Correction to: A comparative genomic analysis of putative pathogenicity genes in the host-specific sibling species Colletotrichum graminicola and Colletotrichum sublineola.

Following the publication of this article [1], the authors informed us of the following error.

A comparative genomic analysis of putative pathogenicity genes in the host-specific sibling species Colletotrichum graminicola and Colletotrichum sublineola.

BACKGROUND: Colletotrichum graminicola and C. sublineola cause anthracnose leaf and stalk diseases of maize and sorghum, respectively. In spite of their close evolutionary relationship, the two species are completely host-specific. Host specificity is often attributed to pathogen virulence factors, including specialized secondary metabolites (SSM), and small-secreted protein (SSP) effectors. Genes relevant to these categories were manually annotated in two co-occurring, contemporaneous strains of C. graminicola and C. sublineola. A comparative genomic and phylogenetic analysis was performed to address the evolutionary relationships among these and other divergent gene families in the two strains. RESULTS: Inoculation of maize with C. sublineola, or of sorghum with C. graminicola, resulted in rapid plant cell death at, or just after, the point of penetration. The two fungal genomes were very similar. More than 50% of the assemblies could be directly aligned, and more than 80% of the gene models were syntenous. More than 90% of the predicted proteins had orthologs in both species. Genes lacking orthologs in the other species (non-conserved genes) included many predicted to encode SSM-associated proteins and SSPs. Other common groups of non-conserved proteins included transporters, transcription factors, and CAZymes. Only 32 SSP genes appeared to be specific to C. graminicola, and 21 to C. sublineola. None of the SSM-associated genes were lineage-specific. Two different strains of C. graminicola, and three strains of C. sublineola, differed in no more than 1% percent of gene sequences from one another. CONCLUSIONS: Efficient non-host recognition of C. sublineola by maize, and of C. graminicola by sorghum, was observed in epidermal cells as a rapid deployment of visible resistance responses and plant cell death. Numerous non-conserved SSP and SSM-associated predicted proteins that could play a role in this non-host recognition were identified. Additional categories of genes that were also highly divergent suggested an important role for co-evolutionary adaptation to specific host environmental factors, in addition to aspects of initial recognition, in host specificity. This work provides a foundation for future functional studies aimed at clarifying the roles of these proteins, and the possibility of manipulating them to improve management of these two economically important diseases.

Genetic and physical mapping of a rice blast resistance locus, Pi-CO39(t), that corresponds to the avirulence gene AVR1-CO39 of Magnaporthe grisea.

We have identified, genetically mapped and physically delineated the chromosomal location of a new rice blast resistance locus, designated Pi-CO39(t). This locus confers resistance to Magnaporthe grisea isolates carrying the AVR1-CO39 avirulence locus. The AVR1-CO39 locus is conserved in non-rice (cereals and grasses)-infecting isolates of M. grisea, making Pi-CO39(t) useful for engineering M. grisea resistance in rice and other cereals. The resistance in the rice line CO39 was inherited as a single dominant locus in segregating populations derived from F(2) and F(3) crosses between disease-resistant (CO39) and susceptible (51583) rice genotypes. Microsatellite, RFLP and resistance gene analog (RGA) markers were used to map the Pi-CO39(t) locus to a 1.2-cM interval between the probenazole-responsive ( RPR1) gene (0.2 cM) and RFLP marker S2712 (1.0 cM) on the short arm of rice chromosome 11. RFLP markers G320 and F5003, and resistance gene analogs RGA8, RGA38 and RGACO39 were tightly linked to the Pi-CO39(t) locus (no recombination detected in a sample of ~2400 gametes). A large-insert genomic library of CO39 was constructed in the binary plant transformation vector pCLD04541. A library screen using RGA8, RGA38 and probes derived from the ends of CO39 clones, as well as BAC end probes from the corresponding locus in the rice cv. Nipponbare, resulted in the assembly of three CO39 contigs of 180 kb, 110 kb and 145 kb linked to the Pi-CO39(t) locus. A 650-kb contig was also constructed representing the susceptible locus, pi-CO39(t), in the Nipponbare genome. The two genomes are highly divergent with respect to additions, deletions and translocations at the Pi-CO39(t) locus, as revealed by the presence or absence of mapping markers.

Reusing nylon membranes for radioactive hybridizations.

We describe a procedure for recycling nylon hybridization membranes, enabling their repeated use for radioactive Southern hybridization analysis of different DNA samples. Following hybridization and probe removal, nylon membranes containing covalently linked DNAs were treated with 0.55% sodium hypochlorite. This destroyed the DNA, thereby preventing it from participating in further hybridization and enabling the membranes to be used subsequently for binding new DNA samples. With this procedure, we were able to reuse a single membrane as many as 13 times, with no detectable loss in signal. This method was shown to be effective for membranes supplied by three different manufacturers.

Chromosome walking to the AVR1-CO39 avirulence gene of Magnaporthe grisea: discrepancy between the physical and genetic maps.

The avrCO39 gene conferring avirulence toward rice cultivar CO39 was previously mapped to chromosome 1 of Magnaporthe grisea between cosegregating markers CH5-120H and 1.2H and marker 5-10-F. In the present study, this region of the chromosome was physically mapped using RecA-mediated Achilles' cleavage. Cleavage of genomic DNA sequences within CH5-120H and 5-10-F liberated a 610-kb restriction fragment, representing the physical distance between these markers. Chromosome walking was initiated from both markers but was curtailed due to the presence of repetitive DNA sequences and the absence of overlapping clones in cosmid libraries representing several genome equivalents. These obstacles were overcome by directly subcloning the target region after release by Achilles' cleavage and a contig spanning avrCO39 was thus assembled. Transformation of two cosmids into a virulent recipient strain conferred a cultivar-specific avirulence phenotype thus confirming the cloning of avrCO39. Meiotic crossover points were unevenly distributed across this chromosomal region and were clustered around the avrCO39 locus. A 14-fold variation in the relationship between genetic and physical distance was measured over the avrCO39 chromosomal region. Thus the poor correlation of physical to genetic distance previously observed in M. grisea appears to be manifested over relatively short distances.

New cosmid vectors for library construction, chromosome walking and restriction mapping in filamentous fungi.

New cosmid vectors were constructed for the ascomycete fungus, Magnaporthe grisea and the basidiomycete fungus, Ustilago maydis. These vectors are capable of transforming M. grisea at frequencies of up to 5 transformants/micrograms linear DNA and U. maydis at up to 25 transformants/microgram circular DNA for integrative transformation. In addition, 2800 transformants/microgram DNA are possible when using an autonomously replicating vector. Since the promoters used in these vectors function in other ascomycete and basidiomycete fungi, we anticipate that these vectors will be widely applicable.

MAGGY, a retrotransposon in the genome of the rice blast fungus Magnaporthe grisea.

Full-length copies of a previously described repetitive DNA sequence (CH2-8) were isolated from the genome of the Magnaporthe grisea strain 2539. One copy of the complete element was sequenced and found to resemble a gypsy-like LTR retrotransposon. We named this element MAGGY (MAGnaporthe GYpsy-like element). MAGGY contains two internal ORFs putatively encoding Gag, Pol and Env-like proteins which are similar to peptides encoded by retroelements identified in other filamentous fungi. MAGGY was found to be widely distributed among M. grisea isolates from geographically dispersed locations and different hosts. It was present in high copy number in the genomes of all nine rice-pathogenic isolates examined. By contrast, M. grisea strains isolated from other Gramineae were found to possess varying copy numbers of MAGGY and in some cases the element was completely absent. The wide distribution of MAGGY suggests that this element invaded the genome of M. grisea prior to the evolution of rice-specific form(s). It may since have been horizontally transmitted to other sub-specific groups. One copy of MAGGY, corresponding to the element we sequenced, was located at identical locations in the genomes of geographically dispersed strains, suggesting that this copy of the element is a relatively ancient insertion.

The Magnaporthe grisea DNA fingerprinting probe MGR586 contains the 3' end of an inverted repeat transposon.

The Magnaporthe grisea repeat (MGR) sequence MGR586 has been widely used for population studies of the rice blast fungus, and has enabled classification of the fungal population into hundreds of genetic lineages. While studying the distribution of MGR586 sequences in strains of M. grisea, we discovered that the plasmid probe pCB586 contains a significant amount of single-copy DNA. To define precisely the boundary of the repetitive DNA in pCB586, this plasmid and four cosmid clones containing MGR586 were sequenced. Only 740 bp of one end of the 2.6-bp insert in the pCB586 plasmid was common to all clones. DNA sequence analysis of cosmid DNA revealed that all the cosmids contained common sequences beyond the cloning site in pCB586, indicating that the repetitive DNA in the fingerprinting clone is part of a larger element. The entire repetitive element was sequenced and found to resemble an inverted repeat transposon. This putative transposon is 1.86 kb in length and has perfect terminal repeats of 42 bp, which themselves contain direct repeats of 16 bp. The internal region of the transposon possesses one open reading frame which shows similarity at the peptide level to the Pot2 transposon from M. grisea and Fot1 from Fusarium oxysporum. Hybridization studies using the entire element as a probe revealed that some strains of M. grisea, whose DNA hybridized to the pCB586 probe, entirely lacked MGR586 transposon sequences.

Genetic and physical mapping of telomeres in the rice blast fungus, Magnaporthe grisea.

Telomeric restriction fragments were genetically mapped to a previously described linkage map of Magnaporthe grisea, using RFLPs identified by a synthetic probe. (TTAGGG)3. Frequent rearrangement of telomeric sequences was observed in progeny isolates creating a potential for misinterpretation of data. Therefore a consensus segregation data set used to minimize mapping errors. TWelve of the 14 telomeres were found to be genetically linked to existing RFLP markers. Second-dimensional electrophoresis of restricted chromosomes confirmed these linkage assignments and revealed the chromosomal location of the two unlinked telomeres. We were thus able to assign all 14 M. grisea telomeres to their respective chromosome ends. The Achilles' cleavage (AC) technique was employed to determine that chromosome 1 markers 11 and CH5-120H were approximately 1.8 Mb and 1.28 Mb, respectively, from their nearest telomeres. RecA-AC was also used to determine that unlinked telomere 6 was approximately 530 kb from marker CH5-176H in strain 2539 and 580 kb in Guy11. These experiments indicated that large portions of some chromosome ends are unrepresented by genetic markers and provided estimates of the relationship of genetic to physical distance in these regions of the genome.

Genome organization of Magnaporthe grisea: genetic map, electrophoretic karyotype, and occurrence of repeated DNAs.

A genetic map of Magnaporthe grisea (anamorph=Pyricularia oryzae and P. grisea), the causal agent of rice blast disease, was generated from segregation data utilizing 97 RFLP markers, two isoenzyme loci and the mating type locus among progeny of a cross between parental strains Guy 11 and 2539. Of the seven chromosomes of M. Grisea, three were resolved by contour-clamped homogeneous electric field (CHEF) electrophoresis, while the remaining four migrated as two doublet bands. By utilizing differences between CHEF mobilities of unresolved chromosomes from the parental strains, Southern analysis with selected markers allowed the chromosomal assignment of all linkage groups. A small translocation involving 1 marker was found in the parental strains used to produce the segregating population from which the map was constructed. Nine classes of repetitive DNA elements were found in the genome of a fungal isolate pathogenic to rice. These occurred only a few times or not at all in the genomes of isolates showing reduced virulence on rice. One repetitive DNA was shown to have structural similarity to the Alu sequences found in primates, a sequence similarity to the copia-like elements of Drosophila, and peptide similarity to transposable elements found in Drosophila, other fungi, and higher plants.

Transformation frequencies are enhanced and vector DNA is targeted during retransformation of Leptosphaeria maculans, a fungal plant pathogen.

Leptosphaeria maculans, a fungal pathogen of Brassica spp., was successfully transformed with the vector pAN8-1, encoding phleomycin resistance. Protoplasts of a vigorous Phleor transformant were then retransformed using the partially homologous vector, pAN7-1 which encodes hygromycin B resistance. Retransformation of this strain to hygromycin resistance occurred at frequencies that were consistently twofold higher than with the original recipient strain. Linearised pAN7-1 DNA transformed phleomycin-resistant protoplasts at higher frequencies still. All the transformants that were tested retained a phleomycin-resistant phenotype (20/20). Molecular analysis of five transformants generated with circular pAN7-1 DNA indicated that in four cases the pAN7-1 vector had integrated into pAN8-1 sequences. These results suggest that transformation frequencies in L. maculans are limited by the ability of vector DNA to integrate into the genome. Hence, construction of strains with target sites for integration may prove to be a generally useful method for improving transformation frequencies of poorly characterised filamentous fungi, particularly when using heterologous vectors. This would greatly facilitate the identification of genes by transfer of gene libraries and the standardisation of chromosomal location effects in studies of expression of nested promoter deletions.