Sr36- and Sr5-Mediated Resistance Response to Puccinia graminis f. sp. tritici Is Associated with Callose Deposition in Wheat Guard Cells.

Race-specific resistance of wheat to Puccinia graminis f. sp. tritici is primarily posthaustorial and often involves the induction of a hypersensitive response (HR). The aim of this study was to investigate host defense responses induced in interactions between P. graminis f. sp. tritici races and wheat lines carrying different race-specific stem rust resistance (Sr) genes. In incompatible interactions between wheat lines carrying Sr36 in three genetic backgrounds (LMPG, Prelude, or W2691) and avirulent P. graminis f. sp. tritici races MCCFC or RCCDM, callose accumulated within 24 h in wheat guard cells contacted by a P. graminis f. sp. tritici appressorium, and P. graminis f. sp. tritici ingress was inhibited following appressorium formation. Accordingly, the expression of transcripts encoding a callose synthase increased in the incompatible interaction between LMPG-Sr36 and avirulent P. graminis f. sp. tritici race MCCFC. Furthermore, the inhibition of callose synthesis through the infiltration of 2-deoxy-D-glucose (DDG) increased the ability of P. graminis f. sp. tritici race MCCFC to infect LMPG-Sr36. A similar induction of callose deposition in wheat guard cells was also observed within 24 h after inoculation (hai) with avirulent P. graminis f. sp. tritici race HKCJC on LMPG-Sr5 plants. In contrast, this defense response was not induced in incompatible interactions involving Sr6, Sr24, or Sr30. Instead, the induction of an HR and cellular lignification were noted. The manifestation of the HR and cellular lignification was induced earlier (24 hai) and was more extensive in the resistance response mediated by Sr6 compared with those mediated by Sr24 or Sr30. These results indicate that the resistance mediated by Sr36 is similar to that mediated by Sr5 but different from those triggered by Sr6, Sr24, or Sr30. Resistance responses mediated by Sr5 and Sr36 are prehaustorial, and are a result of very rapid recognition of molecules derived from avirulent isolates of P. graminis f. sp. tritici, in contrast to the responses triggered in lines with Sr6, Sr24, and Sr30.

Marker-based cloning of the region containing the UhAvr1 avirulence gene from the basidiomycete barley pathogen Ustilago hordei.

Race-cultivar specialization during the interaction of the basidiomycete smut pathogen Ustilago hordei with its barley host was described in the 1940s. Subsequent genetic analyses revealed the presence of dominant avirulence genes in the pathogen that conform to the gene-for-gene theory. This pathosystem therefore presents an opportunity for the molecular genetic characterization of fungal genes controlling avirulence. We performed a cross between U. hordei strains to obtain 54 progeny segregating for three dominant avirulence genes on three differential barley cultivars. Bulked segregant analysis was used to identify RAPD and AFLP markers tightly linked to the avirulence gene UhAvr1. The UhAvr1 gene is located in an area containing repetitive DNA and this region is undetectable in cosmid libraries prepared from the avirulent parental strain. PCR and hybridization probes developed from the linked markers were therefore used to identify cosmid clones from the virulent (Uhavr1) parent. By walking on Uhavr1-linked cosmid clones, a nonrepetitive, nearby probe was found that recognized five overlapping BAC clones spanning 170 kb from the UhAvr1 parent. A contig of the clones in the UhAvr1 region was constructed and selected probes were used for RFLP analysis of the segregating population. This approach genetically defined an approximately 80-kb region that carries the UhAvr1 gene and provided cloned sequences for subsequent genetic analysis. UhAvr1 represents the first avirulence gene cloned from a basidiomycete plant pathogen.

Use of inter-simple sequence repeats and amplified fragment length polymorphisms to analyze genetic relationships among small grain-infecting species of ustilago.

ABSTRACT In the smut fungi, few features are available for use as taxonomic criteria (spore size, shape, morphology, germination type, and host range). DNA-based molecular techniques are useful in expanding the traits considered in determining relationships among these fungi. We examined the phylogenetic relationships among seven species of Ustilago (U. avenae, U. bullata, U. hordei, U. kolleri, U. nigra, U. nuda, and U. tritici) using inter-simple sequence repeats (ISSRs) and amplified fragment length polymorphisms (AFLPs) to compare their DNA profiles. Fifty-four isolates of different Ustilago spp. were analyzed using ISSR primers, and 16 isolates of Ustilago were studied using AFLP primers. The variability among isolates within species was low for all species except U. bullata. The isolates of U. bullata, U. nuda, and U. tritici were well separated and our data supports their speciation. U. avenae and U. kolleri isolates did not separate from each other and there was little variability between these species. U. hordei and U. nigra isolates also showed little variability between species, but the isolates from each species grouped together. Our data suggest that U. avenae and U. kolleri are monophyletic and should be considered one species, as should U. hordei and U. nigra.

The mating-type and pathogenicity locus of the fungus Ustilago hordei spans a 500-kb region.

The fungal pathogen Ustilago hordei causes the covered smut disease of barley and oats. Mating and pathogenicity in this fungus are controlled by the MAT locus, which contains two distinct gene complexes, a and b. In this study, we tagged the a and b regions with the recognition sequence for the restriction enzyme I-SceI and determined that the distance between the complexes is 500 kb in a MAT-1 strain and 430 kb in a MAT-2 strain. Characterization of the organization of the known genes within the a and b gene complexes provided evidence for nonhomology and sequence inversion between MAT-1 and MAT-2. Antibiotic-resistance markers also were used to tag the a gene complex in MAT-1 strains (phleomycin) and the b gene complex in MAT-2 strains (hygromycin). Crosses were performed with these strains and progeny resistant to both antibiotics were recovered at a very low frequency, suggesting that recombination is suppressed within the MAT region. Overall, the chromosome homologues carrying the MAT locus of U. hordei share features with primitive sex chromosomes, with the added twist that the MAT locus also controls pathogenicity.

The pheromone cell signaling components of the Ustilago a mating-type loci determine intercompatibility between species.

The MAT region of Ustilago hordei, a bipolar barley pathogen, harbors distinct mating functions (a and b loci). Here, we show that the b locus is essential for mating and pathogenicity, and can induce pathogenicity when introduced into a strain carrying a b locus of opposite specificity. Transformation experiments using components of the a1 locus and analysis of resulting dual mating phenotypes revealed that this locus harbors a pheromone receptor gene (Uhpra1) and a pheromone gene (Uhmfa1). These U. hordei a1 genes, when introduced by transformation, are necessary and sufficient to make U. maydis, a tetrapolar corn pathogen, intercompatible with U. hordei MAT-2, but not MAT-1, strains. U. hordei strains transformed with the U. maydis a1 locus also become intercompatible with U. maydis a2, but not a1, strains. The interspecies hybrids produced dikaryotic hyphae but were not fully virulent on either corn or barley. Partial, natural intercompatibility was shown to exist between the sugarcane smut U. scitaminea and both U. hordei and U. maydis. These results show that the signal transduction pathway for mating responses is conserved between different smut species. We conclude that, apart from intraspecies compatibility, the Ustilago a locus also dictates intercompatibility in this group of fungi.

Linkage of mating-type loci distinguishes bipolar from tetrapolar mating in basidiomycetous smut fungi.

Sexual compatibility requires self vs. non-self recognition. Genetically, two compatibility or mating-type systems govern recognition in heterothallic basidiomycete fungi such as the edible and woodrotting mushrooms and the economically important rust and smut phytopathogens. A bipolar system is defined by a single genetic locus (MAT) that can have two or multiple alleles. A tetrapolar system has two loci, each with two or more specificities. We have employed two species from the genus Ustilago (smut fungi) to discover a molecular explanation for the genetic difference in mating systems. Ustilago maydis, a tetrapolar species, has two genetically unlinked loci that encode the distinct mating functions of cell fusion (a locus) and subsequent sexual development and pathogenicity (b locus). We have recently described a b locus in a bipolar species, Ustilago hordei, wherein the existence of an a locus has been suspected, but not demonstrated. We report here the cloning of an allele of the a locus (a1) from U. hordei and the discovery that physical linkage of the a and b loci in this bipolar fungus accounts for the distinct mating system. Linkage establishes a large complex MAT locus in U. hordei; this locus appears to be in a region suppressed for recombination.

Three selectable markers for transformation of Ustilago maydis.

Although Ustilago maydis is readily amenable to molecular genetic experimentation, few antibiotic-resistance markers are available for DNA-mediated transformation. This poses constraints on experiments involving targeted gene disruption and complementation. To address this problem, we constructed vectors using one of three additional genes as dominant selectable markers for transformation. Two genes, sat-1 (encoding streptothricin acetyltransferase) and Sh-ble (encoding a phleomycin-resistance polypeptide), are of bacterial origin and have been engineered for expression in Ustilago sp. The third gene encodes an allele of U. maydis beta-tubulin that confers resistance to the fungicide benomyl.

Conservation of the b mating-type gene complex among bipolar and tetrapolar smut fungi.

In the phytopathogenic fungus Ustilago hordei, one locus with two alternate alleles, MAT-1 and MAT-2, controls mating and the establishment of the infectious dikaryon (bipolar mating). In contrast, for U. maydis, these functions are associated with two different gene complexes, called a and b (tetrapolar mating); the a complex has two alternate specificities, and the b gene complex is multiallelic. We have found homologs for the b gene complex in U. hordei and have cloned one from each mating type using sequences from one bEast allele of U. maydis as a probe. Sequence analysis revealed two divergent open reading frames in each b complex, which we called bW (bWest) and bE (bEast) in analogy with the b gene complex of U. maydis. The predicted bW and bE gene products from the two different mating types showed approximately 75% identity when homologous polypeptides were compared. All of the characterized bW and bE gene products have variable amino-terminal regions, conserved carboxy-terminal regions, and similar homeodomain motifs. Sequence comparisons with the bW1 and bE1 genes of U. maydis showed conservation in organization and structure. Transformation of the U. hordei b gene complex into a U. hordei strain of opposite mating type showed that the b genes from the two mating types are functional alleles. The U. hordei b genes, when introduced into U. maydis, rendered the haploid transformants weakly pathogenic on maize. These results indicate that structurally and functionally conserved b genes are present in U. hordei.

The a and b loci of Ustilago maydis hybridize with DNA sequences from other smut fungi.

The smut fungi are obligately parasitic during the sexual phase of their life cycle, and the mating-type genes of these fungi play key roles in both sexual development and pathogenicity. Among species of smut fungi it is common to find a bipolar mating system in which one locus with two alternate alleles is believed to control cell fusion and establishment of the infectious cell type. Alternatively, several species have a tetrapolar mating system in which two different genetic loci, one of which has multiple alleles, control fusion and subsequent development of the infection hyphae. Cloned sequences from the a and b mating-type loci of the tetrapolar smut fungus Ustilago maydis were used as hybridization probes to DNAs from 23 different fungal strains, including smut fungi with both tetrapolar and bipolar mating systems. In general, all of the smut fungi hybridized with the mating-type genes from U. maydis, suggesting conservation of the sequences involved in mating interactions. A selection of DNAs from other ascomycete and basidiomycete fungi failed to hybridize with the U. maydis mating-type sequences. Exceptions to this finding include hybridization of DNA from the a1 idiomorph of U. maydis to DNA from one strain of U. violacea and hybridization of both a idiomorphs to DNA from Saccharomyces cerevisiae.

T-DNA integration: a mode of illegitimate recombination in plants.

Transferred DNA (T-DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T-DNA-tagged genomic loci showed that T-DNA integration resulted in target site deletions of 29-73 bp. In those cases where integrated T-DNA segments turned out to be smaller than canonical ones, the break-points of target deletions and T-DNA insertions overlapped and consisted of 5-7 identical nucleotides. Formation of precise junctions at the right T-DNA border, and DNA sequence homology between the left termini of T-DNA segments and break-points of target deletions were observed in those cases where full-length canonical T-DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T-DNA segments showed no homology to break-points of target sequence deletions. Homology between short segments within target sites and T-DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T-DNA integration results from illegitimate recombination. The data suggest that while the left T-DNA terminus and both target termini participate in partial pairing and DNA repair, the right T-DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.

Recovery of Agrobacterium tumefaciens T-DNA molecules from whole plants early after transfer.

A system for the analysis of independent T-DNA transfer events from Agrobacterium to plants is described. The complete T-DNA except for the 25 bp border sequences was replaced by one genome of a plant virus so that upon transfer to the plant, a viable replicon is produced by circularization. Rescue of virus from such infected plants allowed analysis of DNA sequences at or close to the ends of T-DNA molecules. A rather conserved right border remnant of three nucleotides was found, whereas the sequences remaining at the left end were more variable. A point deletion in the left 25 bp sequence results in even less precise processing at the left end. In addition, many rescued T-DNA molecules carry small direct repeats between the joined T-DNA ends; linear T-DNA molecules are therefore transported to the plant.

Characteristics of trypanosome variant antigen genes active in the tsetse fly.

Trypanosoma brucei contains a repertoire of more than 100 different genes for Variant Surface Glycoproteins (VSGs). A small and strain-specific fraction of these genes is expressed in the salivary glands of the tsetse fly (M-genes), giving rise to metacyclic Variable Antigen Types (M-VATs). Antibodies produced in a chronic trypanosome infection initiated by syringe inoculation of bloodstream forms into mammals (i.e. against B-VATs), will react with most of the M-VATs suggesting that these B-VATs express VSG genes that are similar or identical to M-genes. We have cloned DNA complementary to the VSG mRNA of four of such B-VATs and used this to characterize the corresponding VSG genes. In three of the four VATs we find a single VSG gene hybridizing with the cDNA probe and we provide supporting evidence that this gene is expressed as an M-gene. In the bloodstream repertoire these genes appear to be activated by duplicative translocation to another telomere. In all four variants the putative M-genes are telomeric and in the three cases where the location of the genes on chromosome-sized DNA molecules could be determined, the genes were located in large DNA, whereas the majority of the telomeric VSG genes are in chromosomes less than 1000 kb. Our results are best explained by models for M-gene activation involving telomeric expression sites for these genes which are separate from those used by bloodstream forms. The implications of these results for vaccination are discussed.

Molecular cloning and functional characterization of Rhizobium leguminosarum structural nif-genes by site-directed transposon mutagenesis and expression in Escherichia coli minicells.

In order to study the structural organization and regulation of the expression of the nitrogenase gene cluster in Rhizobium leguminosarum PRE we selected relevant subfragments of the sym-plasmid from clone banks by homology with R. meliloti nif-genes. Site-directed Tn5 mutagenesis was applied to a nif DH-specific clone and subsequently the transposon insertions were transferred back into the wild-type rhizobial genome by homologous recombination. Phenotypic effects of Tn5 mutations in the region of the structural nif-genes were determined by measuring acetylene reduction in nodulated plants and by immunological analysis of bacteroid-specific proteins. The localization of Tn5 insertion sites was in accordance with observed consequences: two genotypically different Tn5-induced mutations within nif D caused repression of CI alpha and beta synthesis and a strong reduction of CII production, thus resulting in a Fix- phenotype. Expression of different cloned Rhizobium DNA inserts, bearing nif K, nif D, nif H, or nif DH, was achieved in Escherichia coli minicells dependent upon the presence of a strong upstream vector promoter sequence. Gene products were identified by immunoprecipitation with specific antisera. Endogenous rhizobial transcriptional start signals in one case (nif H) seemed to be recognized at a low rate by the E. coli system; in contrast, Rhizobium ribosome binding sites for all three structural nif-genes functioned normally in minicells. The approximate location of the coding regions for nif KDH genes was determined and found to be contiguous.