A series of vectors for fungal transformation

Jim Sweigard, Forrest Chumley, Anne Carroll, Lennie Farrall, and Barabara Valent - Central Research and Development, DuPont, P. O. Box 80402, Wilmington, DE 19880-0402 USA

We report a new fungal selectable marker that confers resistance to chlorimuron ethyl, a sulfonylurea herbicide. This gene as well as genes that confer resistance to hygromycin and bialaphos have been engineered to be compact and to eliminate sites for most common restriction enzymes. These three selectable markers have been used to construct a series of vectors for fungal transformation.

We have modified three dominant selectable markers for fungal transformation. First, we cloned a sulfonylurea resistant allele of the Magnaporthe grisea ILV1 gene using the Saccharomyces cerevisiae ILV2 gene as a heterologous probe. These genes encode acetolactate synthase, an enzyme involved in isoleucine and valine synthesis. Sulfonylureas (specifically chlorimuron ethyl, the active ingredient of the herbicide Classicreg.) inhibit acetolactate synthase. The sulfonylurea resistant allele of M. grisea ILV1 has been subcloned as a 2.8 kb fragment and modified by the elimination of eight restriction enzyme sites (GenBank AF013601). Second, a chimeric gene for bialaphos resistance (Pall and Brunelli 1993 Fungal Genet. Newsl. 40:59-63) has been further modified by eliminating five restriction enzyme sites and by removing the transcription terminator (GenBank AF013602). Third, a chimeric gene conferring hygromycin resistance (GenBank) has been reported previously (Carroll et al. 1994 Fungal Genet. Newsl. 41:22) and is included here for completeness. All three selectable markers have had SalI sites introduced at both ends.

The three selectable markers were cloned into various plasmids both within and outside the polylinker (Table 1). First, the genes were cloned as SalI fragments into the polylinker of pUC, pBluescriptII, and pBC vectors. They were also cloned as SalI fragments into the XhoI site of a modified polylinker in pBluescript II and pBC (pCB1519 and pCB1520, respectively) where the XhoI site is flanked on both sides by SmaI sites. Second, the selectable markers were cloned into common cloning vectors outside the polylinker, thus leaving the lacZ gene intact. Most of the restriction enzyme sites in these polylinkers are unique (Table 2). We find that these vectors allow flexible and facile cloning options due to the presence of three fungal selectable markers available as many different restriction fragments including blunt fragments, a range of polylinker choices with blue/white screening, and a choice of bacterial selection for ampicillin or chloramphenicol resistance. All of these plasmids have been deposited in the FGSC.

We have successfully used these vectors to transform M. grisea as described previously for hygromycin selection (Sweigard et al. 1995 Plant Cell 7:1221-1233). Defined complex medium [yeast nitrogen base without amino acids (Difco) 1.7 (g/l); asparagine, 2; NH4NO3, 1; glucose, 10; pH to 6.0 with Na2HPO4) was used to select for bialaphos and sulfonylurea resistance. Bialaphos (25 ug/ml) and chlorimuron ethyl (100 ug/ml) were dissolved in water and dimethylformamide, respectively, and added to media after autoclaving. Chlorimuron ethyl can be purchased from Chem Service, P. O. Box 3108, West Chester, PA 19381-3108, 610-692-3026. We have not tested whether the formulated herbicide Classicreg. can be used for selection instead of the technical material.

Table 1. Plasmids with bialaphos, hygromycin and sulfonylurea resistance

Base plasmid E /cloning site for         Baialaphos     Hygromycin     Sulfonylurea   
fungal selectable marker                 resistance     resistance     resistance     
Selectable marker in polylinker                                                       
pUC19& or pUC118*/SalI                   pCB1517&       pCB1003 & A     pCB 1528*      
pBluescript II SK-/ SalI                 pCB1635        pCB1636        pCB1637(NOT AVAILABLE)        
pBC KS-/ SalI                            pCB1546        pCB1490        pCB1551        
pCB1519/XhoI                             pCB1524B             C         not made       
pCB1520/XhoI                             pCB1525B             D         pCB1550B       
Selectable marker outside lacZ gene                                                   
pBluescript II SK-& or KS+*/SspIF        pCB1530&       pCB1179*       pCB1532&       
pBC KS-& or pBC SK+/BclIG                pCB1531        pCB1004A*       pCB1533&        
pLitmus 28/HpaIF                         pCB1534        pCB1535        pCB1536        
pLitmus 39/HpaIF                         pCB1537        pCB1538        pCB1539        

AReported previously (Carroll et al. 1994 Fungal Genet. Newsl. 41:22)
B Digestion of these plasmids with SmaI yields the respective selectable marker as a blunt restriction fragment
C, D The hygromycin resistant equivalents of these plasmids were not constructed because a blunt fragment can be obtained by HpaI digestion of pCB1003 and pCB1004, respectively.
E pBluescript II and pBC, Stratagene; pLitmus, New England Biolabs; pCB#, this paper. All the base plasmids are ampicillin resistant except pBC KS- and pCB1520 which are chloramphenicol resistant.
F Selectable marker obtained as SmaI fragment from pCB1525 or pCB1550 or as HpaI fragment from pCB1004.
G Selectable marker cloned as SalI fragment (half-site filled in) into the BclI site (half-site filled in)

Table 2. Restriction enzyme sites in the polylinkers of plasmids with intact lacZ geneA

Vector Polylinker   Polylinker Restriction EnzymesB                                        
SK or KS in pBC     SstI ^ SstII* NotI XbaI SpeI BamHI SmaI PstI EcoRI EcoRV HindIII       
or pBluescript      ClaI SalI  XhoI ApaI KpnI                                              

Litmus 28           SnaBI SpeI BglII NsiI BssHII^ BsiWI XhoI EcoRI PstI EcoRV BamHI        
                    HindIII NcoI^# AatII^# AgeI^ XbaI AvrII SstI^ KpnI StuI^ AflII*#       

Litmus 39           SnaBI SpeI SalI SphI*^ I BsrGI BspEI# MluI^ EagI*# NheI^ EcoRI BamHI   
                    EcoRV PstI HindIII KasI^ NgoMI*^ MfeI^ ApaI StuI^ AflII*#              

A These are the plasmids in the second part of Table 1
B All restriction enzyme sites in the vector polylinker are unique for the entire plasmid including the fungal selectable marker except for those sites indicated where a restriction site is also present in the fungal selectable marker for bialaphos (*), hygromycin (#), or sulfonylurea (^) resistance.

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