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Applied Microbiology and Biotechnology

, Volume 97, Issue 1, pp 283–295 | Cite as

Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts

  • Erika P. Abbott
  • Giuseppe Ianiri
  • Raffaello Castoria
  • Alexander Idnurm
Applied genetics and molecular biotechnology

Abstract

The red yeasts of the Pucciniomycotina have rarely been transformed with DNA molecules. Transformation methods were recently developed for a species of Sporobolomyces, based on selection using uracil auxotrophs and plasmids carrying the wild-type copies of the URA3 and URA5 genes. However, these plasmids were ineffective in the transformation of closely related species. Using the genome-sequenced strain of Rhodotorula graminis as a starting point, the URA3 and URA5 genes were cloned and tested for the transformation ability into different Pucciniomycotina species by biolistic and Agrobacterium-mediated transformations. Transformation success depended on the red yeast species and the origin of the URA3 or URA5 genes, which may be related to the high G + C DNA content found in several species. A new vector was generated to confer resistance to nourseothricin, using a native promoter from R. graminis and the naturally high G + C nourseothricin acetyltransferease gene. This provides a second selectable marker in these species. Targeted gene disruption was tested in Sporobolomyces sp. IAM 13481 using different lengths of homologous DNA with biolistic and Agrobacterium transformation methods. Both DNA delivery methods were effective for targeted replacement of a gene required for carotenoid pigment biosynthesis. The constructs also triggered transgene silencing. These developments open the way to identify and manipulate gene functions in a large group of basidiomycete fungi.

Keywords

Basidiomycete Rhodotorula slooffiae Rhodosporidium kratochvilovae β-Carotene RNAi T-DNA 

Notes

Acknowledgments

We thank Joseph Heitman, James Fraser, and the FGSC for providing strains. This research was supported by grants from the United States National Science Foundation (MCB-0920581), the Italian Ministry of Education, University and Scientific Research (PRIN 2008, 2008JKH2MM), and the Italian Ministry of Foreign Affairs (joint research project LS-7, within the executive programme of cooperation in the field of science and technology between Italy and USA 2008–2010). G. I. was also supported by a scholarship from the Department of Agricultural, Environmental and Food Sciences, University of Molise, Italy.

Supplementary material

253_2012_4561_MOESM1_ESM.pdf (216 kb)
ESM 1 (PDF 216 kb)

References

  1. Aime MC, Matheny PB, Henk DA, Frieders EM, Nilsson RH, Piepenbring M, McLaughlin DJ, Szabo LJ, Begerow D, Sampaio JP, Bauer R, Weiß M, Oberwinkler F, Hibbett D (2006) An overview of the higher level classification of Pucciniomycotina based on combined analyses of nuclear large and small subunit rDNA sequences. Mycologia 98:896–905CrossRefGoogle Scholar
  2. Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346CrossRefGoogle Scholar
  3. Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas PJJ (1995) Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14:3206–3214Google Scholar
  4. Castoria R, De Curtis F, Lima G, De Cicco V (1997) β-1 ,3-glucanase activity of two saprophytic yeasts and possible mode of action involved as biocontrol agents against postharvest diseases. Postharvest Biol Technol 12:293–300CrossRefGoogle Scholar
  5. Castoria R, Caputo L, De Curtis F, De Cicco V (2003) Resistance of postharvest biocontrol yeasts to oxidative stress: a possible new mechanism of action. Phytopathology 93:564–572CrossRefGoogle Scholar
  6. Castoria R, Morena V, Caputo L, Panfili G, De Curtis F, De Cicco V (2005) Effect of the biocontrol yeast Rhodotorula glutinis strain LS11 on patulin accumulation in stored apples. Phytopathology 95:1271–1278CrossRefGoogle Scholar
  7. Castoria R, Mannina L, Durán-Patrón R, Maffei F, Sobolev AP, De Felice DV, Pinedo-Rivilla C, Ritieni A, Ferracane R, Wright SAI (2011) Conversion of the mycotoxin patulin to the less toxic desoxypatulinic acid by the biocontrol yeast Rhodosporidium kratochvilovae strain LS11. J Agric Food Chem 59:11571–11578CrossRefGoogle Scholar
  8. Catalanotto C, Azzalin G, Macino G, Cogoni C (2002) Involvement of small RNAs and role of the qde genes in the gene silencing pathway in Neurospora. Genes Dev 16:790–795CrossRefGoogle Scholar
  9. Chattoo BB, Sherman F, Azubalis DA, Fjellstedt TA, Mehnert D, Ogur M (1979) Selection of lys2 mutants of the yeast Saccharomyces cerevisiae by the utilization of α-aminoadipate. Genetics 93:51–65Google Scholar
  10. Coelho MA, Sampaio JP, Gonçalves P (2010) A deviation from the bipolar-tetrapolar mating paradigm in an early diverged basidiomycete. PLoS Genet 6:e1001052CrossRefGoogle Scholar
  11. Covert SF, Kapoor P, Lee M-H, Briley A, Nairn CJ (2001) Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol Res 105:259–264CrossRefGoogle Scholar
  12. Davidson RC, Blankenship JR, Kraus PR, de Jesus BM, Hull CM, D’Souza C, Wang P, Heitman J (2002) A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148:2607–2615Google Scholar
  13. Edman JC (1992) Isolation of telomere-like sequences from Cryptococcus neoformans and their use in high-efficiency transformation. Mol Cell Biol 12:2777–2783Google Scholar
  14. Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A (2000) Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. Int J Syst Evol Microbiol 50:1351–1371CrossRefGoogle Scholar
  15. Fraser JA, Lim SM, Diezmann S, Wenink EC, Arndt CG, Cox GM, Dietrich FS, Heitman J (2006) Yeast diversity sampling on the San Juan Islands reveals no evidence for the spread of the Vancouver Island Cryptococcus gattii outbreak to this locale. FEMS Yeast Res 6:620–624CrossRefGoogle Scholar
  16. Hamamoto M, Sugiyama J, Komagata K (1986) DNA base composition of strains in the genera Rhodosporidium, Cystofilobasidium, and Rhodotorula determined by reverse-phase high performance liquid chromatograph. J Gen Appl Microbiol 32:215–223CrossRefGoogle Scholar
  17. Hood ME, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218CrossRefGoogle Scholar
  18. Ianiri G, Wright SAI, Castoria R, Idnurm A (2011) Development of resources for the analysis of gene function in Pucciniomycotina red yeasts. Fungal Genet Biol 48:685–695CrossRefGoogle Scholar
  19. Idnurm A, Reedy JL, Nussbaum JC, Heitman J (2004) Cryptococcus neoformans virulence gene discovery through insertional mutagenesis. Eukaryot Cell 3:420–429CrossRefGoogle Scholar
  20. Idnurm A, Verma S, Corrochano LM (2010) A glimpse into the basis of vision in the kingdom Mycota. Fungal Genet Biol 47:881–892CrossRefGoogle Scholar
  21. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168Google Scholar
  22. Klassen JL (2010) Phylogenetic and evolutionary patterns in microbial carotenoid biosynthesis are revealed by comparative genomics. PLoS One 5:e11257CrossRefGoogle Scholar
  23. Krügel H, Fiedler G, Smith C, Baumberg S (1993) Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei. Gene 127:127–131CrossRefGoogle Scholar
  24. Lawrence GJ, Dodds PN, Ellis JG (2010) Transformation of the flax rust fungus, Melampsora lini: selection via silencing of an avirulence gene. Plant J 61:364–369CrossRefGoogle Scholar
  25. Lima G, Spina AM, Castoria R, De Curtis F, De Cicco V (2005) Integration of biocontrol agents and food-grade additives for enhancing protection of stored apples from Penicillium expansum. J Food Prot 68:2100–2106Google Scholar
  26. Liu Y, Koh CMJ, Sun L, Hlaing MM, Du M, Peng N, Ji L (2012) Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1 and development of genetic transformation method by dominant selection in oleaginous yeast Rhodosporidium toruloides. Appl Microbiol Biotechnol. doi: 10.1007/s00253-012-4223-9.
  27. Marchand G, Fortier E, Neveu B, Bolduc S, Belzile F, Bélanger RR (2007) Alternative methods for genetic transformation of Pseudozyma antarctica, a basidiomycetous yeast-like fungus. J Microbiol Methods 70:519–527CrossRefGoogle Scholar
  28. McClelland CM, Chang YC, Kwon-Chung KJ (2005) High frequency transformation of Cryptococcus neoformans and Cryptococcus gattii by Agrobacterium tumefaciens. Fungal Genet Biol 42:904–913CrossRefGoogle Scholar
  29. McCluskey K, Wiest A, Plamann M (2010) The Fungal Genetics Stock Center: a repository for 50 years of fungal genetics research. J Biosci 35:119–126CrossRefGoogle Scholar
  30. McCluskey K, Wiest A (2011) The Fungal Genetics Stock Center in the context of a world wide community of ex situ fungal germplasm repositories. Fungal Biol Rev 25:143–150CrossRefGoogle Scholar
  31. Romano N, Macino G (1992) Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6:3343–3353CrossRefGoogle Scholar
  32. Sampaio JP, Gadanho M, Santos S, Duarte FL, Pais C, Fonseca A, Fell JW (2001) Polyphasic taxonomy of the basidiomycetous yeast genus Rhodosporidium: Rhodosporidium kratochvilovae and related anamorphic species. Int J Syst Evol Microbiol 51:687–697Google Scholar
  33. Toffaletti DL, Rude TH, Johnston SA, Durack DT, Perfect JR (1993) Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J Bacteriol 175:1405–1411Google Scholar
  34. Toyn JH, Gunyuzlu PL, White WH, Thompson LA, Hollis GF (2000) A counterselection for the tryptophan pathway in yeast: 5-fluoroanthranilic acid resistance. Yeast 16:553–660CrossRefGoogle Scholar
  35. Tully M, Gilbert HJ (1985) Transformation of Rhodosporidium toruloides. Gene 36:235–240CrossRefGoogle Scholar
  36. Tuon FF, Costa SF (2008) Rhodotorula infection. A systematic review of 128 cases from literature. Rev Iberoam Micol 25:135–140CrossRefGoogle Scholar
  37. Valério E, Gadanho M, Sampaio JP (2008) Reappraisal of the Sporobolomyces roseus species complex and description of Sporidiobolus metaroseus. Int J Syst Evol Microbiol 58:736–741CrossRefGoogle Scholar
  38. Walton FJ, Idnurm A, Heitman J (2005) Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans. Mol Microbiol 57:1381–1396CrossRefGoogle Scholar
  39. Wang X, Wang P, Sun S, Darwiche S, Idnurm A, Heitman J (2012) Transgene induced co-suppression during vegetative growth in Cryptococcus neoformans. PLoS Genet 8:e1002885CrossRefGoogle Scholar
  40. Winston F, Dollard C, Ricupero-Hovasse SL (1995) Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11:53–55CrossRefGoogle Scholar
  41. Xin G, Glawe D, Doty SL (2009) Characterization of three endophytic, indole-3-acetic acid-producing yeasts occurring in Populus trees. Mycol Res 113:973–980CrossRefGoogle Scholar
  42. Yamazaki M, Komagata K (1983) An electrophoretic comparison of enzymes of ballistosporogenous yeasts. J Gen Appl Microbiol 29:115–143CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Erika P. Abbott
    • 1
  • Giuseppe Ianiri
    • 1
    • 2
  • Raffaello Castoria
    • 2
  • Alexander Idnurm
    • 1
  1. 1.Division of Cell Biology and Biophysics, School of Biological SciencesUniversity of Missouri—Kansas CityKansas CityUSA
  2. 2.Dipartimento di Agricoltura, Ambiente e AlimentiUniversità del MoliseCampobassoItaly

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