Gene expression profiling of candidate virulence factors in the laminated root rot pathogen Phellinus sulphurascens

BMC Genomics

Isabel Leal¹  Abul K M Ekramoddoullah¹  Craig Hammett¹  Muhammad A Islam¹  Rona N Sturrock¹  Holly L Williams¹ 

[1] Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, Victoria V8Z 1M5, BC, Canada

关键词: Douglas-fir;    Phellinus sulphurascens;    Laminated root rot (LRR);    Pathogenicity;    Virulence;   

Others  :  1216469
DOI  :  10.1186/1471-2164-15-603

 received in 2014-02-08, accepted in 2014-07-02,  发布年份 2014

【 摘 要 】

Background

Phellinus sulphurascens is a fungal pathogen that causes laminar root rot in conifers, one of the most damaging root diseases in western North America. Despite its importance as a forest pathogen, this fungus is still poorly studied at the genomic level. An understanding of the molecular events involved in establishment of the disease should help to develop new methods for control of this disease.

Results

We generated over 4600 expressed sequence tags from two cDNA libraries constructed using either mycelia grown on cellophane sheets and exposed to Douglas-fir roots or tissues from P. sulphurascens-infected Douglas-fir roots. A total of 890 unique genes were identified from the two libraries, and functional classification of 636 of these genes was possible using the Functional Catalogue (FunCat) annotation scheme. cDNAs were identified that encoded 79 potential virulence factors, including numerous genes implicated in virulence in a variety of phytopathogenic fungi. Many of these putative virulence factors were also among 82 genes identified as encoding putatively secreted proteins. The expression patterns of 86 selected fungal genes over 7 days of infection of Douglas-fir were examined using real-time PCR, and those significantly up-regulated included rhamnogalacturonan acetylesterase, 1,4-benzoquinone reductase, a cyclophilin, a glucoamylase, 3 hydrophobins, a lipase, a serine carboxypeptidase, a putative Ran-binding protein, and two unknown putatively secreted proteins called 1 J04 and 2 J12. Significantly down-regulated genes included a manganese-superoxide dismutase, two metalloproteases, and an unknown putatively secreted protein called Ps0058.

Conclusions

This first collection of Phellinus sulphurascens EST sequences and its annotation provide an important resource for future research aimed at understanding key virulence factors of this forest pathogen. We examined the expression patterns of numerous fungal genes with potential roles in virulence, and found a collection of functionally diverse genes that are significantly up- or down-regulated during infection of Douglas-fir seedling roots by P. sulphurascens.

【 授权许可】

   
2014 Williams et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Thies WG, Sturrock RN: Laminated root rot in Western North America. Portland, OR: USDA Forest Service, Pacific Northwest Research Station General Technical Report; 1995. PNW-GTR-349:32 p
• [2]Nelson EE, Sturrock RN: Susceptibility of western conifers to laminated root rot (Phellinus weirii) in Oregon and British Columbia field tests. West J Appl For 1993, 8:67-70.
• [3]Wallis GW, Reynolds G: The initiation and spread of Poria weirii root rot of Douglas-fir. Can J Bot 1965, 43:1-9.
• [4]Buckland DC, Molnar AC, Wallis GW: Yellow laminated root rot of Douglas-fir. Can J Bot 1954, 32:69-81.
• [5]Sturrock RN, Zeglen S, Turner J: Laminated root rot forest health stand establishment decision aid. BC J Ecosyst Manage 2006, 7:41-43.
• [6]Cook RJ, Edmonds RL, Klopfenstein NB, Littke W, McDonald G, Omdal D, Ripley K, Shaw CG, Sturrock RN, Zambino P: Opportunities for addressing laminated root rot caused by Phellinus sulphurascens in Washington’s forests. Olympia, WA: Washington State Academy of Sciences; 2013. [A report from the Washington State Academy of Sciences, In cooperation with the Washington State Department of Natural Resources]
• [7]Islam MA, Sturrock RN, Williams HL, Ekramoddoullah AKM: Identification, characterization, and expression analyses of class II and IV chitinase genes from Douglas-fir seedlings infected by Phellinus sulphurascens. Phytopathology 2010, 100:356-366.
• [8]Islam MA, Sturrock RN, Ekramoddoullah AKM: Gene expression profiling of a compatible interaction between Douglas-fir and the root rot fungal pathogen Phellinus sulphurascens. Phytopathology 2013, 103:585-593.
• [9]Islam MA, Sturrock RN, Ekramoddoullah AKM: A proteomics approach to identify proteins differentially expressed in Douglas-fir seedlings infected by Phellinus sulphurascens. J Proteomics 2008, 71:425-438.
• [10]Lim YW, Sturrock RN, Leal I, Pellow KW, Yamaguchi T, Breuil C: Distinguishing homokaryons and heterokaryons in Phellinus sulphurascens using pairing tests and ITS polymorphisms. Antonie Van Leeuwenhoek 2008, 93:99-110.
• [11]Shimizu M, Yuda N, Nakamura T, Tanaka H, Wariishi H: Metabolic regulation at the tricarboxylic acid and glyoxylate cycles of the lignin-degrading basidiomycete Phanerochaete chrysosporium against exogenous addition of vanillin. Proteomics 2005, 5:3919-3931.
• [12]Ross-Davis AL, Steward JE, Hanna JW, Kim M-S, Knaus BJ, Cronn R, Rai H, Richardson BA, McDonald GI, Klopfenstein NB: Transcriptome of an Armillaria root disease pathogen reveals candidate genes involved in host substrate utilization at the host-pathogen interface. For Path 2013, 43:468-477.
• [13]DiGuistini S, Ralph SG, Lim YW, Holt R, Jones S, Bohlmann J, Breuil C: Generation and annotation of lodgepole pine and oleoresin-induced expressed sequences from the blue-stain fungus Ophiostoma clavigerum, a Mountain Pine Beetle-associated pathogen. FEMS Microbiol Lett 2007, 267:151-158.
• [14]Hesse-Orce U, DiGuistini S, Keeling CI, Wang Y, Li M, Henderson H, Docking TR, Liao NY, Robertson G, Holt RA, Jones SJM, Bohlmann J, Breuil C: Gene discovery for the bark beetle-vectored fungal tree pathogen Grosmannia clavigera. BMC Genomics 2010, 11:536. BioMed Central Full Text
• [15]Karlsson M, Olson Å, Stenlid J: Expressed sequences from the basidiomycetous tree pathogen Heterobasidion annosum during early infection of Scots pine. Fungal Genet Biol 2003, 39:51-59.
• [16]Olson Å, Aerts A, Asiegbu F, Belbahri L, Bouzid O, Broberg A, Canbäck B, Coutinho PM, Cullen D, Dalman K, Deflorio G, van Diepen LTA, Dunand C, Duplessis S, Durling M, Gonthier P, Grimwood J, Fossdal CG, Hansson D, Henrissat B, Hietala A, Himmelstrand K, Hoffmeister D, Högberg N, James TY, Karlsson M, Kohler A, Kües U, Lee Y-H, Lin YC, et al.: Insight into trade-off between wood decay and parasitism from the genome of a fungal forest pathogen. New Phytol 2012, 194:1001-1013.
• [17]Yakovlev IA, Hietala AM, Steffenrem H, Solheim H, Fossdal CG: Identification and analysis of differentially expressed Heterobasidion parviporum genes during natural colonization of Norway spruce stems. Fungal Genet Biol 2008, 45:498-513.
• [18]Asiegbu FO, Adomas A, Stenlid J: Conifer root and butt rot caused by Heterobasidion annosum (Fr.) Bref. s.l. Mol Plant Pathol 2005, 6:395-409.
• [19]Karlsson M, Hietala AM, Kvaalen H, Solheim H, Olson Å, Stenlid J, Fossdal CG: Quantification of host and pathogen DNA and RNA transcripts in the interaction of Norway spruce with Heterobasidion parviporum. Physiol Mol Plant Pathol 2007, 70:99-109.
• [20]Tudzynski P, Sharon A: Fungal pathogenicity genes. Appl Mycol Biotechnol 2003, 3:187-212.
• [21]Wessels JGH: Fungal hydrophobins: proteins that function at an interface. Trends Plant Sci 1996, 1:9-15.
• [22]Whiteford JR, Spanu PD: Hydrophobins and the interactions between fungi and plants. Mol Plant Pathol 2002, 3:391-400.
• [23]Talbot NJ, Ebbole DJ, Hamer JE: Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 1993, 5:1575-1590.
• [24]Kim S, Ahn I-P, Rho H-S, Lee Y-H: MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization. Mol Microbiol 2005, 57:1224-1237.
• [25]Mgbeahuruike AC, Kovalchuk A, Chen H, Ubhayasekera W, Asiegbu FO: Evolutionary analysis of hydrophobin gene family in two wood-degrading basidiomycetes, Phlebia brevispora and Heterobasidion annosum s.l. BMC Evol Biol 2013, 13:240. BioMed Central Full Text
• [26]Karlsson M, Stenlid J, Olson A: Two hydrophobin genes from the conifer pathogen Heterobasidion annosum are expressed in aerial hyphae. Mycologia 2007, 99:227-231.
• [27]Andreeva L, Heads R, Green CJ: Cyclophilins and their possible role in the stress response. Int J Exp Pathol 1999, 80:305-315.
• [28]Viaud MC, Balhadère PV, Talbot NJ: A Magnaporthe grisea cyclophilin acts as a virulence determinant during plant infection. Plant Cell 2002, 14:917-930.
• [29]Chen MM, Jiang M, Shang J, Lan X, Yang F, Huang J, Nuss D, Chen B: CYP1, a hypovirus-regulated cyclophilin, is required for virulence in the chestnut blight fungus. Mol Plant Pathol 2011, 12:239-246.
• [30]Voigt CA, Schäfer W, Salomon S: A secreted lipase of Fusarium graminearum is a virulence factor required for infection of cereals. Plant J 2005, 42:364-375.
• [31]Duplessis S, Cuomo CA, Lin YC, Aerts A, Tisserant E, Veneault-Fourrey C, David L, Joly DL, Stéphane Hacquard S, Amselem J, Cantarel BL, Chiu R, Coutinho PM, Feau N, Field M, Frey P, Gelhaye E, Goldberg J, Grabherr MG, Kodira CD, Kohler A, Kües U, Lindquist EA, Lucas SM, Mago R, Mauceli E, Morin E, Murat C, Pangilinan JL, Park R, et al.: Obligate biotrophy features unraveled by the genomic analysis of rust fungi. Proc Natl Acad Sci U S A 2011, 108:9166-9171.
• [32]Tyler BM, Tripathy S, Zhang X, Dehal P, Jiang RHY, Aerts A, Arredondo FD, Baxter L, Bensasson D, Beynon JL, Chapman J, Damasceno CMB, Dorrance AE, Dou D, Dickerman AW, Dubchak IL, Garbelotto M, Gijzen M, Gordon SG, Govers F, Grunwald NJ, Huang W, Ivors KL, Jones RW, Kamoun S, Krampis K, Lamour KH, Lee M-K, McDonald WH, Medina M, et al.: Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis. Science 2006, 313:1261-1266.
• [33]Zabel RA, Morrell JJ: Wood microbiology: decay and its prevention. San Diego: Academic Press; 1992.
• [34]Daniel GF, Asiegbu F, Johansson M: The saprotrophic wood-degrading abilities of Heterobasidium annosum intersterility groups P and S. Mycol Res 1998, 1028:991-997.
• [35]Hemsworth GR, Henrissat B, Davies GJ, Walton PH: Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat Chem Biol 2014, 10:122-126.
• [36]Sarkar P, Bosneaga E, Auer M: Plant cell walls throughout evolution: towards a molecular understanding of their design principles. J Exp Bot 2009, 60:3615-3635.
• [37]Kauppinen S, Christgau S, Kofod LV, Halkier T, Dörreich K, Dalbøge H: Molecular cloning and characterization of a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. J Biol Chem 1995, 270:27172-27178.
• [38]ten Have A, Tenberge KB, Benen JAE, Tudzynski P, Visser J, van Jan AL, Kan JAL: The contribution of cell wall degrading enzymes to pathogenesis of fungal plant pathogens. In Agricultural Applications. Berlin Heidelberg: Springer; 2002:341-358.
• [39]Cervone F, Hahn MG, De Lorenzo G, Darvill A: Albersheim P. Host-pathogen interactions XXXIII. a plant protein converts a fungal pathogenesis factor into an elicitor of plant defense responses. Plant Physiol 1989, 90:542-548.
• [40]Khare KB, Bompeix G: Activités proteolytiques des Sclerotinia sclerotiorum et S minor: rôle possible lors de la pathogénèse. Rev Mycol 1976, 40:65-84.
• [41]St. Leger RJ, Bidochka MJ, Roberts DW: Characterization of a novel carboxypeptidase produced by the entomopathogenic fungus Metarhizium anisopliae. Arch Biochem Biophys 1994, 314:392-398.
• [42]Kim ST, Yu S, Kim SG, Kim HJ, Kang SY, Hwang DH, Jang YS, Kang KY: Proteome analysis of rice blast fungus (Magnaporthe grisea) proteome during appressorium formation. Proteomics 2004, 4:3579-3587.
• [43]Dalman K, Himmelstrand K, Olson A, Lind M, Brandström-Durling M, Stenlid J: A genome-wide association study identifies genomic regions for virulence in the non-model organism Heterobasidion annosum s.s. PLoS One 2013, 8(1):e53525.
• [44]Sutherland MW: The generation of oxygen radicals during host plant responses to infection. Physiol Mol Plant Pathol 1991, 39:79-93.
• [45]Tenhaken R, Levin A, Brisson LF, Dixon RA, Lamb C: Function of the oxidative burst in hypersensitive disease resistance. Proc Natl Acad Sci U S A 1995, 92:4158-4163.
• [46]Rolke Y, Liu S, Quidde T, Williamson B, Schouten A, Weltring KM, Siewers V, Tenberge KB, Tudzynski B, Tudzynski P: Functional analysis of H2O2-generating systems in Botrytis cinerea: the major Cu-Zn-superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable. Mol Plant Pathol 2004, 5:17-27.
• [47]Karlsson M, Stenlid J, Olsen Å: Identification of a superoxide dismutase gene from the conifer pathogen Heterobasidion annosum. Physiol Mol Plant P 2005, 66:99-107.
• [48]Dow JM, Davies HA, Daniels MJ: A metalloprotease from Xanthomonas campestris that specifically degrades proline/hydroxyproline-rich glycoproteins of the plant extracellular matrix. MPMI 1998, 11:1085-1093.
• [49]Orbach MJ, Farrall L, Sweigard JA, Chumley FG, Valent B: A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta. Plant Cell 2000, 12:2019-2032.
• [50]Islam MA, Sturrock RN, Holmes TA, Ekramoddoullah AKM: Ultrastructural studies of Phellinus sulphurascens infection of Douglas-fir roots and immunolocalization of host pathogenesis-related proteins. Mycol Res 2009, 113:700-712.
• [51]Sturrock RN, Islam MA, Ekramoddoullah AKM: Host-pathogen interactions in Douglas-fir seedlings infected by Phellinus sulphurascens. Phytopathology 2007, 97:1406-1414.
• [52]Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402.
• [53]Open reading frame finder http://www.ncbi.nlm.nih.gov/projects/gorf/ webcite
• [54]SignalP 3.0 Server http://www.cbs.dtu.dk/services/SignalP/ webcite
• [55]WoLF PSORT http://wolfpsort.org/ webcite
• [56]Ruepp A, Zollner A, Maier D, Albermann K, Hani J, Mokrejs M, Tetko I, Güldener U, Mannhaupt G, Münsterkötter M, Mewes HW: The FunCat, a functional annotation scheme for systematic classification of proteins from whole genomes. Nucleic Acids Res 2004, 32:5539-5545.
• [57]Consortium for the Functional Genomics of Microbial Eukaryotes (COGEME) http://cogeme.ex.ac.uk/index.html webcite
• [58]Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3:1-11.
• [59]Anderson CL, Jensen JL, Orntoft TF: Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004, 64:5245-5250.
• [60]Khanlou KM, Van Bockstaele E: A critique of widely used normalization software tools and an alternative method to identify reliable reference genes in red clover (Trifolium pratense L.). Planta 2012, 236:1381-1393.
• [61]Yan HZ, Liou RF: Selection of internal control genes for real-time quantitative RT-PCR assays in the oomycete plant pathogen Phytophthora parasitica. Fungal Genet Biol 2006, 43:430-438.
• [62]Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001, 29:2002-2007.