James A. Sweigard

Improved Vectors for Selecting Resistance to Hygromycin

Resistance to hygromycin B is an important dominant selectable marker in fungal transformation. Our goal was to improve vectors for hygromycin selection by making the gene more compact, by eliminating sites for commonly used restriction enzymes, and by subcloning the modified gene into convenient vectors. These improvements were made by modifying pCSN43 (Staben et al. 1989 Fungal Genetics Newsl. 36:79-81) through three rounds of megaprimer mutagenesis (Aiyar and Leis, 1993 Biotechniques 14:366-368 ), a technique based on polymerase chain reaction amplification. Plasmid pCSN43 has a 2.4 kb SalI fragment containing the bacterial hph gene (Gritz and Davies, 1983 Gene 25:179-188), encoding hygromycin B phosphotransferase, under control of the Aspergillus nidulans trpC promoter and terminator (Mullaney et al. 1985 MGG 199:37-45) (Fig. 1a).

Identification, cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus.

Genetic analysis of host specificity in the rice blast fungus (Magnaporthe grisea) identified a single gene, PWL2 (for Pathogenicity toward Weeping Lovegrass), that exerts a major effect on the ability of this fungus to infect weeping lovegrass (Eragrostis curvula). The allele of the PWL2 gene conferring nonpathogenicity was genetically unstable, with the frequent appearance of spontaneous pathogenic mutants. PWL2 was cloned based on its map position. Large deletions detected in pathogenic mutants guided the gene cloning efforts. Transformants harboring the cloned PWL2 gene lost pathogenicity toward weeping lovegrass but remained fully pathogenic toward other host plants. Thus, the PWL2 host species specificity gene has properties analogous to classical avirulence genes, which function to prevent infection of certain cultivars of a particular host species. The PWL2 gene encodes a glycine-rich, hydrophilic protein (16 kD) with a putative secretion signal sequence. The pathogenic allele segregating in the mapping population, pwl2-2, differed from PWL2 by a single base pair substitution that resulted in a loss of function. The PWL2 locus is highly polymorphic among rice pathogens from diverse geographic locations.

Magnaporthe grisea Pth11p Is a Novel Plasma Membrane Protein That Mediates Appressorium Differentiation in Response to Inductive Substrate Cues

Mutagenesis of Magnaporthe grisea strain 4091-5-8 led to the identification of PTH11, a pathogenicity gene predicted to encode a novel transmembrane protein. We localized a Pth11–green fluorescent protein fusion to the cell membrane and vacuoles. pth11 mutants of strain 4091-5-8 are nonpathogenic due to a defect in appressorium differentiation. This defect is reminiscent of wild-type strains on poorly inductive surfaces; conidia germinate and undergo early differentiation events, but appressorium maturation is impaired. Functional appressoria are formed by pth11 mutants at 10 to 15% of wild-type frequencies, suggesting that the protein encoded by PTH11 (Pth11p) is not required for appressorium morphogenesis but is involved in host surface recognition. We assayed Pth11p function in multiple M. grisea strains. These experiments indicated that Pth11p can activate appressorium differentiation in response to inductive surface cues and repress differentiation on poorly inductive surfaces and that multiple signaling pathways mediate differentiation. PTH11 genes from diverged M. grisea strains complemented the 4091-5-8 pth11 mutant, indicating functional conservation. Exogenous activation of cellular signaling suppressed pth11 defects. These findings suggest that Pth11p functions at the cell cortex as an upstream effector of appressorium differentiation in response to surface cues.

Magnaporthe grisea Pathogenicity Genes Obtained Through Insertional Mutagenesis

We have initiated a mutational analysis of pathogenicity in the rice blast fungus, Magnaporthe grisea, in which hygromycin-resistant transformants, most generated by restriction enzyme-mediated integration (REMI), were screened for the ability to infect plants. A rapid primary infection assay facilitated screening of 5,538 transformants. Twenty-seven mutants were obtained that showed a reproducible pathogenicity defect, and 18 of these contained mutations that cosegregated with the hygromycin resistance marker. Analysis of eight mutants has resulted in the cloning of seven PTH genes that play a role in pathogenicity on barley, weeping lovegrass, and rice. Two independent mutants identified the same gene, PTH2, suggesting nonrandom insertion of the transforming DNA. These first 7 cloned PTH genes are described.

The CPKA gene of Magnaporthe grisea is essential for appressorial penetration

The rice blast fungus Magnaporthe grisea uses appressoria to penetrate into plant cells. Appressorium formation occurs following conidial germination on hydrophobic surfaces and may involve a cyclic AMP (cAMP)-dependent signaling mechanism. Recently, gene replacement mutants of CPKA, a gene encoding a proposed catalytic subunit of cAMP-dependent protein kinase A, were shown to be defective in appressorium formation, cAMP responsiveness, and lesion formation (T. K. Mitchell and R. A. Dean, Plant Cell, 7:1869–1878, 1995). Here we report a detailed phenotypic characterization of three cpkA mutants. cpkA mutants are dramatically reduced in pathogenicity toward healthy plants. However, the reduced pathogenicity does not appear to be due to a loss of appressorium formation. cpkA mutants are delayed in appressorium formation but form appressoria at the same level as wild-type strains over a 24-h period. Appressoria formed by cpkA mutants are fully melanized but are smaller than wild type and are defective in pen...

A series of vectors for fungal transformation

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. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol44/iss1/19 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 Classic reg .) 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, 610-692Published by New Prairie Press, 2017 3026. We have not tested whether the formulated herbicide Classic reg . 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 pCB1524 C not made pCB1520/XhoI pCB1525 D

The PWL host specificity gene family in the blast fungus Magnaporthe grisea.

The PWL2 gene, isolated from a Magnaporthe grisea rice pathogen, prevents this fungus from infecting a second host grass, weeping lovegrass. We have investigated the distribution of sequences homologous to PWL2 in M. grisea strains isolated from diverse grass species. Multiple PWL2 homologs with varying degrees of sequence homology were identified. The presence of PWL2 homologs does not correlate with an avirulent phenotype on weeping lovegrass in many cases: some strains were fully pathogenic on weeping lovegrass although they carry multiple PWL2 homologs. Three weakly hybridizing PWL2 homologs were cloned and characterized. One of these, the PWL1 gene previously identified by genetic analysis, functioned to prevent infection of weeping lovegrass. Cloned PWL3 and PWL4 genes were nonfunctional, although PWL4 became functional if its expression was driven by either the PWL1 or the PWL2 promoter. The PWL1, PWL2, and PWL3/PWL4 genes map to different genomic locations. The amino acid sequences of the predicted PWL1, PWL3, and PWL4 proteins have 75, 51, and 57% identity, respectively, to the PWL2 protein. Our studies indicate that PWL genes are members of a dynamic, rapidly evolving gene family.

Disruption of a Magnaporthe grisea cutinase gene.

SummaryUsing a one-step strategy to disrupt CUT1, a gene for cutinase, cut1− mutants were generated in two strains of Magnaporthe grisea. One strain, pathogenic on weeping lovegrass and barley and containing the arg3–12 mutation, was transformed with a disruption vector in which the Aspergillus nidulans ArgB+ gene was inserted into CUT1. Prototrophic transformants were screened by Southern hybridization, and 3 of 53 tested contained a disrupted CUT1 gene (cut1 : : ArgB+). A second strain, pathogenic on rice, was transformed with a disruption vector in which a gene for hyg B resistance was inserted into CUT1. Two of the 57 transformants screened by Southern hybridization contained a disrupted CUT1 gene (cut1:. Hyg). CUT1 mRNA was not detectable in transformants that contained a disrupted gene. Transformants with a disrupted CUT1 gene failed to produce a cutin-inducible esterase that is normally detected by activity staining on non-denaturing polyacrylamide gels. Enzyme activity, measured either with tritiated cutin or with p-nitrophenyl butyrate as a substrate, was reduced but not eliminated in strains with a disrupted CUT1 gene. The infection efficiency of the cut1− disruption transformants was equal to that of the parent strains on all three host plants. Lesions produced by these mutants had an appearance and a sporulation rate similar to those produced by the parent strains. We conclude that the M. grisea CUT1 gene is not required for pathogenicity.

Reef coral fluorescent proteins for visualizing fungal pathogens.

The fluorescent proteins AmCyan, ZsGreen, ZsYellow, and AsRed, modified versions of proteins identified recently from several Anthozoa species of reef corals, were expressed for the first time in a heterologous system and used for imaging two different fungal plant pathogens. When driven by strong constitutive fungal promotors, expression of these reef coral fluorescent proteins yielded bright cytoplasmic fluorescence in Fusarium verticillioides and Magnaporthe grisea, and had no detectable effect on either growth rate or the ability to cause disease. Differential intracellular localization of the fluorescent proteins resulted in the discrimination of many subcellular organelles by confocal and multi-photon microscopy, and facilitated monitoring of such details as organelle dynamics and changes in the permeability of the nuclear envelope. AmCyan and ZsGreen were sufficiently excited at 855 and 880 nm, respectively, to allow for time-resolved in planta imaging by two-photon microscopy.

Cloning and analysis of CUT1, a cutinase gene from Magnaporthe grisea

SummaryA gene from Magnaporthe grisea was cloned using a cDNA clone of the Colletotrichum gloeosporioides cutinase gene as a heterologous probe; the nucleotide sequence of a 2 kb DNA segment containing the gene has been determined. DNA hybridization analysis shows that the M. grisea genome contains only one copy of this gene. The predicted polypeptide contains 228 amino acids and is homologous to the three previously characterized cutinases, showing 74% amino acid similarity to the cutinase of C. gloeosporioides. Comparison with previously determined cutinase sequences suggests that the gene contains two introns, 115 and 147 bp in length. The gene is expressed when cutin is the sole carbon source but not when the carbon source is cutin and glucose together or glucose alone. Levels of intracellular and extracellular cutinase activity increase in response to growth in the presence of cutin. The activity level is higher in a transformant containing multiple copies of the cloned gene than in the parent strain. Non-denaturing polyacrylamide gels stained for esterase activity show a single major band among intracellular and extracellular proteins from cutin-grown cultures that is not present among intracellular and extracellular proteins prepared from glucose-grown or carbon-starved cultures. This band stains more intensely in extracts from the multicopy transformant than in extracts from the parent strain. We conclude that the cloned DNA contains a M. grisea gene for cutinase, which we have named CUT1.