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Honey Bee Research Unit, USDA-ARS, Weslaco, Texas 78596
J.H. de León
Beneficial Insects Research Unit, USDA-ARS, Weslaco, Texas 78596
X. Qin
G.M. Weinstock
Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The genome of the honeybee fungal pathogen Ascosphaera apis (Maassen) encodes three putative high mobility group (HMG-box) transcription factors. The predicted proteins (MAT1-2, STE11 and HTF), each of which contain a single strongly conserved HMG-box, exhibit high similarity to mating type proteins and STE11-like transcription factors previously identified in other ascomycete fungi, some of them important plant and human pathogens. In this study we characterized the A. apis HMG-box containing genes and analyzed the structure of the mating type locus (MAT1-2) and its flanking regions. The MAT1-2 locus contains a single gene encoding a protein with an HMG-box. We also have determined the transcriptional patterns of all three HMG-box containing genes in both mating type idiomorphs and discuss a potential role of these transcription factors in A. apis development and reproduction. A multiplex PCR method with primers amplifying mat1-2-1 and Ste11 gene fragments is described. This new method allows for identification of a single mating type idiomorph and might become an essential tool for applied and basic research of chalkbrood disease in honeybees.
Key words: Ascosphaera apis, gene expression, HMG-box, honeybee, idiomorph, mating type, transcription factor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Chorbinski and Rypula 2003). A. apis belongs to the heterothallic ascomycetes, where sexual reproduction typically is controlled by a single mating type locus (MAT), represented by two alternate idiomorphs (Kronstad and Staben 1997, Poggeler 2001). Both MAT idiomorphs encode proteins with putative DNA-binding motifs that are found in a family of transcription factors which play an essential role in fungal development and the progression of disease (Coppin et al 1997). One of the MAT idiomorphs encodes a protein containing a conserved high-mobility-group domain (HMG-box), and the other encodes a protein containing an alpha box domain and sometimes additional HMG-box proteins (Kronstad and Staben 1997, Cozijnsen and Howlett 2003, Rau et al 2005).
In fungi, insects and vertebrates HMG-box proteins control crucial developmental events (van Beest et al 2000). HMG-box proteins are classified by sequence, number of HMG-boxes and DNA-binding characteristics. A subfamily of these proteins, the MATA-HMG-box (NCBI Conserved Domain Database accession number: cd01389.2), contains a single HMG-box and includes the fungal mating type determinants and STE11 transcription factors (Kjaerulff et al 1997, van Beest et al 2000). The HMG-box of these proteins binds to a minor groove of DNA in a sequence-specific manner to regulate the transcription of target genes. Only a few target genes have been identified in ascomycete fungi. They are genes encoding pheromones and pheromone receptors, which are needed for mating and fertilization (Coppin and Debuchy 2000). In addition the mating type transcription factors (MAT) also have been implicated in regulation of genes involved in the vegetative incompatibility (Coppin et al 1997) and possibly in differential virulence of fungal strains (Kwon-Chung et al 1992). In some ascomycete fungi multiple full or partial HMG-box genes have been found at and away from the MAT locus (Debuchy et al 1993, Ferreira et al 1996, Ferreira et al 1998, Yun et al 2000, Varga 2003, Paoletti et al 2005). The function of the additional putative HMG-box transcription factors encoded outside the MAT locus is poorly understood and might or might not be directly related to mating (Lovell-Badge 1995, Bustin 1999).
Knowledge of the molecular basis of the mating types in A. apis will provide insights into fundamental processes such as sexual and asexual development and also can aid applied research on this agriculturally important fungal species. For example understanding the mechanisms of reproduction in pathogenic fungi could suggest new approaches for developing improved disease management strategies. Therefore in this study we identified and characterized three HMG-box genes in the A. apis genome. One of these genes was found at the MAT locus of the A. apis mating type idiomorph represented by our fungal isolate called 0.5–1A. The mating systems of heterothallic ascomycetes historically were named in a number of ways (Arnaise et al 1993, Coppin et al 1997). In this study we have adopted the nomenclature proposed by Turgeon and Yoder (2000) and designated the two A. apis mating type idiomorphs MAT1-1 and MAT1-2 and refer to the HMG-box mating type gene as mat1-2-1. The other two A. apis HMG-box genes were present in the genome of both idiomorphs and were designated Ste11-like, based on homology of the predicted protein with other STE11 proteins, and Htf for a hypothetical transcription factor.
We also characterized the transcription of these three HMG-box genes in the presence vs. absence of the mating partner to confirm which is the mating type determinant. In addition a new multiplex PCR technique was developed that relies on the amplification of idiomorph-specific sequences and therefore enables molecular distinction of the A. apis idiomorphs. Along with the PCR-based method described by Murray et al (2005) this might become an essential tool for applied and basic studies of chalkbrood disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) were grown 6–9 d both separately and mixed on solid and liquid YGPS media (Bailey 1981). Solid cultures were grown at 35 C under 6% CO2. Liquid cultures were grown at 35 C with shaking at 225 rpm under normal atmosphere in 2 mL volumes containing ampicillin (100 µg/mL) and streptomycin (12 µg/mL). Fungal cultures were examined routinely with light microscopy to monitor development of spore cysts. Mating and sexual reproduction was never detected in a culture with a single mating type idiomorph or in a mixed liquid culture of the two idiomorphs. Two A. apis isolates used in this study and in the genome sequencing project have been deposited in the USDA-ARSEF culture collection (Murray et al 2005).
Bioinformatics, PCR amplification and cloning.— –
The draft sequence of the A. apis genome was assembled into long scaffolds (N50 of 44 kb) and produced 7x genome coverage, which was sufficient contiguity to allow for gene predictions (Qin et al 2006). We have identified three MATA-HMG-box containing genes with a TBLASTN homology search of the preliminary A. apis genome sequences at the Baylor College of Medicine Human Genome Sequencing Center (BCM-HGSC) Website (http://www.hgsc.bcm.tmc.edu/projects/microbial/Aapis). For validation of these newly identified sequences a reciprocal analysis was performed with the deduced A. apis protein sequences as query for a BLASTP search at the NCBI nonredundant protein database (http://www.ncbi.nlm.nih.gov/BLAST). The Basic Local Alignment Search Tool program compared A. apis ORFs to the database sequences and calculated the statistical significance of matches. The program identified fungal mating type sequences with the calculated values as low as 7e-49. The sequences with a value 3e-07 or higher were eukaryotic sex determinants belonging to a different class (SOX-TCF_HMG-box) of the HMG-box protein superfamily.
Gene-specific primers were used to generate PCR fragments of A. apis HMG-box containing genes: (i) mat1-2-1 (MAT2Scaf74F/R); (ii) Ste11-like (MAT2Scaf474F/R); and (iii) Htf (MAT2Scaf173F/R). An additional pair of primers (SLA2F1 and DNALR1) was used to amplify the A. apis MAT1-2 locus. All PCR fragments were cloned into the pCR2.1-TOPO plasmid vector with the TOPO TA Kit (Invitrogen, Carlsbad, California) and inserts were sequenced by SeqWright (Houston, Texas). For amplification of gene fragments we used Taq Polymerase (Promega Inc., Madison, Wisconsin) and these PCR conditions: after an initial denaturation step at 94 C for 3 min, 35 cycles of denaturation (94 C for 45 s), annealing (62 C for 45 s) and extension (72 C for 45 s). The final extension step was performed at 72 C for 10 min. All PCR primers used in this study are shown (TABLE I).
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Genomic DNA and RNA extraction and single-stranded DNA (ssDNA) production.— – Mycelial masses were removed from the cultures and after squeezing out most of the liquid were separated into aliquots, weighed and stored at –80 C until further processing. Fungal genomic DNA was isolated from the mycelia by the method of Borges et al (www.fgsc.net/fgn37/borges.html). Samples collected for RNA isolation were placed in TRIzol (Invitrogen, Carlsbad, California) before freezing and the manufacturer’s protocol was followed for isolating total RNA. The RNA pellet was resuspended in diethyl pyrocarbonate (DEPC) treated water, incubated 30 min at 37 C with 2 units of DNase I (Ambion, Austin, Texas), and stored at –80 C. RNA integrity and quantity was examined on a 1.0% denaturing agarose/formaldehyde gel. Quantification was verified with Eppendorf’s BioPhotometer with samples that had been diluted 50-fold in TE (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) and incubated 10 min at 65 C. cDNA was synthesized with the SuperScriptTM First-Strand Synthesis system for reverse transcription-PCR (RT-PCR) (Invitrogen, Carlsbad, California), with oligo-dT primers, approximately 1 µg of total RNA, following the manufacturer’s protocol. ssDNA was produced separately from both mating type idiomorphs and from mixed fungal culture, grown in liquid and on solid culture medium as described above. A PCR approach was used to determine expression profiles of the three A. apis HMG-box genes with the amplification conditions described above. ssDNA concentration in each sample was normalized with respect to A. apis Actin gene expression. The resulting ssDNA dilutions were used in all PCR amplifications.
Phylogenetic analysis of putative A. apis HMG-box proteins.— –
The phylogenetic relationships of putative A. apis HMG-box proteins was analyzed with both neighbor joining and parsimony methods. The DNA sequencing program Sequencher (Gene Codes Corp., Ann Arbor, Michigan) was used to process the raw DNA sequences, and the Clustal W program (DNAStar Inc, Madison, Wisconsin) was used initially for multiple alignment of DNA and deduced protein sequences. The NCBI accession numbers of the HMG-box proteins used in this study are shown (TABLE II). The alignment program Clustal X (Thompson et al 1997) and the phylogenetic program PAUP version 4.0b10 for Macintosh (PPC) (Swofford 2002) were used for alignment, bootstrapping (as percentage of 1000 replications) (Felsenstein 1985) and reconstruction of trees as described in de León et al (2006). Phylogenetic trees were constructed with both distance and maximum parsimony methods. For distance analysis the neighbor joining (NJ) algorithmic method was performed with the uncorrected ‘p’ genetic distance parameter, which is the proportion of nucleotide sites differing between two compared sequences (Saitou and Nei 1987). For parsimony analysis heuristic searches for the most parsimonious trees were conducted with closest addition sequence and the branch-swapping algorithm by tree bisection-reconnection (TBR). Characters were unordered and unweighted, gaps were treated as missing data and topological constraints were not enforced.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A. apis mat1-2-1 was identified from the preliminary genomic sequence (Scaffold 74) of a single idiomorph (0.5–1A) only. A sequence analysis of this 1098 bp gene and its putative 338 amino acid (aa) protein showed that it had the hallmarks of MAT1-2 family genes—the presence of a single MATA-HMG-box and two small introns (Yun et al 2000, Scherrer et al 2005). The first intron is present upstream of the MATA-HMG-box, and the second intron is located at a conserved position within the MATA-HMG-box (FIG. 1). This mating type gene was PCR amplified from 0.5–1A genomic DNA with mat1-2-1 gene-specific primers (not shown). In contrast it could not be amplified from the genome of the second mating type idiomorph represented in this study by the isolate A10. This gene was designated mat1-2-1, according to standard nomenclature (Turgeon and Yoder 2000).
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MATI-2 locus analysis.— –
From the genomic sequence the size of the A. apis MAT1-2 locus was determined to be about 3.6 kb; this was confirmed by PCR amplification of 0.5–1A genomic DNA with PCR primers (SLA2F1/DNALR1) designed to anneal within the MAT1-2 flanking regions (TABLE I
). The MAT1-2 locus contains a single mat1-2-1 gene encoding a protein with a MATA-HMG-box. Two genes were found in the flanking regions of the A. apis mating type locus. The Sla2 (cytoskeleton assembly control protein) gene is located upstream of mat1-2-1, and the DNA lyase gene is located downstream of mat1-2-1 and is transcribed in the opposite direction (FIG. 2).
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). The phylogram revealed three major clades supported by high confidence or bootstrap values: (i) a large clade (clade I) comprising mating type transcription factors; (ii) a clade (II) comprising STE11-like transcription factors; and (iii) a clade (III) comprising the remaining hypothetical HMG-box proteins. This phylogeny is consistent with the results published by Varga et al (2003). Clade I includes mating type proteins previously identified in various species of filamentous fungi. A. apis MAT1-2-1 is a member of this protein family and showed high identity (62% within the 82 aa of their HMG-boxes) to MAT-2 from Aspergillus fumigatus (XP_754989
[GenBank]
). Clade II includes STE11-like proteins and hypothetical proteins containing an MATA-HMG-box. A putative A. apis STE11-like protein shares high amino acid similarity with STE11-like transcription factors and hypothetical proteins from other Ascomycete fungi, such as A. fumigatus (XP_751590
[GenBank]
), P. carnini (AAP13349
[GenBank]
), C. immitis (EAS31563
[GenBank]
) and A. nidulans (XP_661271
[GenBank]
). Separation of clade II from the rest of the HMG-box transcription factors was supported by 100% confidence. And finally clade III includes other MATA-HMG-box hypothetical proteins and is represented by the A. apis HTF putative protein. Separation of clades I and III was supported by high bootstrap values, although delineation of mating type transcription factors vs. other hypothetical HTF proteins is less certain. Therefore we analyzed gene expression to determine which of the three A. apis MATA-HMG-box putative proteins is in fact a mating type determinant.
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). The other two genes, Ste11-like and Htf, were expressed in both mating type idiomorphs. Stell-like and mat1-2-1 gene expression appeared to be lower in fungus cultured in liquid medium (FIG. 4). The third, the Htf gene, was highly expressed in all tested samples under all growth conditions. The A. apis Actin gene was highly expressed in both idiomorphs and served as a control in this study.
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to detect the presence of A. apis in a mixed fungal culture of closely related fungal species that often are isolated from infected bee samples. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The HMG-box containing genes have been characterized in several species of filamentous ascomycetes that are highly diverged from A. apis (Coppin et al 1997, Varga 2003). Evolutionarily diverged members of this large protein family were shown to activate different signaling pathways that are involved in processes including both vegetative and sexual reproduction. Some of these proteins were shown to control processes other then mating, such as postfertilization events, internuclear recognition and vegetative incompatibility (Arnaise et al 1993, Coppin et al 1997, Coppin and Debuchy 2000). Because of a relatively low amino acid sequence similarity between the HMG-box proteins, gene function may not be conserved among HMG-box orthologues from highly diverged species.
The phylogenetic analysis of the predicted amino acid sequences of the HMG-box showed similarity to putative transcription factors found in filamentous ascomycetes, including plant and human pathogens (Burt et al 1996, Yokoyama et al 2004). Of note, the Aspergillus fumigatus genome also encodes three MATA-HMG-box proteins (XP_754989
[GenBank]
, XP_751745
[GenBank]
, XP_751590
[GenBank]
) with high amino acid sequence similarity to the A. apis putative HMG-box proteins. Two of the three A. apis genes studied here (mat1-2-1 and Htf ) exhibited significant similarity (30–39% identity of deduced amino acid sequences) with mating type (MAT1-2) genes of other filamentous ascomycetes (FIG. 3). The third gene Ste11-like is found to be most closely related (~56% identity of deduced amino acid sequence) to genes that encode STE11-like transcription factors. Analysis of the A. apis 0.5–1A isolate’s mating type locus revealed that it contains a single gene encoding a protein with the MATA-HMG motif. Two genes (Sla2 and DNA lyase) were found within flanking regions of the A. apis MAT1-2 locus, with homology to genes encoding a putative SLA-2 (cytoskeleton assembly control protein) from A. fumigatus (Galagan et al 2005) and a DNA lyase from Xanthoria polycarpa (Scherrer et al 2005, Yokoyama et al 2003). Of interest, both these genes (Sla2 and DNA lyase) frequently are found in the flanking regions of mating type loci of other ascomycete fungi (Cozijnsen and Howlett 2003, Galagan et al 2005).
Determining the expression profiles of the putative A. apis transcription factors and their target genes (e.g. mating pheromones and pheromone receptors) is essential for understanding their role in fungal development. Therefore we used the RT-PCR approach to investigate expression profiles of the three A. apis genes encoding the putative transcription factors: MAT1-2-1, STE11-like and HTF. Gene expression analysis showed that all three genes were expressed at the RNA level. Similarly to the A. fumigatus mating type gene (Paoletti et al 2005) the A. apis mat1-2-1 was expressed exclusively in the MAT1-2 idiomorph. When a mixed culture of the two idiomorphs was tested, the mat1-2-1 expression was detected both before (FIG. 4, liquid culture) and after mating (FIG. 4, solid medium). Expression of the mating type genes during the vegetative phase is in agreement with their role in fertilization and also was reported by Coppin and Debuchy (2000). An apparently increased level of mat1-2-1 transcript in solid culture containing both mycelia and spore cysts is suggestive of a role in sexual reproduction (FIG. 4). It is likely that components other then mating type proteins (e.g. transcription factors, pheromones and pheromone receptors) are needed for sexual reproduction of A. apis in a liquid culture, where sexual structures were never detected. For example the ascomycete yeast S. cerevisiae produces mating type-specific agglutinins that mediate close physical interaction and adherence and are necessary for mating in liquid. These factors have not been found in filamentous fungi (Kronstad 1997). It is also possible that the oxygen-poor environment might contribute to inhibition of A. apis reproduction in liquid culture.
The sizes of the mat1-2-1 amplicons from genomic DNA vs. cDNA confirmed the presence and processing of the two small introns within this gene (not shown). As expected no transcript was detected with mat1-2-1 sequence-specific primers in a culture containing only the MAT1-1 idiomorph (FIG. 4, A10 lane). That mat1-2-1 of A. apis is expressed in a mating type-specific manner further supports the finding that the gene encodes a mating type protein. The other two HMG-box genes (Ste11-like and Htf ) were expressed in both mating type idiomorphs and during all life stages of the fungus (FIG. 4), suggesting that these two putative transcription factors might activate signaling pathways other then mating. For example the MATA-HMG-box protein of the S. cerevisiae (Rox1) represses transcription of the hypoxic genes in response to change in the oxygen levels (Zitomer et al 1997).
On the other hand STE11 transcription factors have been implicated in sexual development and filamentous growth. Several lines of evidence suggest that the STE11 transcription factor is a master regulator of the sexual differentiation program in fission yeast (Xue-Franzen et al 2006). In fission yeast, STE11 was indispensable for mating type determination (van Beest et al 2000, Qin et al 2003). It was determined that STE11 directs sexual differentiation by controlling expression of genes required for yeast mating and meiosis. In S. pombe the M-cell specific mating type gene Mat1-Mc controls expression of the target genes with the assistance of Ste11 binding to a DNA fragment called the consensus "response element" or TR-box (Sugimoto et al 1991, Kjaerulff et al 1997, van Beest et al 2000). The TR-boxes of ubiquitously expressed genes and those of mating type-specific genes differ in sequence, which could be the basis for the specificity of the target gene activation by the MATA-HMG-box transcription factors (Kjaerulff et al 1997). Of note, the addition of only one "T" at the 5' of the target gene promoter region converts binding specificity from mating type genes to ubiquitously expressed genes (Kjaerulff et al 1997).
The amino acid analysis of the predicted HTF protein from A. apis revealed a characteristic long C-terminus similar to an A. fumigatus HMG-box protein (XP_751745
[GenBank]
). The acidic C-terminal domain might be needed for maximum transcription activation (Moss et al 1998). The HMG-box flanking sequences have been shown to enhance the DNA-binding properties of the HMG-box by providing additional DNA contacts and are required for optimal DNA bending, implying that sharp bending is required for maximum transcriptional activation (Bustin 1999, Li et al 2006, Stott et al 2006).
Partial sequences of the mat1-2-1 and Ste11-like genes were used to develop a multiplex PCR assay for identification of single mating type idiomorphs. This new PCR-based method should significantly simplify identification of mating type idiomorphs in this honeybee pathogen, which currently is accomplished by either attempting mating with a known strain or by an attempt to detect sexual structures by microscopy.
The identification of other key molecules involved in sexual reproduction (e.g. alpha box mating type transcription factor, mating pheromones and pheromone receptors) soon will be possible due to an imminent completion of A. apis genome sequencing project by BCM-HGSC (Qin et al 2006) and will provide a unique opportunity to further elucidate insights into reproduction and pathogenesis of this important honeybee fungal pathogen.
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FOOTNOTES |
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1 Corresponding author. E-mail: karonstein{at}weslaco.ars.usda.gov
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