| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Forest Mycology & Pathology, Swedish University of Agricultural Sciences, P.O. 7026, SE-750 07, Uppsala, Sweden
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Two hydrophobin genes (HAH1 and HAH2) have been identified in a Heterobasidion annosum infection-stage cDNA-library. Comparisons of their nucleotide and amino acid sequences show similarity to the coh1 hydrophobin from Coprinopsis cinerea and the sc3 hydrophobin from Schizophyllum commune. Both HAH1 and HAH2 display the amino acid consensus pattern of class I hydrophobins, including the spacing of eight conserved cysteine residues. Real-time quantitative RT-PCR showed high expression of both genes in aerial hyphae but low expression in submerged hyphae and during in vitro infection of pine seedlings. Segregation analysis of HAH1 and HAH2 in a defined cross of Heterobasidion annosum localised HAH1 to linkage group 3 but did not positioned HAH2 in the genetic linkage map. Sequence characteristics and expression patterns of HAH1 and HAH2 suggest a role in aerial growth of mycelia, but not during pathogenesis.
Key words: conifer root rot, forest pathology, gene expression, gene structure
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
, Elliot and Talbot 2004). These proteins self assemble into an amphipatic film at hydrophilic/hydrophobic interfaces (Wösten 2001). Hydrophobins are divided into two different classes (I or II) based on the characteristic spacing of conserved cystine residues and hydrophobicity patterns (Kershaw and Talbot 1998, Wösten 2001). These proteins are involved in many important functions during the fungal life cycle, including aerial hyphae formation, spore production and dispersal, stabilization of fruiting body structures, infection structure attachment and as toxins (Talbot et al 1996, Kershaw and Talbot 1998, Del Sorbo et al 2000, Wösten 2001).
Heterobasidion annosum (Fr.) Bref. sensu lato is a major cause of conifer root rot which is economically the most devastating disease of coniferous forests in northern temperate regions (Korhonen and Stenlid 1998). The H. annosum s.l. species complex consists of several different species and intersterility (IS) groups. In Europe three species with partly overlapping distributions and host specificities have been identified (Korhonen 1978, Capretti et al 1990), H. annosum sensu stricto, H. parviporum Niemelä & Korhonen and H. abietinum Niemelä & Korhonen, with a preference for pine, spruce and fir, respectively (Niemelä and Korhonen 1998). In North America two IS groups are present, P which preferentially infect Pinus spp, and S, which has been found on Picea, Abies and Tsuga (Harrington et al 1989). The establishment of the disease in a forest stand depends on aerial spread of basidiospores (Risbeth 1951). Once established H. annosum s.l. readily forms infection structures such as appresoria and infection pegs on conifer seedling roots (Asiegbu et al 1993, 2005). Both the formation of aerial structures for spore spread and infection structures indicate a potential role for hydrophobins in H. annosum s.l. pathogenicity.
In the current investigation we are discussing two ways in which hydrophobins can be important in the life cycle of H. annosum, either through attachment of infection structures or in formation of aerial structures for spore spread. The aim is to test which function is most probable by looking at sequence similarity, linkage to virulence QTLs and gene expression.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
) was grown in 60 mL of liquid Hagem-medium (Stenlid 1985) at 21 C for 10 d and thereafter homogenized with a mixer (Philips HR 1340/00) for 20 s. The resulting mycelial suspension was used for in vitro infection of 2 wk old, aseptically grown Pinus sylvestris (provenance Saleby) seedlings as described be Karlsson et al (2005). Mycelial suspension also was used to inoculate (1 : 10) 50 mL Hagem-medium in E-flasks or 10 mL Hagem-medium in Petri dishes. Mycelia were incubated at 21 C in the dark and harvested after 6 d (E-flasks) or 10 d (Petri dishes), frozen with liquid N2 and freeze-dried.
Nucleic acid processing.— –
Extraction of nucleic acids was done with 3% hexadecyl-tri-methyl-ammonium bromide (CTAB) and phenol/chloroform as described by Karlsson et al (2005). PCR, PCR clean-up, oligo(dT) primed reverse transcription of total RNA (including kanamycin positive control synthetic mRNA) and nucleic acid concentrations was performed as described by Karlsson et al (2005). Amplification of two different cDNA with similarity to hydrophobins, hereafter referred to as HAH1 and HAH2 respectively, was done with the 5'
TriplEx2 / 3'TriplEx2 primer pair (Clontech, Palo Alto, California) while amplification of genomic DNA for segregation analysis was done with primers Hah1aF (5'-agcttcatctgcaatccttac-3'), Hah1aR (5'-acattctcagtgctcaacatcaag-3'), Hah2aF (5'-tcttcgcactcgc tctcattcaca-3') and Hah2aR (5v-atcccaacgtatttttccgctcat-3') for the respective genes.
Sequencing and sequence analysis.— –
Sequences were determined with a CEQ 2000 using the Dye Terminator Cycle Sequencing Chemistry (Beckman Coulter, Fullerton, California) and protocols, except that the total reaction volume was 10 µL with 3 µL PCR product as template. Sequencing was achieved with the same primers that were used for amplification. Nucleotide sequence data were reported to GenBank (NCBI) with accession Nos. DQ198364
[GenBank]
and DQ198365
[GenBank]
. Hydrophobicity plots were made with Protein Hydrophobicity Plots (http://arbl.cvmbs.colostate.edu/molkit/hydropathy/) using the KYTE-DOOLITTLE settings and a 7 bp window size. Prediction of signal peptides was made with Sigcleave (von Heijne 1986) and PROSITE (release 19.10, http://www.expasy.org/prosite/) was used to search for protein domains.
Real-time quantitative PCR.— –
Transcript levels were quantified by real-time quantitative RT-PCR with the SYBR Green PCR Master Mix Kit (Applied Biosystems, Foster City, California) as described by Karlsson et al (2005). Amplification of HAH1 transcripts were achieved with primers HAH1 forward (5'-gctccggccaccaccacgactat-3') and HAH1 reverse (5'-agacgggctgctgaacg-3'), while amplification of HAH2 transcripts were achieved with primers HAH2 forward (5'-ggcctcgcgctgatggtcta-3') and HAH2 reverse (5'-tggctggcagggatggtat-3'). Amplification of H. annosum 
-tubulin gene (TUB1) transcripts was achieved with primers a-Tub forward/reverse for normalization purposes (Karlsson et al 2005). Amplification of a kanamycin positive control was achieved with primers Km-1 and Km-2 (Baeriswyl 2002) and was used as a control of the efficiencies of the reverse transcriptase reactions. Cycle threshold (Ct) values for the PCR product growth curve were determined for at least three biological replicates based on two technical replicates. Expression levels were calculated according to the 2–
Ct method (Livak and Schmittgen 2001). ANOVA was performed on expression data of individual genes.
Genetic linkage analysis.— –
A genetic linkage map has been published for a H. annosum s.l. cross between a North American P (32-1, the isolate used in the present study) and a North American S isolate (Lind et al 2005). The presence of either of the two parental sequence variants for the two hydrophobin genes were detected in the mapping population after amplification and subsequent restriction fragment length polymorphism analysis. The PCR product of HAH1 and HAH2 were respectively cut with the restriction enzymes AciI and HinfI. Positioning of the two hydrophobin genes in the genetic linkage map was tested with JoinMap 3.0 (Stam 1993, van Ooijen and Voorips 2001).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
) two clones with similarity to hydrophobins were amplified by PCR and sequenced. The HAH1 open reading frame (ORF) consisted of 420 bp with a 5' untranslated region (UTR) of 51 bp and a 3' UTR of 113 bp, while the HAH2 ORF consisted of 369 bp with a 5' UTR of 84 bp and a 3' UTR of 133 bp. The DNA sequence at the start codons of both HAH1 and HAH2 resembles a typical translational initiation site of eukaryotic mRNAs in that they have an adenine base in their –3 position, although both genes have a thymine base at position +4 (Kozak 1991). Comparison of cDNA and genomic DNA revealed the presence of three introns in HAH1, while HAH2 contained only two introns that corresponded to introns two and three in HAH1. The translated hah1 sequence, consisting of 139 amino acids with a predicted molecular mass of 14.0 kDa, displayed high similarity to hydrophobins sc3 from Schizophyllum commune (64.7% identity) and coh1 from Coprinus cinerea (62.8 identity). The hah2 sequence consisted of 122 amino acids with a predicted molecular mass of 12.4 kDa and showed high similarity to hydrophobins poh2 from Pleurotus ostreatus (58.2% identity) and coh1 from C. cinerea (56.6% identity). The similarity between HAH1 and HAH2 was 60% at the DNA level and 59% at the protein level. Both deduced protein sequences display eight characteristic hydrophobin cysteine residues (FIG. 1). The spacing of these cystine residues in hah1/hah2, X56/39-C-X6-C-C-X32-C-X13-C-X5-C-C-X12-C-X7, are consisted with the class I hydrophobin consensus C-X5–7-C-C-X19–39-C-X8–23-C-X5-C-C-X6–18-C-X2–13 (Kershaw and Talbot 1998). The hydrophobicity plots of both proteins were consistent with class I hydrophobins (data not shown). The first 21 amino acids of hah1 and the 22 first of hah2 were predicted to be secretion signal peptides. In addition both hah1 and hah2 were predicted to contain an N-myristoylation site.
|
To analyse the expression of the hydrophobin genes in aerial hyphae and during infection of pine seedling roots total RNA were prepared from aerial hyphae separated from liquid cultures and from aseptically inoculated seedling roots 72 h post inoculation. Total RNA from vegetative mycelia grown submerged in liquid Hagem media also were isolated. Transcript levels in the different treatments of both hydrophobin genes were normalized by –tubulin gene transcript levels and compared with the 2–Ct method. An Fmax test was performed that concluded that the variances were homogeneous. Both HAH1 and HAH2 showed significant (ANOVA, P < 0.05) higher transcript levels in aerial hyphae compared with submerged hyphae or in H. annosum seedling root infection (TABLE I). However the expression of HAH2 was much higher than HAH1 in aerial hyphae.
|
). The segregation at the two hydrophobin loci are shown (TABLE II). Progeny with ambiguous result was not included in the following mapping analysis. HAH1 did not segregate according to the expected 1:1 (TABLE II). The HAH1 locus was positioned on linkage group 3 (Lind et al 2005), while HAH2 did not link to any marker. No linkage to any known quantitative trait loci (QTL) was established, either for virulence or growth rate on agar (personal communication M. Lind).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
), as well as pathogenic and mutualistic interactions (Ochman and Moran 2001).
The identification of two hydrophobin genes in an infection-stage H. annosum cDNA-library could suggest an involvement of these gene products in pathogenesis. Other hydrophobins contribute to virulence either by attaching appressoria to plant surfaces, such as the class I Mpg1 hydrophobin from the rice blast fungus Magnaporthe grisea (Talbot et al 1993, Talbot et al 1996, Kershaw et al 1998, Soanes et al 2002), or by function as toxins, such as the class II CU hydrophobin from Ophiostoma novoulmi and O. ulmi (Del Sorbo et al 2000). Neither hah1 nor hah2 show any extensive similarity with these proteins, but rather with coh1 and sc3 which are involved in the formation of aerial hyphae, as shown by the absence of SC3 and COH1 transcripts in Schizophyllum commune and Coprinus cinereus mutant strains, which have lost the ability to produce aerial hyphae (van Wetter et al 1996, Asgeirsdottir et al 1997). This is interesting as the ability to form aerial structures is vital for H. annosum s.l. to complete its life cycle and to establish in a forest stand (Risbeth 1951, Redfern and Stenlid 1998). The higher expression of HAH1 and especially HAH2 in aerial hyphae, as compared to submerged hyphae or during early infection of pine seedling roots, supports the involvement of these gene products during aerial hyphae formation, rather than during pathogenesis. The similar, but not identical, expression patterns indicate partial functional redundancy of HAH1 and HAH2, a situation that seem to be common for hydrophobins (Kershaw et al 1998, Lugones et al 1998).
Segregation analysis showed no linkage between the two hydrophobins and known QTLs for virulence. It is still possible that not all QTLs for virulence are identified in the linkage map due to a large proportion of unexplained genetic variation for virulence and that the parental isolates do not display polymorphism for all aspects of virulence. The HAH1 gene showed a segregation that deviated from the assumed 1 : 1, which also have been found for 39% of the AFLP markers in the Heterobasidion genetic linkage map (Lind et al 2005).
Here we report on the identification of two hydrophobin genes from the conifer pathogen H. annosum. The sequence characteristics and expression data of these genes suggest that they encode class I hydrophobins with a putative role in aerial hyphae, but not during pathogenesis, although more extensive research is needed to address the precise functions of HAH1 and HAH2 in H. annosum biology.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Corresponding author. E-mail: Magnus.Karlsson{at}mykopat.slu.se
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Asiegbu F, Daniel G, Johansson M. 1993. Studies on the infection of Norway spruce roots by Heterobasidion annosum. Can J Bot 71:1552–1561.[CrossRef]
———, Adomas A, Stenlid J. 2005. Conifer root and butt rot caused by Heterobasidion annosum (Fr.) Bref. s.l. Mol Plant Pathol 6(4):395–409.[CrossRef]
Baeriswyl T. 2002. Chilling-induced 1-aminocyclopropane-1-carboxylic acid synthase gene expression in the tomato (Lycopersicum esculentum) [Doctoral dissertation]. University of Basel. 62 p.
Capretti P, Korhonen K, Mugnai L, Romagnoli C. 1990. An intersterility group of Heterobasidion annosum specialized to Abies alba. Eur J For Path 20:231–240.[CrossRef]
Chase TE. 1985. [Doctoral dissertation] Burlington: University of Vermont. 244 p.
Del Sorbo G, Scala F, Parrella G, Lorito M, Comparini C, Ruocco M, Scala A. 2000. Functional expression of the gene cu, encoding the phytotoxic hydrophobin ceratoulmin, enables Ophiostoma quercus, a nonpathogen on Elm, to cause symptoms of Dutch elm disease. Mol Plant-Microbe Interac 13:43–53.[CrossRef]
Dunham MJ, Badrane H, Ferea T, Adams J, Brown PO, Rosenzweig F, Botstein D. 2002. Characteristic genome rearrangements in experimental evolution of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 99:16144–16149.
Elliot MA, Talbot NJ. 2004. Building filaments in the air: aerial morphogenesis in bacteria and fungi. Curr Opin Microbiol 7(6):594–601.[CrossRef][Medline]
Harrington TC, Worrall JJ, Rizzo DM. 1989. Compatibility among host-specialized isolates of Heterobasidion annosum from western North America. Phytopathol 79(3):290–296.[CrossRef]
Karlsson M, Olson Å, Stenlid J. 2003. Expressed sequences from the basidiomycetous tree pathogen Heterobasidion annosum during early infection of Scots pine. Fungal Genet Biol 39:51–59.[CrossRef][Medline]
———, Stenlid J, Olson Å. 2005. Identification of a superoxide dismutase gene from the conifer pathogen Heterobasidion annosum. Physiol Mol Plant Pathol 66(3):99–107.[CrossRef]
Kershaw MJ, Talbot NJ. 1998. Hydrophobins and Repellents: proteins with fundamental roles in fungal morpogenesis. Fungal Genet Biol 23:18–23.[CrossRef][Medline]
———, Wakley G, Talbot NJ. 1998. Complementation of the Mpg1 mutant phenotype in Magnaporthe grisea reveals functional relationships between fungal hydrophobins. EMBO J 17(14):3838–3849.[CrossRef][Medline]
Korhonen K. 1978. Intersterility groups of Heterobasidion annosum. Comm Inst For Fenn 94(6):1–25.
———, Stenlid J. 1998. Biology of Heterobasidion annosum. In: Woodward S, Stenlid J, Karjalainen R, Huttermann A, eds. Heterobasidion annosum, Biology, Ecology, Impact and Control. Wallingford, Oxon, UK: CAB International. p 43–70.
Kozak M. 1991. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 266(30):19867–19870.
Lind M, Olson Å, Stenlid J. 2005. An AFLP-markers based genetic linkage map of Heterobasidion annosum locating intersterility genes. Fungal Genet Biol 42(6):519–527.[CrossRef][Medline]
Livak K, Schmittgen T. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2–Ct method. Methods 25:402–408.[CrossRef][Medline]
Lugones L, Wosten H, Wessels J. 1998. A hydrophobin (ABH3) specifically secreted by vegetatively growing hyphae of Agaricus bisporus (common white button mushroom). Microbiol 144(8):2345–2353.
Niemelä T, Korhonen K. 1998. Taxonomy of the Genus Heterobasidion. In: Woodward S, Stenlid J, Karjalainen R, Huttermann A, eds. Heterobasidion annosum, Biology, Ecology, Impact and Control. Wallingford, Oxon, UK: CAB International. p 27–33.
Ochman H, Moran NA. 2001. Genes lost and genes found: evolution of bacterial pathogenesis and symbiosis. Science 292:1096–1098.
Redfern DB, Stenlid J. 1998. Spore dispersal and infection. In: Woodward S, Stenlid J, Karjalainen R, Huttermann A, eds. Heterobasidion annosum, Biology, Ecology, Impact and Control. Wallingford, Oxon, UK: CAB International. p 105–124.
Risbeth J. 1951. Observations on the biology of Fomes annosus with particular reference to East Anglian pine plantations. II. Spore production, stump infection, and saprophytic activity in stumps. Ann Bot 15:1–21.[Medline]
Soanes DM, Kershaw MJ, Cooley RN, Talbot NJ. 2002. Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea. Mol Plant-Microbe Interac 15(12):1253–1267.[CrossRef]
Stam P. 1993. Construction of integrated genetic-linkage maps by means of a new computer package—Joinmap. Plant J 3(5):739–744.
Stenlid J. 1985. Population structure of Heterobasidion annosum as determined by somatic incompatibility, sexual incompatibility, and isoenzyme patterns. Can J Bot 63:2268–2273.
Talbot NJ, Ebbole DJ, Hamer JE. 1993. Identification and characterization of Mpg1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5(11):1575–1590.[Abstract]
———, Kershaw MJ, Wakley GE, de Vries O, Wessels J, Hamer JE. 1996. MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related development of Magnaporthe grisea. Plant Cell 8(6):985–999.[Abstract]
van Ooijen JW, Voorips RE. 2001. Joinmap 3.0 Software for the calculation of genetic linkage maps. Wageningen: Plant Research International.
van Wetter MA, Schuren FHJ, Schuurs TA, Wessels JGH. 1996. Targeted mutation of the SC3 hydrophobin gene of Schizophyllum commune affects formation of aerial hyphae. FEMS Microbiol Lett 140(2–3):265–269.
von Heijne G. 1986. A new method for predicting signal sequence cleavage sites. Nucl Acid Res 14(11):4683–4690.
Wösten H. 2001. Hydrophobins: multipurpose proteins. Annu Rev Microbiol 55:625–646.[CrossRef][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
