Generation of Trichoderma atroviride mutants with constitutively activated G protein signaling by using geneticin resistance as selection marker

BMC Research Notes

Susanne Zeilinger²  Theresa Radebner²  Carolina Escobar Rodrìguez²  Markus Omann¹  Sabine Gruber² 

[1] Current address: Zuckerforschung Tulln GmbH, Josef-Reiter-Strasse 21-23, Tulln, Austria;Research Area Molecular Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, Gumpendorferstrasse 1a, Wien, Austria

关键词: Mycoparasitism;    G protein signaling;    Geneticin;    Genetic transformation;    Trichoderma;    Fungi;   

Others  :  1165188
DOI  :  10.1186/1756-0500-5-641

 received in 2012-06-14, accepted in 2012-11-12,  发布年份 2012

【 摘 要 】

Background

Species of the fungal genus Trichoderma are important industrial producers of cellulases and hemicellulases, but also widely used as biocontrol agents (BCAs) in agriculture. In the latter function Trichoderma species stimulate plant growth, induce plant defense and directly antagonize plant pathogenic fungi through their mycoparasitic capabilities. The recent release of the genome sequences of four mycoparasitic Trichoderma species now forms the basis for large-scale genetic manipulations of these important BCAs. Thus far, only a limited number of dominant selection markers, including Hygromycin B resistance (hph) and the acetamidase-encoding amdS gene, have been available for transformation of Trichoderma spp. For more extensive functional genomics studies the utilization of additional dominant markers will be essential.

Results

We established the Escherichia coli neomycin phosphotransferase II-encoding nptII gene as a novel selectable marker for the transformation of Trichoderma atroviride conferring geneticin resistance. The nptII marker cassette was stably integrated into the fungal genome and transformants exhibited unaltered phenotypes compared to the wild-type. Co-transformation of T. atroviride with nptII and a constitutively activated version of the Gα subunit-encoding tga3 gene (tga3^Q207L) resulted in a high number of mitotically stable, geneticin-resistant transformants. Further analyses revealed a co-transformation frequency of 68% with 15 transformants having additionally integrated tga3^Q207L into their genome. Constitutive activation of the Tga3-mediated signaling pathway resulted in increased vegetative growth and an enhanced ability to antagonize plant pathogenic host fungi.

Conclusion

The neomycin phosphotransferase II-encoding nptII gene from Escherichia coli proved to be a valuable tool for conferring geneticin resistance to the filamentous fungus T. atroviride thereby contributing to an enhanced genetic tractability of these important BCAs.

【 授权许可】

   
2012 Gruber et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

【 参考文献 】

• [1]Druzhinina IS, Seidl-Seiboth V, Herrera-Estrella A, Horwitz BA, Kenerley CM, Monte E, Mukherjee PK, Zeilinger S, Grigoriev IV, Kubicek CP: Trichoderma: the genomics of opportunistic success. Nat Rev Microbiol 2011, 9(10):749-759.
• [2]Harman GE, Howell CR, Viterbo A, Chet I, Lorito M: Trichoderma species–opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2004, 2(1):43-56.
• [3]Kubicek CP, Herrera-Estrella A, Seidl-Seiboth V, Martinez DA, Druzhinina IS, Thon M, Zeilinger S, Casas-Flores S, Horwitz BA, Mukherjee PK, Mukherjee M, Kredics L, Alcaraz LD, Aerts A, Antal Z, Atanasova L, Cervantes-Badillo MG, Challacombe J, Chertkov O, McCluskey K, Coulpier F, Deshpande N, von Döhren H, Ebbole DJ, Esquivel-Naranjo EU, Fekete E, Flipphi M, Glaser F, Gómez-Rodríguez EY, et al.: Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma. Genome Biol 2011, 12(4):R40. BioMed Central Full Text
• [4]Verma M, Brar SK, Tyagi RD, Surampalli RY, Valero JR, et al.: Antagonistic fungi, Trichodrema spp.: panoply of biological control. Biochem Eng 2007, 37:1-20.
• [5]Brunner K, Omann M, Pucher ME, Delic M, Lehner SM, Domnanich P, Kratochwill K, Druzhinina I, Denk D, Zeilinger S: Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride. Curr Genet 2008, 54(6):283-299.
• [6]Mukherjee PK, Latha J, Hadar R, Horwitz BA: Role of two G-protein alpha subunits, TgaA and TgaB, in the antagonism of plant pathogens by Trichoderma virens. Appl Environ Microbiol 2004, 70:542-549.
• [7]Omann M, Lehner S, Escobar C, Brunner K, Zeilinger S: The 7-transmembrane receptor Gpr1 governs processes relevant for the antagonistic interaction of Trichoderma atroviride with its host. Microbiology 2012, 158:107-118.
• [8]Rocha-Ramirez V, Omero C, Chet I, Horwitz BA, Herrera-Estrella A: Trichoderma atroviride G-protein alpha-subunit gene tga1 is involved in mycoparasitic coiling and conidiation. Eukaryot Cell 2002, 1:594-605.
• [9]Reithner B, Brunner K, Schuhmacher R, Peissl I, Seidl V, Krska R, Zeilinger S: The G protein alpha subunit Tga1 of Trichoderma atroviride is involved in chitinase formation and differential production of antifungal metabolites. Fungal Genet Biol 2005, 42(9):749-760.
• [10]Zeilinger S, Reithner B, Scala V, Peissl I, Lorito M, Mach RL: Signal transduction by Tga3, a novel G protein alpha subunit of Trichoderma atroviride. Appl Environ Microbiol 2005, 71(3):1591-1597.
• [11]Schuster A, Schmoll M: Biology and Biotechnology of Trichoderma. Appl Microbiol Biotechnol 2010, 87(3):787-799.
• [12]Ruis-Diez B: Strategies for the transformation of filamentous fungi. J Appl Microbiol 2002, 92(2):189-195.
• [13]Padilla IMG, Burgos L: Aminoglycoside antibiotics: structure, functions and effects on in vitro plant culture and genetic transformation protocols. Plant Cell Rep 2010, 29:1203-1213.
• [14]Son H, Seo YS, Min K, Park AR, Lee J, Jin JM, Lin Y, Cao P, Hong SY, Kim EK, Lee SH, Cho A, Lee S, Kim MG, Kim Y, Kim JE, Kim JC, Choi GJ, Yun SH, Lim JY, Kim M, Lee YH, Choi YD, Lee YW: A phenome-based functional analysis of transcription factors in the cereal head blight fungus Fusarium graminearum. PLoS Pathog 2011, 7(10):e1002310.
• [15]Shimizu T, Ito T, Kanematsu S: Transient and multivariate system for transformation of a fungal plant pathogen, Rosellinia necatrix, using autonomously replicating vectors. Curr Genet 2012, 58(3):129-138.
• [16]Weiland JJ: Transformation of Pythium aphanidermatum to geneticin resistance. Current Genet 2003, 42:344-352.
• [17]Rodríguez-Sáiz M, Lembo M, Bertetti L, Muraca R, Velasco J, Malcangi A, de la Fuente JL, Barredo JL: Strain improvement for cephalosporin production by Acremonium chrysogenum using geneticin as a suitable transformation marker. FEMS Microbiol Lett 2004, 235(1):43-49.
• [18]Vijn I, Govers F: Agrobacterium tumefaciens-mediated transformation of the oomycete plant pathogen Phytophtora infestans. Mol Plant Pathol 2003, 4(6):459-467.
• [19]Namiki F, Matsunaga M, Okuda M, Inoue I, Nishi K, Fujita Y, Tsuge T: Mutation of an arginine biosynthesis gene causes reduced pathogenicity in Fusarium oxysporum f. sp. melonis. Mol Plant Microbe Interact. 2001, 14:580-584.
• [20]Masters SB, Miller RT, Chi MH, Chang FH, Beiderman B, Lopez NG, Bourne HR: Mutations in the GTP-binding site of GS alpha alter stimulation of adenylyl cyclase. J Biol Chem 1989, 264:15467.
• [21]Bae YS, Knudsen GR: Cotransformation of Trichoderma harzianum with beta-glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils. Appl Environ Microbiol 2000, 66:810.
• [22]Bowen J, Crowhurst R, Templeton M, Stewart A: Molecular markers for a Trichoderma harzianum biological control agent: Introduction of the hygromycin B resistance gene and the β‐glucuronidase gene by transformation. New Zeal J Crop Hort Sci 1996, 24:219-228.
• [23]Inglis PW, Queiroz PR, Valadares-Inglis MC: Transformation with green fluorescent protein of Trichoderma harzianum 1051, a strain with biocontrol activity against Crinipellis perniciosa, the agent of witches'-broom disease of cocoa. J Gen Appl Microbiol 1999, 45:63-67.
• [24]Sánchez-Torres P, González R, Pérez-González JA, González-Candelas L, Ramón D: Development of a transformation system for Trichoderma longibrachiatum and its use for constructing multicopy transformants for the egl1 gene. Appl Microbiol Biotechnol 1994, 41:440-446.
• [25]Steyaert JM, Weld RJ, Stewart A: Isolate-specific conidiation in Trichoderma in response to different nitrogen sources. Fungal Biol 2010, 114(2–3):179-188.
• [26]Do Nascimento Silva R, Steindorff AS, Ulhoa CJ, Felix CR: Involvement of G-alpha protein GNA3 in production of cell wall-degrading enzymes by Trichoderma reesei (Hypocrea jecorina) during mycoparasitism againstPythium ultimum. Biotechnol Lett 2009, 31:531-536.
• [27]Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A laboratory Manual. 2nd edition. New York: Cold Spring Harbor Laboratory Press; 1989.
• [28]Mullaney ER, Hamer JE, Roberti KA, Yelton MM, Timberlake WE: Primary structure of the trpC gene of Aspergillus nidulans. Mol Gen Genet 1985, 199:37-45.
• [29]Peterbauer CK, Lorito M, Hayes CK, Harman GE, Kubicek CP: Molecular cloning and expression of the nag1 gene (N-acetyl-β-D-glucosaminidase-encoding gene) from Trichoderma harzianum P1. Current Genet 1996, 30:325-331.
• [30]Berman DM, Wilkie TM, Gilman AG: GAIP and RGS4 Are GTPase-activating proteins for the Gi Subfamily of G protein α subunits. Cell 1996, 86:445-452.
• [31]Regenfelder E, Spellig T, Hartmann A, Lauenstein S, Bölker M, Kahmann R: G proteins in Ustilago maydis: transmission of multiple signals? EMBO J 1997, 16:1934-1942.
• [32]Segers GC, Nuss DL: Constitutively activated G [alpha] negatively regulates virulence, reproduction and hydrophobin gene expression in the chestnut blight fungus Cryphonectria parasitica. Fungal Genet Biol 2003, 38:198-208.
• [33]Peterbauer C, Litscher D, Kubicek C: The Trichoderma atroviride seb1 (stress response element binding) gene encodes an AGGGG-binding protein which is involved in the response to high osmolarity stress. Mol Genet Genomics 2002, 268:223-231.
• [34]Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CAMJJ: Transformation of Aspergillus nidulans based on the hygromycin B marker from Escherichia coli. Gene 1987, 56:117-124.