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 pCB1539AReported previously (Carroll et al. 1994 Fungal Genet. Newsl. 41:22)
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*#
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