1. Introduction
Site-directed and random mutagenesis have been useful tools in molecular biology. The application of directed mutagenesis in medically important fungi has been limited by the availability of molecular genetic techniques. Even species in which efficient genetic transformation methodologies exist, mutagenesis approaches were sparsely used due to diploidism. Lack of genetic tools hindered understanding of virulence mechanisms of medically important fungi. With the arrival of whole-genome sequencing, as well as improved techniques of genetic manipulation, the ability to address these questions is improving. A comprehensive review of mutagenesis in pathogenic fungi is outside the scope of this review, so not all studies were included. The intent of this review is to educate the reader on applications of site-directed and random insertional mutagenesis in medically important fungi in order to provide ideas for novel approaches to address major issues in pathogenic fungal research.
2. Site-directed mutagenesis
Site-directed mutagenesis has been exploited to understand signaling pathways, mechanisms of drug resistance, and identification of promoter DNA binding sites. Applications used less frequently have included protein localization and function of specific genes. In most instances, the site of the mutation was selected due to homology to model species or mammalian genes.
2.1. Signaling pathways
The most commonly reported application of site-directed mutants is the construction of dominant-negative and dominant-active alleles. The ability to make dominant-active alleles is particularly useful in diploid strains, since both endogenous alleles do not have to be disrupted. The amino acids chosen for mutation are often based on homology to
2.1.1. Phosphomimetics
The introduction of an amino acid substitution so the residue acts as constitutively phosphorylated or non-phosphorylated is a common technique to study cellular processes. Phosphomimetics have been used to study MAPK, cAMP-PKA, calcineurin, and two–component signaling, as well as cytokinesis and the heat shock response, in medically important fungi (Bockmühl and Ernst, 2001; Fox and Heitman, 2005; Hicks et al., 2005; Li et al., 2008; Menon et al., 2006; Nicholls et al., 2011).
The cAMP-PKA pathway regulates multiple cellular processes in eukaryotes. cAMP levels are regulated by phosphodiesterases (
Putative phosphorylated residues have not always been identified previously in model fungi. Consequently,
Unlike previous studies, target residues in the two-component response regulator, Ssk1p, were identified by sequence comparison to a bacterial response regulator (Menon et al., 2006). Invariant aspartic acid residues were substituted using site-directed mutagenesis. This study demonstrated that phosphorylation of two different residues affects regulation of different cellular processes involved in virulence (Menon et al., 2006).
2.1.2. G protein signaling
Another common application for site-directed mutagenesis has been G protein signaling. In
As in
2.2. Mechanisms of drug resistance
Many fungal species present antifungal drug resistance
The azoles interfere with ergosterol biosynthesis by targeting lanosterol 14 alpha-demethylase (
The most studied gene is
2.3. Promoter response elements
Mutagenesis is a common approach to identify DNA binding sites in promoters. Nested deletions are probably the most commonly reported method used in the medically important fungi. Site-directed mutagenesis has been used to introduce point mutations in the
In another study, the authors wanted to identify the promoter binding sites in the pH responsive gene,
Site-directed mutagenesis and ChIP have also been used to identify the genes that a specific transcription factor binds. A good illustration is the gain-of-function allele of the transcription factor
2.4. Gene function-essential genes
Surprisingly, site-directed mutagenesis has not been used extensively to determine the function of a gene. Gene disruption is routinely the method of choice to study gene function. However, this is not possible when studying the function of essential genes. The use of conditional promoters is often used. Results can sometimes be misleading, since phenotypic testing is performed under suboptimal growth conditions due to promoter-dependent nutritional constraints. Even so, expression of an essential gene under a conditional reporter does not allow complete analysis of multifunctional genes. Very few studies have taken advantage of directed mutagenesis of a specific gene.
The first report of the use of site-directed mutagenesis of an essential and multifunctional gene was the signaling regulatory gene,
Site-directed mutagenesis was also used to decipher the role of the hemoglobin response gene (
Essential genes are often prospective drug targets. One such gene is
2.5. Gene function – genes with multiple functions
The transcriptional regulator,
2.6. Other applications
Site-directed mutagenesis has been used to determine how GPI-tagged proteins discriminate between localization to the plasma membrane and cell wall (Mao et al., 2008). N and C termini of cell wall or plasma proteins were fused to GFP. The termini were subjected to truncation and mutagenesis. Localization of mutant alleles was examined by microscopy. One potential pitfall, however, is that the GFP tag itself can cause protein mislocalization. These experiments identified the omega cleavage site. Further domain exchange and mutagenesis studies identified which residues dictated cell wall or plasma membrane localization (Mao et al., 2008).
2.7. Methodology
Site-directed mutagenesis (
3. Insertional mutagenesis
Insertional mutagenesis methods are commonly used in model fungi species. Although genomes are similar between model and medically important fungal species, there are still significant differences. Forward screens (screens for new genes that are involved in a phenotype, often using homologs) in model fungi will not identify genes important for pathogenesis, since these species are usually attenuated in virulence or are avirulent. Signaling pathways are shared among fungi, but downstream targets and regulation vary. It is estimated that only 61% of the essential genes in
3.1. Selection of insertion mutants by complementation of auxotrophy
Initial studies used complementation of auxotrophy as a ‘mutagen.’ Auxotrophic strains were transformed with plasmids carrying an auxotrophic marker (
3.2. Signature-tagged mutagenesis (STM)
Signature-tagged mutagenesis is a method originally designed to identify genes required for pathogenesis (Hensel et al., 1995). A large number of mutants were created by insertional mutagenesis. The inserted DNA includes a unique oligonucleotide tag that resembles a ‘barcode.’ In principle, up to 96 mutants can be inoculated into one host; strains not recovered are thought to harbor a mutation specific for
The first studies were performed prior to the identification of useful transposons. In order to identify virulence factors in
Many of the medically important fungi can cause different types of infections and/or colonize and infect multiple organs. Unlike bacteria, they do not have true ‘virulence factors’; but they do have virulence “attributes.“ Since
Another important parameter is the number of strains that can be injected into a mouse and have an equal opportunity to survive. Nelson et al. (2001) did a prescreen with hygromycin and G418 resistant strains (100:1) and ascertained that it was possible to inoculate 100 strains. However, studies with hybridization signals showed that they could not reliably detect more than 80 strains. Experiments were performed with pools of 48 strains. Six hundred seventy-two mutants were screened, and 39 gave different output signals. Twenty-four of the mutants were tested singly in the mouse, and 6 of these had significant changes in virulence (Nelson et al., 2001). Brown et al. (2000) determined that subsequent hybridization efficiency was 80%, so, although they used pools of 96, they always inoculated 2 mice per pool (Brown et al., 2000). In total 4648 tagged strains were screened, and 35 strains (0.8%) gave weak signals in the output pool after two rounds of STM. These strains were tested in a competitive inhibition infection, in which the attenuated strain was present as 50% of the inoculum. Nine strains showed a competitive disadvantage, and two of these demonstrated significantly reduced virulence. The site of the mutation of one strain was not identifiable; the second mutation was upstream of the PABA synthetase gene. Further analysis confirmed that
Cormack et al. (1999) exploited STM to construct a mutant library in
3.3. Transposon-mediated insertional mutagenesis
Transposon technology has been used in pathogenic fungi to construct libraries, add epitope tags, and understand cellular processes. The technology has been adapted for diploid organisms using the parasexual cycle, haploid insufficiency, and homologous recombination (Carr et al., 2010; Davis et al., 2002; Firon et al., 2003; Juarez-Reyes et al., 2011; Spreghini et al., 2003; Uhl et al., 2003). The use of transposons has superseded auxotrophic and STM approaches.
Essential genes are often considered good drug targets. Firon et al. (2003) exploited the parasexual cycle to develop a transposon-mediated insertional mutagenesis protocol to identify essential genes in
Uhl et al. (2003) developed a transposon mutant library in
Davis et al. (2002) constructed a transposon mutant library in
Spreghini et al. (2003) exploited transposon mutagenesis to add an epitope to the putative cell wall protein, Dfg5p. Since conventional epitope tagging of amino and carboxyl termini was not an option, they wanted to identify an internal site which, when disrupted with a tag, did not compromise function. The Tn
In the transposon examples above, mutagenesis was performed
A novel use of random insertion was the analysis of subtelomeric silencing of
3.4. Agrobacterium
T -DNA
Prior to T-DNA mutagenesis, insertional mutagenesis was attempted by electroporation or biolistic transformation of naked DNA. Researchers have developed protocols that have improved the efficiency of transformation using T-DNA in
T-DNA was used to identify genes in
Marion et al. (2006) performed a more comprehensive analysis of insertional mutagenesis in
The use of T-DNA in
3.5. Methodologies to identify the site of insertion
3.5.1,. Two-step PCR
In the first step of two-step PCR (Chun et al., 1997), sequence on one side of the insertion site is amplified with a degenerate primer and a primer homologous to the sequence in one of the ends of the inserted DNA. (There are two end-specific primers. A primer specific for only one end is used. Note that a tranposon can insert in either orientation.) The degenerate primer contains 20 nucleotides of defined sequence at the 5’-end, 10 nucleotides of degenerate sequence (
3.5.2. Thermal asymmetric interlaced PCR (TAIL PCR)
TAIL PCR (Liu and Whittier, 1995) is another method to identify sequences flanking insertions. It is a modified version of hemispecific (one-sided) PCR. The purpose is to favor amplification of the desired product. It uses specific primers homologous to DNA in the integrating cassette or plasmid and a degenerate primer that can anneal to the gDNA flanking the insertion. The strategy is that the specific primers are long, nested, and have a high Tm; the degenerate primer is short and has a low Tm. The first five cycles are high stringency cycles to favor annealing to and linear amplification from the specific primer. Then there is one low stringency cycle to allow the degenerate primer to anneal. Because there are now several copies of the gDNA adjacent to the insertion, the chance of the degenerate primer annealing to the desired product is increased. However, other products might form from the primers finding additional annealing sites in the genome. Using a second and a third primer completely homologous to the inserted DNA will favor the desired product that is made from both the specific and degenerate primers instead of either one alone. This is accomplished by interlacing reduced stringency and high stringency cycles.
4. Closing remarks
Site-directed and insertional mutagenesis are techniques that can be used to advance our understanding of the pathogenesis of medically important fungi. The exploitation of these tools has resulted in a better understanding of drug-resistant mechanisms, transcription factors, signaling pathways and vital cellular processes. Site-directed mutagenesis could be better utilized to decipher the functions of essential and multi-functional genes. While all approaches cannot be used in the always-diploid strains, transposon-mediated insertional mutagenesis can be used to construct libraries. Additionally, T-DNA can be used to improve transformation efficiency in dimorphic fungi and in C. neoformans.
References
- 1.
Abuodeh R. O Orbach M. J Mandel M. A Das A Galgiani J. N 2000 Genetic Transformation of Coccidioides immitis Facilitated by Agrobacterium tumefaciens. Journal of Infectious Diseases. 181,6:2106 EOF 10 EOF - 2.
Le Pape,Alvarez-rueda N Fleury A Morio F Pagniez F Gastinel L 2011 Amino Acid Substitutions at the Major Insertion Loop of Candida albicans Sterol 14alpha-Demethylase Are Involved in Fluconazole Resistance. e21239. - 3.
Bassilana M Arkowitz R. A 2006 Rac1 and Cdc42 Have Different Roles in Candida albicans Development Eukaryotic Cell. 5,2: 321-329. - 4.
Bockmühl D. P Ernst J. F 2001 A Potential Phosphorylation Site for an A-Type Kinase in the Efg1 Regulator Protein Contributes to Hyphal Morphogenesis of Candida albicans. 1523 EOF 30 EOF - 5.
Brakhage A. A Langfelder K 2002 MENACING MOLD: The Molecular Biology of Aspergillus fumigatus. - 6.
Brandhorst T. T Rooney P. J Sullivan T. D Klein B. S 2002 Using new genetic tools to study the pathogenesis of Blastomyces dermatitidis 25 EOF 30 EOF - 7.
Jr., Holden, D. W.,Brown J. S Aufauvre-brown A Brown J Jennings J. M Arst H 2000 Signature-tagged and directed mutagenesis identify PABA synthetase as essential for Aspergillus fumigatus pathogenicity Mol Microbiol. 36,6:1371 EOF 1380 EOF - 8.
Cardoza R. E Vizcaino J. A Hermosa M. R Monte E Gutierrez S 2006 A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation. J Microbiol. 44,4:383 EOF 95 EOF - 9.
M. J.,Carr P. D Tuckwell D Hey P. M Simon L d Enfert C Birch M Oliver J. D Bromley 2010 The Transposon impala Is Activated by Low Temperatures: Use of a Controlled Transposition System To Identify Genes Critical for Viability of Aspergillus fumigatus. 438 EOF 48 EOF - 10.
Chun K. T Edenberg H. J Kelley M. R Goebl M. G 1997 Rapid Amplification of Uncharacterized Transposon-tagged DNA Sequences from Genomic DNA. Yeast. 13,3:233 EOF 40 EOF - 11.
Cognetti D Davis D Sturtevant J 2002 The Candida albicans 14-3-3 gene, BMH1, is essential for growth. Yeast. 19,1: 55-67. - 12.
Cormack B. P Ghori N Falkow S 1999 An adhesin of the yeast pathogen Candida glabrata mediating adherence to human epithelial cells. Science. 285,5427: 578-582. - 13.
Davis D. A Bruno V. M Loza L Filler S. G Mitchell A. P 2002 Candida albicans Mds3p, a Conserved Regulator of pH Responses and Virulence Identified Through Insertional Mutagenesis. Genetics. 162,4: 1573-1581. - 14.
Dobrowolska A Staczek P 2009 Development of transformation system for Trichophyton rubrum by electroporation of germinated conidia. Curr Genet. 55,5: 537-542. - 15.
Edwards J. A Zemska O Rappleye C. A 2011 Discovery of a Role for Hsp82 in Histoplasma Virulence through a Quantitative Screen for Macrophage Lethality. Infect. Immun. 79,8: 3348-3357. - 16.
gene function test in diploid Candida albicans. J Bacteriol. 182,20: 5730-5736.Enloe B Diamond A Mitchell A. P 2000 A Single-transformation - 17.
Erickson T Liu L Gueyikian A Zhu X Gibbons J Williamson P. R 2001 Multiple virulence factors of Cryptococcus neoformans are dependent on VPH1. Mol Microbiol. 42,4: 1121-1131. - 18.
Feng Q Summers E Guo B Fink G 1999 Ras Signaling Is Required for Serum-Induced Hyphal Differentiation in Candida albicans. Journal of Bacteriology. 181,20: 6339-6346. - 19.
C.,Firon A Villalba F Beffa R d Enfert 2003 Identification of Essential Genes in the Human Fungal Pathogen Aspergillus fumigatus by Transposon Mutagenesis. Eukaryotic Cell. 2,2: 247-255. - 20.
Fox D. S Heitman J 2005 Calcineurin-Binding Protein Cbp1 Directs the Specificity of Calcineurin-Dependent Hyphal Elongation during Mating in Cryptococcus neoformans. Eukaryotic Cell. 4,9: 1526-1538. - 21.
Gauthier G. M Sullivan T. D Gallardo S. S Brandhorst T. T Wymelenberg A. J. V Cuomo C. A Suen G Currie C. R Klein B. S 2010 SREB, a GATA transcription factor that directs disparate fates in Blastomyces dermatitidis including morphogenesis and siderophore biosynthesis. PLoS Pathogens. 6,4: 1-16. - 22.
Green B Bouchier C Fairhead C Craig N Cormack B 2012 Insertion site preference of Mu, Tn5, and Tn7 transposons. Mobile DNA. 3,1: 3. - 23.
Hensel M Shea J. E Gleeson C Jones M. D Dalton E Holden D. W 1995 Simultaneous identification of bacterial virulence genes by negative selection. Science. 269,5222: 400-403. - 24.
Phosphodiesterase Modulates Cyclic AMP Levels through a Protein Kinase A-Mediated Negative Feedback Loop in Cryptococcus neoformans. Eukaryotic Cell. 4,12: 1971-1981.Hicks J. K Bahn Y S Heitman J 2005 Pde - 25.
Idnurm A Reedy J. L Nussbaum J. C Heitman J 2004 Cryptococcus neoformans Virulence Gene Discovery through Insertional Mutagenesis. Eukaryotic Cell. 3,2: 420-429. - 26.
De Las Penas, A., Castano, I.,Juarez-reyes A 2011 Analysis of subtelomeric silencing in Candida glabrata. Methods Mol Biol.734 279 301 - 27.
Kakeya H Miyazaki Y Miyazaki H Nyswaner K Grimberg B Bennett J. E 2000 Genetic analysis of azole resistance in the Darlington strain of Candida albicans. Antimicrob Agents Chemother. 44,11: 2985-2990. - 28.
mediates pathways associated with virulence in Candida albicans. Microbiology. 155,PtKelly M. N Johnston D. A Peel B. A Morgan T. W Palmer G. E Sturtevant J. E 2009 Bmh p 5 1536 1546 - 29.
Krysan P Young J Sussman M 1999 T-DNA as an insertionalmutagen in Arabidopsis. Plant Cell. 11,12: 2283- 2290. - 30.
Kummasook A Cooper C. R Vanittanakom N 2010 An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei. Medical Mycology. 48,8: 1066-1074. - 31.
Lamb D. C Kelly D. E Schunck W. H Shyadehi A. Z Akhtar M Lowe D. J Baldwin B. C Kelly S. L 1997 The mutation T315A in Candida albicans sterol 14alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity. J Biol Chem. 272,9: 5682-5688. - 32.
Lamb D. C Kelly D. E Baldwin B. C Kelly S. L 2000 Differential inhibition of human CYP3A4 and Candida albicans CYP51 with azole antifungal agents. Chem Biol Interact. 125,3: 165-175. - 33.
Laskowski M. C Smulian A. G 2010 Insertional mutagenesis enables cleistothecial formation in a non-mating strain of Histoplasma capsulatum. BMC Microbiology. 10. - 34.
Lenardon M Lesiak I Munro C Gow N 2009 Dissection of the Candida albicans class I chitin synthase promoters. Molecular Genetics and Genomics. 281,4: 459-471. - 35.
Li C. R Wang Y. M Wang Y 2008 The IQGAP Iqg1 is a regulatory target of CDK for cytokinesis in Candida albicans. EMBO J. 27,22: 2998-3010. - 36.
Liu Y. G Whittier R. F 1995 Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from1 and YAC clones for chromosome walking. Genomics. 25,3: 674-681. - 37.
Magrini V Goldman W. E 2001 Molecular mycology: a genetic toolbox for Histoplasma capsulatum. Trends in Microbiology. 9,11: 541-546. - 38.
and Downstream Regulation of Asexual Development in Aspergillus fumigatus. Eukaryotic Cell. 5,10: 1585-1595.Mah J H Yu J. -H 2006 Upstream - 39.
Mao Y Zhang Z Gast C Wong B 2008 C-Terminal Signals Regulate Targeting of Glycosylphosphatidylinositol-Anchored Proteins to the Cell Wall or Plasma Membrane in Candida albicans. Eukaryotic Cell. 7,11: 1906-1915. - 40.
Marion C. L Rappleye C. A Engle J. T Goldman W. E 2006 An alpha-(1,4)-amylase is essential for alpha-(1,3)-glucan production and virulence in Histoplasma capsulatum. Mol Microbiol. 62,4: 970-983. - 41.
Mellado E Garcia-effron G Alcazar-fuoli L Melchers W. J Verweij P. E Cuenca-estrella M Rodriguez-tudela J. L 2007 A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations. Antimicrob Agents Chemother. 51,6: 1897-1904. - 42.
Menon V Li D Chauhan N Rajnarayanan R Dubrovska A West A. H Calderone R 2006 Functional studies of the Ssk1p response regulator protein of Candida albicans as determined by phenotypic analysis of receiver domain point mutants. Molecular Microbiology. 62,4: 997-1013. - 43.
Le Pape,Morio F Loge C Besse B Hennequin C 2010 Screening for amino acid substitutions in the Candida albicans Erg11 protein of azole-susceptible and azole-resistant clinical isolates: new substitutions and a review of the literature. Diagn Microbiol Infect Dis. 66,4: 373-384. - 44.
Nelson R. T Hua J Pryor B Lodge J. K 2001 Identification of virulence mutants of the fungal pathogen Cryptococcus neoformans using signature-tagged mutagenesis. Genetics. 157,3: 935-947. - 45.
MacCallum, D. M., Kaffarnik, F. A., Selway, L., Peck, S. C., Brown, A. J.,Nicholls S 2011 Activation of the heat shock transcription factor Hsf1 is essential for the full virulence of the fungal pathogen Candida albicans. Fungal Genet Biol. 48,3: 297-305. - 46.
Noffz C. S Liedschulte V Lengeler K Ernst J. F 2008 Functional Mapping of the Candida albicans Efg1 Regulator. Eukaryotic Cell. 7,5: 881-893. - 47.
Jr., Solis, N., Filler, S. G., Mitchell, A.Norice C. T Smith F. J 2007 Requirement for Candida albicans Sun41 in Biofilm Formation and Virulence. Eukaryotic Cell. 6,11: 2046-2055. - 48.
Palmer G. E Johnson K. J Ghosh S Sturtevant J 2004 Mutant alleles of the essential 14-3-3 gene in Candida albicans distinguish between growth and filamentation. Microbiology. 150,Pt6 1911 1924 - 49.
Palmer G. E Sturtevant J. E 2004 Random mutagenesis of an essential Candida albicans gene. Curr Genet. 46,6: 343-356. - 50.
Park H Liu Y Solis N Spotkov J Hamaker J Blankenship J. R Yeaman M. R Mitchell A. P Liu H Filler S. G 2009 Transcriptional Responses of Candida albicans to Epithelial and Endothelial Cells. Eukaryotic Cell. 8,10: 1498-1510. - 51.
Peterson A. W Pendrak M. L Roberts D. D 2011 ATP Binding to Hemoglobin Response Gene 1 Protein Is Necessary for Regulation of the Mating Type Locus in Candida albicans. Journal of Biological Chemistry. 286,16: 13914-13924. - 52.
Prasannan P Suliman H. S Robertus J. D 2009 Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans. Biochemical and Biophysical Research Communications. 382,4: 730-734. - 53.
Ramon A. M Fonzi W. A 2003 Diverged Binding Specificity of Rim101p, the Candida albicans Ortholog of PacC. Eukaryotic Cell. 2,4: 718-728. - 54.
Sanchez-martinez C Perez-martin J 2002 Gpa2, a G-Protein alpha Subunit Required for Hyphal Development in Candida albicans. Eukaryotic Cell. 1,6: 865-874. - 55.
Sanglard D Ischer F Koymans L Bille J 1998 Amino acid substitutions in the cytochrome450 lanosterol 14alpha-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents. Antimicrob Agents Chemother. 42,2: 241-253. - 56.
Schmalhorst P. S Krappmann S Vervecken W Rohde M Muller M Braus G. H Contreras R Braun A Bakker H Routier F. H 2008 Contribution of Galactofuranose to the Virulence of the Opportunistic Pathogen Aspergillus fumigatus. Eukaryotic Cell. 7,8: 1268-1277. - 57.
Sheng C Chen S Ji H Dong G Che X Wang W Miao Z Yao J Lü J Guo W Zhang W 2010 Evolutionary trace analysis of CYP51 family: implication for site-directed mutagenesis and novel antifungal drug design. Journal of Molecular Modeling. 16,2: 279-284. - 58.
Jr.,Smulian A. G Gibbons R. S Demland J. A Spaulding D. T Deepe G. S 2007 Expression of Hygromycin Phosphotransferase Alters Virulence of Histoplasma capsulatum. Eukaryotic Cell. 6,11: 2066-2071. - 59.
Snelders E Karawajczyk A Verhoeven R. J. A Venselaar H Schaftenaar G Verweij P. E Melchers W. J. G 2011 The structure-function relationship of the Aspergillus fumigatus cyp51A L98H conversion by site-directed mutagenesis: The mechanism of L98H azole resistance. Fungal Genetics and Biology. 48,11. - 60.
Spreghini E Davis D. A Subaran R Kim M Mitchell A. P 2003 Roles of Candida albicans Dfg5p and Dcw1p Cell Surface Proteins in Growth and Hypha Formation. Eukaryotic Cell. 2,4: 746-755. - 61.
Sullivan T Rooney P Klein B 2002 Agrobacterium tumefaciens integrates transfer DNA into single chromosomal sites of dimorphic fungi and yields homokaryotic progeny from multinucleate yeast. Eukaryot Cell. 1,6: 895- 905. - 62.
Uhl M. A Biery M Craig N Johnson A. D 2003 Haploinsufficiency-based large-scale forward genetic analysis of filamentous growth in the diploid human fungal pathogen C. albicans. EMBO J. 22,11: 2668-2678. - 63.
VandenBerg A. L., Ibrahim, A. S., Edwards, J. E., Jr., Toenjes, K. A., Johnson, D. I.,2004 Cdc42p GTPase Regulates the Budded-to-Hyphal-Form Transition and Expression of Hypha-Specific Transcripts in Candida albicans. Eukaryotic Cell. 3,3: 724-734. - 64.
Yamada T Makimura K Satoh K Umeda Y Ishihara Y Abe S 2009 Agrobacterium tumefaciens-mediated transformation of the dermatophyte, Trichophyton mentagrophytes: an efficient tool for gene transfer. Med Mycol. 47,5: 485-494. - 65.
Zhang P Xu B Wang Y Li Y Qian Z Tang S Huan S Ren S 2008 Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the fungus Penicillium marneffei. Mycological Research. 112,8: 943-949. - 66.
M.,Znaidi S Barker K. S Weber S Alarco A M Liu T. T Boucher G Rogers P. D Raymond 2009 Identification of the Candida albicans Cap1p Regulon. Eukaryotic Cell. 8,6: 806-820.