hae III digest size markers; Lanes 1–9 and 11–17 are different band fragments of cDNA insert.
Fig. 2. Comparison of differential screening approach of yeast and mycelium-specific subtracted cDNAs of Ophiostoma piceae. Equal amounts of amplified yeast cDNAs were arrayed onto two sets of membranes and hybridized with two different probes. Panel A: Blots hybridized with forward (yeast) subtracted cDNA probe. Panel B: Blots hybridized with reverse (mycelium) subtracted cDNA probe. Differentially expressed yeast transcripts were shown as clones with dark signals in Panel A. On each membrane, negative hybridization control (boxed); positive hybridization control (shown with arrow).
hybridized only to the forward-subtracted probe were strong candidates for differential expression. As was shown in Panel A of Fig. 2 almost 70% of the clones are strong candidates for differential expression. Clones that fail to hybridize may represent extremely rare transcripts and therefore lie below the sensitivity limit (Fig. 2; Panel A; D2, D10, E6). These clones typically correspond to low abundance transcripts which were enriched during the subtraction. It was found that 192 out of 260 Y specific and 108 out of 151 M specific clones passed this screening test and were strong candidates for differential expression. Identified clones were arranged as O. piceae Y-specific clones (OPYs) and O. piceae M-specific clones (OPMs), respectively, and sequenced.
3.3. Sequence analysis of differentially expressed clones A random selection of 35 OPYs and 15 OPMs were sequenced. All ESTs sequenced from the libraries of
subtracted cDNAs had sequences of adaptor 1 and adaptor 2R on both ends indicating that contaminating background due to non-specific amplification of the tester was very low. The results of sequence matching with known genes are summarized in Table 2. Sequence analysis revealed that 22 out of 35 OPYs sequences matched known genes, whereas the remainder were novel sequences. Sequence analysis of OPMs revealed that 12 out of 15 sequences showed similarity with known genes. While the remainder could be novel sequences. Out of the 15 OPM sequences, 7 gave significant matches with the sequences in the N. crassa genome, With NCBI blastn search OPY94 showed similarity with the morning specific library of N. crassa and OPM82 showed similarity with evening specific library of N. crassa [8]. Yeast specific clone OPY5 showed similarity with glucose repressible gene protein (GRG-1) of Podospora anadian which is very important in ophiostomatoid fungi. Mycelium specific transcript OPM46
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showed similarity to rice xA7/avrX a7 subtracted library ESTs of Oryza sativa (Accession No. CA305759). Since each cDNA clone obtained by subtraction was digested with restriction enzyme before subtractive hybridization [9] they covered a partial region of full-length cDNA.
4. Discussion In terms of growth environment of O. piceae, Y differs from M in many respects. Many genes which are upregulated or downregulated in Y or M could be involved in such differences. Identification of the genes responsible for these phenotypic differences requires methods which rapidly and efficiently compare the transcripts expressed in these two cell forms. An equalizing cDNA subtraction method, termed SSH, provides the technical basis for such a comparison. The availability of differential cDNA libraries allowed us to investigate various aspects of O. piceae metabolism. Isolation of differentially expressed Y and M specific genes would serve as an initial step towards understanding the basic mechanism of sapstain. Through the analysis of reverse Northern blot results, it has been found that mostly differentially expressed cDNAs were enriched during SSH procedure as they were monitored by nested primer PCR at the initial screening step. Furthermore, for high throughput analysis, it is reasonable to limit differential screening by using forward and reverse subtracted cDNA probes, in this case, confirming that differential expression of the candidate clones is probably unnecessary [10–12]. In this study, subtracted libraries generated 30% of new sequences in Y and 20% in M (Table 2) which is consistent with the average percentage of new sequences in SSH subtracted libraries reported by others [11,13,14]. Taken together about 70% of OPYs and OPMs encoded aa sequences similar to functionally defined proteins (Table 2). The remainder had no predictable function, based on their sequences. Since each cDNA clone obtained by subtraction was digested with restriction enzyme before subtractive hybridization [9], they generally covered a partial region of full-length cDNA. Isolation of full-length cDNA and genomic DNA is necessary for functional and expression analysis. These subtracted cDNA fragments will be available as probes for this purpose. In our present study, to obtain longer cDNA clones, we will use an O. piceae EST database as a source for screening of known genes. Among cDNAs isolated by the subtraction, we identified independent clones that encode predicted proteins significantly similar to very important proteins. For example, clone OPY5 has shown similarity with a glucose repressible gene protein (GRG-1) of Podospora anserina [15]. Although, understanding the mechanism of glucose repression in yeast has proved to be a difficult and challenging problem, a multitude of genes in different path-
ways are repressed by glucose at the level of transcription [16]. In S. cerevisiae, the initial signal for glucose repression could be linked to an increased glycolytic flux [17]. Interruption of glycosylation in Ophiostomatoid fungi may thus be an interesting avenue for control. Genes controlling protein glycosylation affect the fungal cell wall carbohydrate composition which may in turn affect a large number of pathogenicity determinants [18]. It is interesting to note that some of the yeast specific transcripts showed similarity with morning specific library of N. crassa and that mycelium specific transcripts showed similarity with evening specific library of N. crassa. Some of the Y and M specific transcripts gave significant matches with known genes or sequences like putative homing endonuclease which are generally associated with unusual DNA splicing and incorporation events [19]. Here, in this study we have shown that SSH used in conjunction with high throughput differential screening allows rapid and easy identification of differentially expressed genes. The system introduced in this paper virtually excludes the possibility of isolating false positive clones. To the best of our knowledge, this is the first report describing an efficient methodology for isolating differentially expressed genes in Ophiostomatoid fungi, opening promising avenues of research into their presumably growth specific functions with regard to sapstain and development of efficient and environmentally acceptable control methods.
Acknowledgements We thank Jennifer Wong for her assistance with this work and Dr. Devki Nandan for reviewing this manuscript. This work was supported by a grant of the Natural Science and Engineering Research Council.
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