Skip Navigation

Genetic Transformation of Coccidioides immitis Facilitated by Agvobactevium tumefaciens

  1. Raed O. Abuodeh1,2,4,a,
  2. Marc J. Orbach1,3,
  3. M. Alejandra Mandel1,3,
  4. Anath Das5 and
  5. John N. Galgiani1,2,4
  1. 1Valley Fever Center for Excellence, Tucson
  2. 2Medical and Research Services, Southern Arizona Veterans Affairs Health Care System, Tucson
  3. 3Department of Plant Pathology, College of Agriculture, Tucson
  4. 4Department of Internal Medicine, College of Medicine, University of Arizona, Tucson
  5. 5Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul
  1. Reprints or correspondence: Dr. John N. Galgiani, Valley Fever Center for Excellence (1-111), 3601 S. Sixth Ave., Tucson, AZ 85723 (spherule{at}u.arizona.edu).

  1. Presented in part: 100th annual meeting of the American Society for Microbiology, Los Angeles, 24 May 2000.

  • ↵a Present affiliation: College of Health Sciences, University of Sharjah, Sharjah, United Arab Emirates.

 
Next Section

Abstract

Agrobacterium tumefaciens was used to facilitate genetic transformation of Coccidioides immitis. A gene cassette containing the gene encoding hygromycin phosphotransferase (hph) was cloned into a T-DNA vector plasmid and introduced into A. tumefaciens, and the resultant strain was used for cocultivation with germinated arthroconidia. This procedure produced numerous colonies 60- to >500-fold more resistant to hygromycin than untransformed mycelia. Both polymerase chain reaction and Southern blot analysis of the transformants indicated that all contained hph, usually as a single genomic copy. A transformation frequency of 1 per 105 arthroconidia was obtained by varying the germination time prior to cocultivation and altering the bacterium : fungus ratio. This approach requires no special equipment that might complicate biocontainment. Furthermore, transformation does not require digestion of fungal cell walls, further simplifying this procedure. A. tumefaciens-facilitated transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for C. immitis and perhaps other fungi of medical importance.

Genetic analysis of Coccidioides immitis would undoubtedly contribute to our understanding of the disease it causes [1]. Progress in this regard, however, has been impeded by the lack of methods to transform this fungus efficiently. For example, although Yu and Cole succeeded in introducing DNA into C. immitis by use of a biolistic method [2], their studies produced only 3 transformants.

We have approached this problem by borrowing a commonly used technique from plant geneticists. Agrobacterium tumefaciens is a Gram-negative bacterium that causes crown galls by transferring a part of its tumor-inducing (Ti) plasmid DNA into the plant nuclear genome [3]. The transferred DNA (T-DNA) is flanked by a 24-bp imperfect direct repeat sequence known as the border sequence, and the intervening DNA can be as much as 150 kb in size. A second region of the Ti-plasmid, the vir region, is also essential for DNA transfer. Recently, A. tumefaciens-facilitated gene transfer was extended to filamentous fungi [4, 5]. Stimulated by these reports, we tested this approach for transforming C immitis. Our success, combined with the relative simplicity of the procedure, makes A. tumefaciens-facilitated transformation appealing for this and perhaps other medically important fungi.

Previous SectionNext Section

Materials and Methods

Fungal and bacterial strains

Arthroconidia (5 × 107 or 5 × 108) from C. immitis (strain Silveira) [6] were inoculated into 100 mL of glucose yeast extract (GYE) liquid medium in 500-mL Erlenmeyer flasks and allowed to germinate at 37°C with agitation for 24 h or other time periods, as indicated in the text. Germlings were collected by centrifugation, resuspended, and used in numbers as indicated in the Results. For selected experiments, spheroplasts were made from germlings by enzymatic digestion of fungal cell walls. All A. tumefaciens strains used were derivatives of A. tumefaciens EHA105 [7].

Plasmid construction

The gene encoding hygromycin phosphotransferase (hph) was cloned into the transforming plasmid in 2 steps. The hph cassette was first cloned between T-DNA borders. A second fragment was then added that contained a wide-hostrange replicon and a mutant virG gene to allow plasmid maintenance in A. tumefaciens and inducer-independent expression of the vir genes, respectively [8]. These steps were carried out as follows.

The plasmid pAD1310 is an intermediate Ti-plasmid vector that contains the octopine Ti-plasmid pTiA6 left and right border regions of the T-DNA. Plasmid pAD1308 was constructed by deleting the EcoRI-BamHI polylinker region of plasmid pUC118. pAD1310 was constructed by cloning the 3.5-kb HindIII fragment of pEndI [9] into plasmid pAD1308.

For the construct used in most studies, the hph cassette was derived from plasmid pMP6 [10]. This cassette, as a 2.9-kb Hin dIII fragment, contained a modified cpc-1 promoter of Neurospora crassa, the hph gene from Escherichia coli, and the Aspergillus nidulans trp C terminator [10]. After its ends were filled in, using the Klenow enzyme, the hph cassette was cloned into the filled-in BamHI site of plasmid pAD1310 to produce pRATi2.9. For comparison, another hph cassette was derived from plasmid pCB1004 and contained the E. coli hph gene under the control of the A. nidulans trp C promoter [11]. This was isolated as a 1.4-kb HpaI fragment and cloned into the filled-in BamHI site of plasmid pAD1310 to produce pRATi1.4.

After pRATi2.9 and pRATil.4 were constructed, a 7.5-kb fragment from plasmid pAD1378 [12] was inserted into each. This fragment contained the wide-host-range replicon 75△2 and the constitutive vir gene mutant virGN54D [8]. The 7.5-kb EcoRI fragment of pAD1378 was cloned into the SalI site of plasmid pRATi2.9 and the XbaI site of plasmid pRATi1.4 to construct plasmids pAD1625 and pAD1624, respectively. Plasmids pAD1625 and pAD1624 were introduced into A. tumefaciens EHA105 by electroporation [13] to construct A. tumefaciens A974 and A973, respectively.

Procedure for transforming C. immitis

A. tumefaciens A973 or A974 was grown overnight in 2 mL of agrobacterium broth (AB) liquid medium with carbenicillin and tetracycline at 28°C with shaking, to A600 1.0–1.5. An aliquot (0.25 mL) of this culture was diluted 15–20-fold in induction medium and grown for another 24 h, until an A600 of 0.5–1.0 was obtained. The bacteria were collected by centrifugation at 4000 rpm for 10 min, washed once, and resuspended in 0.5 mL of induction medium (AB medium plus 25 mM 2-[N-morpholino]ethanesulfonic acid, pH 5.8).

Arthroconidial germlings were mixed in sterile microfuge tubes with A. tumefaciens in varying ratios. The cell mixtures were centrifuged briefly, the supernatant was removed, and the pellet was dispersed with a sterile spatula and placed on sterile 0.45-μm nitrocellulose filters on solid induction medium and incubated for 2 days at room temperature.

Selection of transformants

After incubation, filters were transferred to tubes with 1.0 mL of normal saline. Cells were dislodged and were plated on GYE agar containing 200 μM cefotaxime or 50 μg/mL kanamycin for counterselection against A. tumefaciens and containing hygromycin at concentrations as indicated in Results. Plates were incubated at 37°C until discrete colonies emerged. They were enumerated, and representative colonies were subcultured for further study.

Analysis of transformants

Genomic DNA [6] was analyzed by polymerase chain reaction (PCR), using primers specific for hph or for a specific gene of C. immitis [6]. The 2 reactions were carried out simultaneously. Southern blot analyses were performed with genomic DNA from putative transformants and untransformed C. immitis. The 4 probes used were the hph gene cassette, the p-Bluescript vector, and the T-DNA left and right border fragments.

Previous SectionNext Section

Results

Transformation

Cocultivation of A. tumefaciens A973 or A974 with 1 × 107 C. immitis germlings (grown for 24 h), with or without prior spheroplasting, resulted in the growth of discrete fungal colonies on selection plates containing hygromycin (10 μg/mL). Transformation with A. tumefaciens A973 produced 22 colonies (10 from spheroplasts and 12 from non-spheroplasted germlings), and that with A974 produced 38 colonies (26 from spheroplasts and 12 from nonspheroplasted germlings). When subcultured to solid medium containing higher concentrations of hygromycin, all putative transformants were resistant to⩾150 μg/mL, and 46 were resistant to 500 μg/mL (the highest concentration tested). All 14 putative transformants with lower resistance (150 or 300 μ/mL hygromycin) were derived from cocultivation with A. tumefaciens A973, in which hph is under the control of the trpC promoter. In control experiments, spontaneous resistance to hygromycin (2.5 μg/mL) was not observed.

Mitotic stability of all 60 putative transformants was tested by growth for 3 days in liquid GYE with hygromycin (10 μg/mL, 37°C with shaking), subculture onto GYE agar without hygromycin for 2 weeks, and screening for resistance by growth on GYE agar containing 150, 300, or 500 μg/mL of hygromycin. The level of resistance to hygromycin after this procedure was found to be identical to that obtained previously.

Molecular characterization of transformant strains

To study whether the hph gene was present in the transformants, genomic DNA from 23 putative transformants, obtained using A. tumefaciens A974, was analyzed by PCR. A band specific for hph was observed with DNA from all transformants but not with DNA from untransformed C. immitis (figure 1A).

Figure 1
View larger version:
Figure 1

Molecular analysis of hygromycin-resistant transformants of Coccidioides immitis. A, Polymerase chain reaction (PCR) analysis of transformant DNAs. DNA of the untransformed recipient strain Silveira and 23 hygromycin-resistant strains was subjected to PCR analysis using oligos (forward, 5′-AGTGTCACGTTGCAAGACCTGCCA-3′; reverse, 5′-GTGACGGTGTCGTCCATCACAGTA-3′) that should generate a 221-bp hygromycin phosphotransferase (hph) gene band (open arrowhead) and oligos (forward, 5′-CGTTCTCGTCCGTTAGAC-3′; reverse, 5′-CA-GCACCTGGGAACTCAGCT-3′) that should generate a 731-bp band from the gene encoding a proline-rich antigen specific for C. immitis (dosed arrowhead). Strains were generated through cocultivation of Agrobacterium tumefaciens with 24-h C. immitis germlings, either without (lanes 1–12) or with (lanes 13–23) enzymatic digestion of the fungal cell wall. The 50-μL PCR reaction solution contained 0.2 mM of each dNTP, 1.5 mM MgCl2, 1 nM of each primer, 0.5 U Taq-2000 polymerase (Statagene, La Jolla, CA), and 100 ng of template DNA. The reaction conditions were 94°C for 3 min (94°C for 1 min, 55°C for 2 min, and 72°C for 2 min), 35 cycles, and 72°C for 7 min. B, Probing of hygromycin-resistant C. immitis strain DNAs with the hph gene. DNAs of the same 23 hygromycin-resistant strains in panel A were digested with Eco RI, electrophoresed, and transferred to a nylon membrane. The DNAs in lanes 4 and 6 failed to be digested. The filter was probed with an hph gene probe. Probes were labeled with 32P dCTP. The DNAs are in the same order as in panel A. C, Diagrammatic representation of a transferred DNA (T-DNA) insertion in a C. immitis chromosome. The thin lines represent C. immitis genomic DNA, and the thick black line represents T-DNA sequences. The left border (LB) and right border (RB) are indicated by boxes. The hygromycin-resistance cassette is likewise indicated by boxes: the Neurospora crassa cpc-1 promoter (Vcpc-1) and the Aspergillus nidulans trpC terminator (TtrpC) are indicated by open boxes, and the hygromycin-resistance gene is indicated by a cross-hatched box. Restriction enzyme sites indicated include EcoRI (E), BgIII (Bg), BamHI (B), and HindIII (H). The variability in the positions of the C. immitis-flanking EcoRI sites is represented by the zigzag between them and the T-DNA ends. The lines below the map indicate the size of the predicted bands from the hph gene (as an Hpal fragment of pCB1004) and the LB and RB probes, which were isolated from pAD1310. The LB probe is a 1.6-kb EcoRI-HindIII fragment that corresponds to nucleotides 602–2212 of T-DNA (GenBank accession no. X00493), and the RB probe is a 0.3-kb BamHI-SacI fragment that corresponds to nucleotides 13780–14090 of the T-DNA.

Southern blot hybridization analysis was performed to study the nature of the transforming DNA insertions. If transformation was due to mobilization of the T-DNA from A. tumefaciens, followed by insertion into the C. immitis genome, only those sequences between the T-DNA borders will be transferred. The simplest fate of the transferred DNA would be a single insertion of the T-DNA, as diagrammed in figure 1C. Although all transformant DNAs hybridized to the hph gene probe, none exhibited homology to plasmid vector sequences (data not shown), which indicates that transformation occurred via T-DNA-mediated transfer of DNA. With the hph gene probe (figure 1B), 17 of the 21 samples in which the EcoRI digestion was successful had 2 bands, as would be expected with a single T-DNA insertion (figure 1C). One of the bands was a 2.3-kb internal T-DNA fragment that included most of the hph gene and the terminator (figure 1C). The second band in each transformant represents a junction fragment that includes the cpc-1 promoter, the T-DNA right border, and adjacent C. immitis genomic DNA. The variability in the size of this fragment is due to differences in the size of flanking genomic DNA. That the junction fragments vary in size among the different transformants indicates that the transforming DNA integrated into diverse sites within the C. immitis genome. The hybridization patterns of 4 transformants gave results suggesting a more complex integration event. In 2 of them, 2 bands were present, but the 2.3-kb internal band was absent, which suggests a rearrangement of the T-DNA (figure 1B, lanes 1 and 9). Two other transformants had >2 hybridizing bands, which suggests that >1 copy of the T-DNA had been inserted. The hph probe did not hybridize to DNA from untransformed C. immitis (figure 1B).

Hybridization with the right border fragment identified, in each transformant, ⩾1 bands that comigrated with the variable band(s) seen with the hph gene probe (data not shown). Hybridization with the left border probe (figure 1C) also indicated that the majority of transformants had a single T-DNA insertion, with others having a more complex pattern (data not shown).

Effects of other factors on transformation frequency

Both germination time and the ratio of A. tumefaciens to germlings had a major effect on the transformation frequency (table 1). Arthroconidia germinated for 5 h prior to cocultivation produced no transformants, whereas those germinated for 24 h and 48 h prior to cocultivation produced 2 and 51 colonies, respectively, from cells resulting from germinating 5 × 106 arthroconidia. Also evident was that changing the germling : bacteria ratio from 1 : 1 to 1 : 500 led to a 10-fold increase in transformation efficiency.

Table 1
View larger version:
Table 1

Effect of mycelial germination times and the ratio of bacteria to germlings on the frequency of hygromycin-resistant colonies.

Previous SectionNext Section

Discussion

In this report, we demonstrate a simple and efficient method for transforming C. immitis by use of A. tumefaciens-mediated DNA transfer. Transformation with A. tumefaciens requires a minimum of manipulations and no special equipment. These features make the technique very attractive for studies with C. immitis and potentially with other pathogenic fungi that must be handled with special biocontainment procedures. The plasmid constructs that we used had 2 mutations, one that permits constitutive expression of all vir genes and another that allows high plasmid copy numbers [8]. These features improved transformation efficiency in plants [14] and may be responsible for the greater frequency of transformants and higher degree of resistance produced by A974. A. tumefaciens A974 produced greater hygromycin resistance than did A973. From other studies, we expect this finding is due to differences in promoters used to construct the hyg cassettes [10]. In most transformants, Southern blot analysis demonstrated that the transforming DNA inserted into the C. immitis as a single T-DNA fragment, and, in all cases, transformation resulted in stable genomic integration of hyg and phenotypic resistance to hygromycin.

A significant increase in transformation efficiency was observed by either extending the germination time or increasing the ratio of bacteria to mycelia. Cultures that were germinated for 48 h resulted in mycelial mats that were difficult to disperse into homogeneous suspensions. Therefore, germinating arthroconidia for 24 h may be preferable. On the other hand, we did not determine a maximum ratio of A. tumefaciens to germlings. It is possible that the use of more bacteria may further improve transformation efficiency.

In addition to the introduction of foreign genes, use of A. tumefaciens-facilitated transformation can facilitate other molecular studies. For example, this approach could be the basis of insertional mutagenesis gene tagging in C. immitis, as has been done with other fungi [15]. Also, as with other fungi, the addition of homologous sequences to the transforming DNA may allow targeted gene disruption [3, 5]. Mycelia of C. immitis appear to be haploid [16]. Thus, single disruptions could possibly produce altered phenotypes. Because both of these applications could be useful to the study of other pathogenic fungi, our findings may not be restricted solely to C. immitis.

Previous SectionNext Section

Acknowledgments

We thank Maria Lourdes Lewis (Research Service, Southern Arizona Veterans Affairs Health Care System, Tucson) for maintenance of fungal cultures and production of genomic DNA. We thank also Dr. S. Gelvin (Department of Biological Sciences, Purdue University, West Lafayette, IN) for providing A. tumefaciens strain EHA105 for use in our studies.

Previous SectionNext Section

Footnotes

  • Financial support: US Department of Veterans Affairs; California Health Care Foundation; Purdue University (grant U.S.D.A. Prime 96-34340-2711).

  • Received December 13, 1999.
  • Revision received February 14, 2000.
Previous Section
 

References

  1. 1.↵
    1. Galgiani JN
    . Coccidioidomycosis: a regional disease of national importance; rethinking approaches for control. Ann Intern Med 1999;130:293-300.
    Abstract/FREE Full Text
  2. 2.↵
    1. Yu J-J,
    2. Cole GT
    . Biolistic transformation of the human pathogenic fungus Coccidioides immitis. J Microbiol Methods 1998;33:129-41.
    CrossRefWeb of Science
  3. 3.↵
    1. Biswas BB,
    2. Das A
    1. Das A
    . DNA transfer from Agrobacterium to plant cells in crown gall tumor disease. In: Biswas BB, Das A, editors. Plant microbe interactions. London: Plenum; 1998. p. 343-63.
  4. 4.↵
    1. de-Groot MJ,
    2. Bundock P,
    3. Hooykaas PJ,
    4. Beijersbergen AG
    . Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 1998;16:839-42.
    CrossRefMedlineWeb of Science
  5. 5.↵
    1. Gouka RJ,
    2. Gerk C,
    3. Hooykaas PJJ,
    4. Bundock P,
    5. Musters W,
    6. Verrips CT,
    7. et al
    . Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination. Nat Biotechnol 1999;17:598-601.
    CrossRefMedlineWeb of Science
  6. 6.↵
    1. Peng T,
    2. Orsborn KI,
    3. Orbach MJ,
    4. Galgiani JN
    . Proline-rich vaccine candidate antigen of Coccidioides immitis: conservation among isolates and differential expression with spherule maturation. J Infect Dis 1999;179:518-21.
    Abstract/FREE Full Text
  7. 7.↵
    1. Ritchie S,
    2. Lui C-N,
    3. Sellmer J,
    4. Kononowicz H,
    5. Hodges T,
    6. Gelvin S
    . Agrobacterium tumefaciens-mediated expression of gusA in maize tissues. Transgenic Res 1993;2:252-65.
    CrossRef
  8. 8.↵
    1. Gubba S,
    2. Xie Y-H,
    3. Das A
    . Regulation of Agrobacterium tumefaciens virulence gene expression: isolation of a mutation that restores virGD52E function. Mol Plant Microb Interact 1995;8:788-91.
    MedlineWeb of Science
  9. 9.↵
    1. Klee H,
    2. Yanofsky M,
    3. Nester EW
    . Vectors for transformation of higher plants. Bio/technology 1985;3:637-42.
    CrossRef
  10. 10.↵
    1. Orbach MJ
    . A cosmid with a HyR marker for fungal library construction and screening. Gene 1994;150:159-62.
    CrossRefMedlineWeb of Science
  11. 11.↵
    1. Carroll AM,
    2. Sweigard JA,
    3. Valent B
    . Improved vectors for selecting resistance to hygromycin. Fungal Genet Newslett 1994;41:22-22.
  12. 12.↵
    1. Das A,
    2. Anderson L,
    3. Xie Y-H
    . Delineation of the interaction domains of Agrobacterium tumefaciens Vir B7 and VirB9 by use of the yeast two-hybrid assay. J Bacteriol 1997;179:3404-9.
    Abstract/FREE Full Text
  13. 13.↵
    1. Mersereau M,
    2. Pazour G,
    3. Das A
    . Efficient transformation of Agrobacterium tumefaciens by electroporation. Gene 1990;90:149-51.
    CrossRefMedlineWeb of Science
  14. 14.↵
    1. Hansen G,
    2. Das A,
    3. Chilton M-D
    . Constitutive expression of the virulence genes improves the efficiency of plant transformation by Agrobacterium. Proc Natl Acad Sci USA 1994;91:7603-7.
    Abstract/FREE Full Text
  15. 15.↵
    1. Brown JS,
    2. Holden DW
    . Insertional mutagenesis of pathogenic fungi. Curr Opin Microbiol 1998;1:390-94.
    CrossRefMedlineWeb of Science
  16. 16.↵
    1. Reichard U,
    2. Yu J-J,
    3. Seshan KR,
    4. Cole GT
    . Transformation of Coccidioides immitis and molecular evidence for haploidy of this fungal pathogen [abstract F22]; Proceedings of the general meeting of the American Society for Microbiology; 30 May-3 June 1999; Chicago. Herndon, VA: ASM Press; 1999. p. 299.
| Table of Contents

This Article

  1. J Infect Dis. (2000) 181 (6): 2106-2110. doi: 10.1086/315525
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most