Population Genomic Analyses Reveal Connectivity via Human-Mediated Transport across Populus Plantations in North America and an Undescribed Subpopulation of Sphaerulina musiva.

Domestication of plant species has affected the evolutionary dynamics of plant pathogens in agriculture and forestry. A model system for studying the consequences of plant domestication on the evolution of an emergent plant disease is the fungal pathogen Sphaerulina musiva. This ascomycete causes leaf spot and stem canker disease of Populus spp. and their hybrids. A population genomics approach was used to determine the degree of population structure and evidence for selection on the North American population of S. musiva. In total, 122 samples of the fungus were genotyped identifying 120,016 single-nucleotide polymorphisms after quality filtering. In North America, S. musiva has low to moderate degrees of differentiation among locations. Three main genetic clusters were detected: southeastern United States, midwestern United States and Canada, and a new British Columbia cluster (BC2). Population genomics suggest that BC2 is a novel genetic cluster from central British Columbia, clearly differentiated from previously reported S. musiva from coastal British Columbia, and the product of a single migration event. Phenotypic measurements from greenhouse experiments indicate lower aggressiveness of BC2 on Populus trichocarpa. In summary, S. musiva has geographic structure across broad regions indicative of gene flow among clusters. The interconnectedness of the North American S. musiva populations across large geographic distances further supports the hypothesis of anthropogenic-facilitated transport of the pathogen.

Cryptic Speciation in Western North America and Eastern Eurasia of the Pathogens Responsible for Laminated Root Rot.

Coniferiporia sulphurascens is a facultative fungal pathogen that causes laminated root rot (LRR) in commercially important coniferous species worldwide. This fungus spreads primarily by way of vegetative mycelium transferring at points of contact between infected and healthy roots. Successful intervention to control LRR requires a better understanding of the population structure and genetic variability of C. sulphurascens. In this study, we investigated the population genetic structure and origin of C. sulphurascens populations in western North America and eastern Eurasia collected from multiple coniferous hosts. By analyzing the small and large mitochondrial ribosomal RNA subunit genes combined with six nuclear loci (internal transcribed spacer region, actin, RNA polymerase II largest subunit, RNA polymerase II second-largest subunit, laccase-like multicopper oxidase, and translation elongation factor 1-α), we observed that none of the alleles among the loci were shared between North American (NA) and Eurasian C. sulphurascens populations. In total, 55 multilocus genotypes (MLGs) were retrieved in C. sulphurascens isolates occurring in these two continental regions. Of these, 41 MLGs were observed among 58 isolates collected from widespread locations in British Columbia (Canada) and the northwestern United States, while 14 MLGs were observed among 16 isolates sampled in Siberia and Japan. Our data showed that the levels of genetic differentiation between the NA and Eurasian populations are much greater than the populations from within each continental region; the two continental populations formed clearly divergent phylogenetic clades or lineages since they were separated approximately 7.5 million years ago. Moreover, the Eurasian population could be the source of the NA population. Our study indicates the existence of cryptic diversity in this pathogen species, and strongly suggests that the NA and Eurasian populations represent two lineages, which have progressively diverged from each other in allopatry.

Neonectria bordenii sp. nov., a potential symbiote of the alder bark beetle, and its detection by quantitative PCR.

A taxonomically comprehensive perspective on the fungal associates of bark beetles (Coleoptera: Curculionidae: Scolytinae), and powerful molecular tools for detection of these fungi, are imperative to understanding bark beetle impacts on forest ecosystems. The most common filamentous fungi living alongside bark beetles in infested trees are ophiostomatoids (Ascomycota: Ophiostomatales and Microascales), yet an undescribed species of Neonectria (Neonectria sp. nov.; Ascomycota: Hypocreales) was recently identified cohabitating with the alder bark beetle, Alniphagus aspericollis, in red alder, Alnus rubra. The hardwood-infesting alder bark beetle is found throughout the range of its red alder host in the Pacific Coast region of North America and is associated with Neonectria sp. nov. in southwestern British Columbia, Canada. The aim of this study was to describe and name Neonectria sp. nov. and to develop a quantitative PCR (qPCR) assay to enable rapid detection of Neonectria sp. nov. from individual adult alder bark beetles and to define the distribution of the fungus. Neonectria sp. nov. was phylogenetically and morphologically determined to represent a distinct species closely related to N. ditissima and is described herein as Neonectria bordenii sp. nov. Neonectria bordenii was reliably detected from individual whole-beetle DNA extractions using a probe-based qPCR assay targeting multi-copy internal transcribed spacers (ITS) of nuclear ribosomal DNA. The qPCR assay amplified the fungus from 87.8 % (36/41) of individual alder bark beetle samples and was highly sensitive to N. bordenii, with a lower limit of detection of 1 × 10-6 ng/µL of culture DNA (or ~262 genome copies). Application of the qPCR assay developed in this study will expedite future research evaluating N. bordenii as a potential symbiote of the alder bark beetle. Citation: Wertman DL, Tanney JB, Hamelin RC, Carroll AL (2024). Neonectria bordenii sp. nov., a potential symbiote of the alder bark beetle, and its detection by quantitative PCR. Fungal Systematics and Evolution 13: 15-28. doi: 10.3114/fuse.2024.13.02.

Large-scale genomic analyses with machine learning uncover predictive patterns associated with fungal phytopathogenic lifestyles and traits.

Invasive plant pathogenic fungi have a global impact, with devastating economic and environmental effects on crops and forests. Biosurveillance, a critical component of threat mitigation, requires risk prediction based on fungal lifestyles and traits. Recent studies have revealed distinct genomic patterns associated with specific groups of plant pathogenic fungi. We sought to establish whether these phytopathogenic genomic patterns hold across diverse taxonomic and ecological groups from the Ascomycota and Basidiomycota, and furthermore, if those patterns can be used in a predictive capacity for biosurveillance. Using a supervised machine learning approach that integrates phylogenetic and genomic data, we analyzed 387 fungal genomes to test a proof-of-concept for the use of genomic signatures in predicting fungal phytopathogenic lifestyles and traits during biosurveillance activities. Our machine learning feature sets were derived from genome annotation data of carbohydrate-active enzymes (CAZymes), peptidases, secondary metabolite clusters (SMCs), transporters, and transcription factors. We found that machine learning could successfully predict fungal lifestyles and traits across taxonomic groups, with the best predictive performance coming from feature sets comprising CAZyme, peptidase, and SMC data. While phylogeny was an important component in most predictions, the inclusion of genomic data improved prediction performance for every lifestyle and trait tested. Plant pathogenicity was one of the best-predicted traits, showing the promise of predictive genomics for biosurveillance applications. Furthermore, our machine learning approach revealed expansions in the number of genes from specific CAZyme and peptidase families in the genomes of plant pathogens compared to non-phytopathogenic genomes (saprotrophs, endo- and ectomycorrhizal fungi). Such genomic feature profiles give insight into the evolution of fungal phytopathogenicity and could be useful to predict the risks of unknown fungi in future biosurveillance activities.

Tumours with loss of MSH6 expression are MSI-H when screened with a pentaplex of five mononucleotide repeats.

BACKGROUND: microsatellite instability (MSI) is commonly screened using a panel of two mononucleotide and three dinucleotide repeats as recommended by a consensus meeting on MSI tumours held at the National Cancer Institute (Bethesda, MD, USA). According to these recommendations, tumours are classified as MSI-H when at least two of the five microsatellite markers show instability, MSI-L when only one marker shows instability and MSS when none of the markers show instability. Almost all MSI-H tumours are characterised by alterations in one of the four major proteins of the mismatch repair (MMR) system (MLH1, MSH2, MSH6 or PMS2) that renders them MMR deficient, whereas MSI-L and MSS tumours are generally MMR proficient. However, tumours from patients with a pathogenic germline mutation in MSH6 can sometimes present an MSI-L phenotype with the NCI panel. The MSH6 protein is not involved in the repair of mismatches of two nucleotides in length and consequently the three dinucleotide repeats of the NCI panel often show stability in MSH6-deficient tumours. METHODS: a pentaplex panel comprising five mononucleotide repeats has been recommended as an alternative to the NCI panel to determine tumour MSI status. Several studies have confirmed the sensitivity, specificity and ease of use of the pentaplex panel; however, its sensitivity for the detection of MSH6-deficient tumours is so far unknown. Here, we used the pentaplex panel to evaluate MSI status in 29 tumours known to harbour an MSH6 defect. RESULTS: MSI-H status was confirmed in 15 out of 15 (100%) cases where matching normal DNA was available and in 28 out of 29 (97%) cases where matching DNA was not available or was not analysed. CONCLUSION: these results show that the pentaplex assay efficiently discriminates the MSI status of tumours with an MSH6 defect.

Patterns of colonization and spread in the fungal spruce pathogen Onnia tomentosa.

The basidiomycetous fungus Onnia tomentosa is one of the most widespread root rot pathogens in North America. Although the disease is more severe on spruce and pine trees, this pathogen can infect several coniferous species. To study the population structure of O. tomentosa, we harvested 180 basidiocarps in a 45-year-old white spruce plantation in western Quebec in autumn 1997 and extracted DNA directly from individual basidiocarps. Using a combination of spatial coordinates and molecular data based on the analysis of two mitochondrial and three nuclear loci, we measured the average genet size and molecular diversity and assessed the relative contribution of basidiospores and vegetative growth to the stand colonization. Most of the sampled basidiocarps that clustered spatially belonged to the same genet. A total of 37 discrete multilocus genets of an average size of 3.42 m were obtained. The genet size distribution was skewed towards smaller genets (<3 m) that displayed higher diversity than the larger genets (>3 m). The nuclear loci were in Hardy-Weinberg equilibrium in the larger genets, but not in the smaller genets, which displayed a deficiency of heterozygotes. This suggests a Wahlund effect, whereby different colonization events resulted in expected heterozygosity higher than observed heterozygosity. Using an estimate of the growth rate of the fungus, only a few of the largest genets were approximately the age of the plantation. These observations are consistent with the colonization by basidiospores subsequent to site preparation and tree planting followed by secondary colonization events and vegetative spread.

Evaluation of molecular markers for Phytophthora ramorum detection and identification: testing for specificity using a standardized library of isolates.

Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as beta-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately identified P. ramorum in blind evaluations of 60 field samples that included examples of plant infection by 11 other Phytophthora spp. The SSCP analysis identified eight of these species, with three identified to a species group.

KRAS and BRAF oncogenic mutations in MSS colorectal carcinoma progression.

In sporadic colorectal cancer (CRC), KRAS are alternative to BRAF mutations and occur, respectively, in 30 and 10% of cases. Few reports addressed the association between KRAS-BRAF mutations and tumour progression specifically in sporadic microsatellite-stable (MSS) CRC. We screened KRAS and BRAF in 250 MSS primary CRC and 45 lymph node (LN) metastases and analysed the pathological features of the cases to understand the involvement of KRAS-BRAF activation in progression and metastasis. Forty-five per cent of primary MSS CRCs carried mutations in at least one of these genes and mutations were associated with wall invasion (P=0.02), presence and number of LN metastases (P=0.02 and P=0.03, respectively), distant metastases (P=0.004) and advanced stage (P=0.01). We demonstrated that KRAS and BRAF are alternative events in Tis and T1 MSS CRC and, KRAS rather than BRAF mutations, contributed to the progression of MSS CRC. The frequency of KRAS and/or BRAF mutations was higher in LN metastases than in primary carcinomas (P=0.0002). Mutated LN metastases displayed KRAS associated or not with BRAF mutations. BRAF mutations were never present as a single event. Concomitant KRAS and BRAF mutations increased along progression of MSS CRCs, suggesting that activation of both genes is likely to harbour a synergistic effect.

First Report of Amylostereum areolatum, the Fungal Symbiont of Sirex noctilio, on Pinus spp. in Canada.

Amylostereum areolatum (Fr.) Boidin (Russulales: Stereaceae) is a white rot fungus that has a symbiotic relationship with several woodwasps including Sirex noctilio Fabricius (Hymenoptera: Siricidae). The vectored fungus together with a phytotoxic mucus, both injected during oviposition by the female S. noctilio, rapidly weaken the host tree, rendering it susceptible to larval development (3). Host trees of A. areolatum include species of Pinus (mainly), Abies, Larix, and Picea and Cryptomeria japonica and Pseudotsuga menziesii (Fungal Databases [online]; USDA). The siricid woodwasp is native to Eurasia and North Africa and has been introduced into New Zealand, Australia, South America, and South Africa (1). In July of 2005, the first established North American population of S. noctilio was reported in Oswego, NY. Prompted by this initial discovery, a trap survey of Ontario counties located along the Canada-U.S. border, close to Upstate New York, was conducted in September and October of 2005. S. noctilio females were captured in four locations in southern Ontario. Two additional locations for S. noctilio were also reported in a survey conducted independently (2). In September and October of 2006, logs of Scots pines showing current Sirex oviposition sites were harvested from the Ontario area bordered by Lakes Huron, Erie, and Ontario to determine the presence of the species-specific fungal symbiont of S. noctilio, A. areolatum. Fungal isolates were obtained by surface sterilizing wood chips showing decay columns followed by incubation at 20°C on 2% malt extract agar. Cultures with morphological characteristics typical of A. areolatum-presence of clamp connections and arthrospores-were used for DNA analysis to confirm species identification. DNA sequences of the internal transcribed spacer (ITS) of the ribosomal RNA gene were queried against the NCBI GenBank database. There was a 99 to 100% match between the ITS sequences from the Ontario isolates and sequences from European and Asian A. areolatum isolates (GenBank Accession Nos. EU249343 and EU249344 versus AF454428, AF506405, AY781245, and AF218389). Matches with A. chailletii (Pers.) Boidin, a native related species, were around 97%. These results confirmed the presence of A. areolatum in the infested material. Cultures were deposited in the National Mycological Herbarium of Canada (DAOM 239280-DAOM 239295). To our knowledge, this represents the first report of A. areolatum in Canada. In its natural range, the insect-fungal complex exists in equilibrium with its host trees and parasites, thus, few negative impacts are observed. However, in the Southern Hemisphere where it has been introduced, it has become a major pest, attacking many important commercial North American species planted as exotics (1). Conifer forests in Canada are threatened by the spread of the S. noctilio/A. areolatum complex, particularly plantations and stands of Pinus banksiana, P. contorta, P. ponderosa, P. resinosa, P. strobus, and P. sylvestris. A survey of Eastern Canada to detect the presence of S. noctilio is on going, and genetics work is being conducted to determine the origin of the introduction of A. areolatum. References: (1) W. M. Ciesla. J. For. 101:18, 2003. (2) P. de Groot et al. Gt. Lakes Entomol. 39:49, 2006. (3) B. Slippers et al. S. Afr. J. Sci. 99:70, 2003.

TCF-4 isoforms absent in TCF-4 mutated MSI-H colorectal cancer cells colocalize with nuclear CtBP and repress TCF-4-mediated transcription.

TCF-4 is the main effector of the Wnt/Wingless signalling pathway. As with other TCF/LEF factors, numerous alternative splicings at its 3' end affect its expression. Such a mechanism leads to the synthesis of numerous TCF-4 isoforms among which some contain binding domains for CtBP, an ubiquitous transcriptional corepressor. Of interest, we described a frequent TCF-4 frameshift mutation in mismatch-repair deficient colorectal cancers (MSI-H cancers) that leads to the selective loss of TCF-4 isoforms with CtBP binding abilities. We provide here data that argue for a partial colocalization of CtBP with TCF-4 isoforms containing CtBP binding domains in cellulo, and for a functional role of CtBP in repressing TCF-4 mediated transcription. We also demonstrate that such a colocalization is not observed in MSI-H colorectal cancer cells that harbour the TCF-4 frameshift mutation, and that CtBP is not able to repress TCF-4-mediated transcription in this context. Taken together, our results strongly suggest that CtBP would play a role in regulating TCF-4 mediated transcription upon its binding with some TCF-4 isoforms encoded by alternatively spliced mRNA. They also suggest a role for TCF-4 frameshift mutation during MSI-H colorectal tumour progression, by regulating the relative proportion of the different TCF-4 isoforms.

Microsatellite instability and mutation analysis of candidate genes in urothelial cell carcinomas of upper urinary tract.

A subset of upper urinary tract urothelial cell carcinomas (UUC), arising sporadically or as a manifestation of hereditary non-polyposis colorectal cancer, displays microsatellite instability (MSI). MSI tumours are characterized by defective mismatch repair and accumulation of frameshift mutations in numerous genes harbouring repeats in their coding sequences. We have evaluated the incidence of MSI in UUC and the intratumoral distribution of mutations in 13 candidate target genes. A total of 58 unselected UUC were screened for MSI using the panel of five mononucleotide markers recently recommended by the National Cancer Institute for a precise MSI assessment. Four tumours displayed MSI (7%), among which at least three had alterations in the genes MSH3, BAX, MRE11, RAD50. Mutations in genes involved in key cellular pathways (ATR, DNA-PKcs, MBD4, TCF-4, MSH6, and BLM) were further detected. BAX and MRE11 mutations tend to present homogeneously within the three MSI UUC. Immunohistochemistry (MLH1, MSH2, MSH6) showed that loss of mismatch repair protein expression occurred in all MSI UUC defining the gene defect and that MRE11 and RAD50 mutations were associated with their concomitant loss expression. In conclusion, MSI UUC represent a small proportion of UUC in which BAX and MRE11 mutations are frequent and may play a role early in UUC tumorigenesis.

Genetic Diversity in Poplar Leaf Rust (Melampsora medusae f. sp. deltoidae) in the Zones of Host Sympatry and Allopatry.

ABSTRACT Poplar leaf rust caused by Melampsora medusae f. sp. deltoidae is a widespread disease in North America, where epidemics occur within zones of sympatry and allopatry of telial hosts (Populus spp.) and aecial hosts (Larix spp.). To test the hypothesis that epidemics originate in the zone of sympatry where the rust can complete its life cycle, populations in sympatry and allopatry were analyzed with single-strand conformational polymorphism for codominant detection of alleles directly from uredinia. More alleles were detected in rust populations in the zone of host sympatry than in allopatry. Almost all alleles found in the zone of allopatry were a subset of the allelic diversity present in the zone of host sympatry. Distance analyses clustered populations according to geographic origin, but not sampling year or type of stand (plantation or natural stands). Large differences in allelic and genotypic frequency were observed between years in allopatry but not in sympatry, suggesting new colonizations in allopatric populations. Our results point to a dynamic and complex pattern of inoculum dissemination in polar leaf rust. The hypothesis most consistent with our results is that populations in sympatry represent a source of inoculum for epidemics, with some annual recolonization in allopatry, possibly via intermediate population jumps.

Genetic Diversity and the Presence of Two Distinct Groups in Ophiostoma clavigerum Associated with Dendroctonus ponderosae in British Columbia and the Northern Rocky Mountains.

ABSTRACT The sapstaining fungal pathogen Ophiostoma clavigerum is associated with the mountain pine beetle (Dendroctonus ponderosae), which is currently the most destructive forest pest in North America. The genetic diversity of O. clavigerum populations collected from five sites in Canada and two sites in the United States was estimated with amplified fragment length polymorphism (AFLP) analysis. Genomic DNA from 170 O. clavigerum isolates was digested with EcoRI and PstI and amplified with six primer sets. A total of 469 AFLP markers consisting of 243 monomorphic and 226 polymorphic loci were scored. The overall genetic diversity of the O. clavigerum population was low (Hs = 0.0531) and the differentiation of the seven O. clavigerum populations was moderate (Phi = 0.143). Genetic distances among the populations were not significantly correlated with geographic distance (r = 0.3235, P = 0.074). Two genetically distinct groups in the O. clavigerum populations were shown by unique AFLP profiles and the unweighted pair group method with arithmetic averages. Further work to characterize biological differences between the two groups will be needed to confirm whether cryptic species are present in the O. clavigerum population.

Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons.

ABSTRACT Sudden oak death, caused by Phytophthora ramorum, is a severe disease that affects many species of trees and shrubs. This pathogen is spreading rapidly and quarantine measures are currently in place to prevent dissemination to areas that were previously free of the pathogen. Molecular assays that rapidly detect and identify P. ramorum frequently fail to reliably distinguish between P. ramorum and closely related species. To overcome this problem and to provide additional assays to increase confidence, internal transcribed spacer (ITS), beta-tubulin, and elicitin gene regions were sequenced and searched for polymorphisms in a collection of Phytophthora spp. Three different reporter technologies were compared: molecular beacons, TaqMan, and SYBR Green. The assays differentiated P. ramorum from the 65 species of Phytophthora tested. The assays developed were also used with DNA extracts from 48 infected and uninfected plant samples. All environmental samples from which P. ramorum was isolated by PARP-V8 were detected using all three real-time PCR assays. However, 24% of the samples yielded positive real-time PCR assays but no P. ramorum cultures, but sequence analysis of the coxI and II spacer region confirmed the presence of the pathogen in most samples. The assays based on detection of the ITS and elicitin regions using TaqMan tended to have lower cycle threshold values than those using beta-tubulin and seemed to be more sensitive.

Differential signaling networks of Bcr-Abl p210 and p190 kinases in leukemia cells defined by functional proteomics.

The two major isoforms of the oncogenic Bcr-Abl tyrosine kinase, p210 and p190, are expressed upon the Philadelphia chromosome translocation. p210 is the hallmark of chronic myelogenous leukemia, whereas p190 occurs in the majority of B-cell acute lymphoblastic leukemia. Differences in protein interactions and activated signaling pathways that may be associated with the different diseases driven by p210 and p190 are unknown. We have performed a quantitative comparative proteomics study of p210 and p190. Strong differences in the interactome and tyrosine phosphoproteome were found and validated. Whereas the AP2 adaptor complex that regulates clathrin-mediated endocytosis interacts preferentially with p190, the phosphatase Sts1 is enriched with p210. Stronger activation of the Stat5 transcription factor and the Erk1/2 kinases is observed with p210, whereas Lyn kinase is activated by p190. Our findings provide a more coherent understanding of Bcr-Abl signaling, mechanisms of leukemic transformation, resulting disease pathobiology and responses to kinase inhibitors.

Molecular and Morphological Evidence for Interspecific Hybridization Between Cronartium ribicola and C. comandrae on Pinus flexilis in Southwestern Alberta.

In May 2003, a survey was conducted in southwestern Alberta, east of the Rocky Mountains, to determine the extent of the spread and genetic diversity of white pine blister rust, which is caused by Cronartium ribicola J.C. Fisch. Aeciospores were sampled from white pine blister rust cankers in three infected limber pine (Pinus flexilis James) stands separated from one another by 100 to 215 km. DNA genotypes were determined for 12 codominant PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) loci representing genes derived from an EST library. At each site sampled, some aecia displayed DNA genotypes that were heterozygous at all loci and possessed novel alleles (GenBank Accession Nos. DQ009533-DQ009611). At Waterton Lakes, Kananaskis County, and Porcupine Hills, 29%, 11%, and 3% of sampled aecia and 38%, 33%, and 10% of sampled trees, respectively, possessed these unusual profiles. In May 2004, similar genetic profiles were found at two of these sites, Waterton Lakes and Kananaskis County, at 17 and 25% of sampled aecia (25% of sampled trees). In each of these aecia, genotyping and sequence analysis revealed this pattern was due to the presence of one C. ribicola and one C. comandrae Peck. allele at each of the 12 loci. Scanning electron microscopy (SEM) revealed aeciospore morphology that was intermediate between C. ribicola and C. comandrae. Aeciospores were longer (16 to 20 × 25 to 40 µm) than the expected range for C. ribicola (18 to 20 × 22 to 31 µm) (3). They were also fusiform, obovoid or short-to-long ellipsoid, but not pyriform-acuminate as in C. comandrae, and without a true conspicuous smooth spot as in C. ribicola. This provides evidence for interspecific hybridization between C. ribicola and C. comandrae, the causal agent of comandra blister rust. We hypothesize that the presence of nearby C. comandrae-infected lodgepole pine (P. contorta Dougl.) could have led to spermatization of C. ribicola receptive hyphae by C. comandrae pycniospores, resulting in the formation of hybrid aecia. An important question is whether these hybrids have a different host range that could potentially extend its geographic range in areas where the telial host, Ribes spp. L., is not abundant. The hybrid rust Melampsora × columbiana Newcombe was shown to exhibit virulence against certain hybrid poplar clones that had previously been reported as resistant against both parental rusts (M. medusae Thuem. and M. occidentalis Jacks) and abundant pathogenic variation has been observed (2). Furthermore, the ability to colonize unexpected hosts could provide fitness advantages over parental species, as was observed in Phytophthora spp. pathogenic on alder (1). Host range and virulence assays should be conducted to assess the potential impact of this hybrid. References: (1) C. M. Brasier et al. Proc. Natl. Acad. Sci. USA 96:5878, 1999. (2) G. Newcombe et al. Phytopathology 91:981, 2001. (3) W. G. Ziller. The Tree Rusts of Western Canada. Can. For Serv. No. 1329. Pacific Forestry Center, Victoria, BC, 1974.

The human T-cell transcription factor-4 gene: structure, extensive characterization of alternative splicings, and mutational analysis in colorectal cancer cell lines.

The human T cell transcription factor-4 (hTCF-4) interacts functionally with beta-catenin in the Wnt signaling pathway, which regulates many developmental processes. Moreover, inappropriate reactivation of this pathway attributable to APC or beta-catenin mutations has been described in colorectal cancers. Because only the human TCF-4 cDNA sequence was known, we determined its genomic structure. A total of 17 exons, of which 5 were alternative, were identified. Moreover, four alternative splice sites were observed either experimentally or in silico by a BLAST approach in expressed sequence tag databases. The alternative use of three consecutive exons localized in the 3' part of the hTCF-4 gene changes the reading frames used in the last exon, leading to the synthesis of a number of hTCF-4 isoforms with short, medium, or long-size COOH-terminal ends. We next screened the entire hTCF-4 gene for mutations in a series of 24 colorectal cancer cell lines by denaturing gradient gel electrophoresis and/or direct sequencing. Besides an already described deletion of an A in an (A)9 coding repeat in four cases, we found DNA variants in eight cases for a total of 12 variants, of which 8 were coding. These include one frameshift mutation in the beta-catenin binding domain (exon 1), and one missense mutation in exon 4. In the remaining six cases, nonsense or frameshift mutations were localized in the 3' part of the gene. These latter alterations have as a common consequence to decrease the proportion of the long COOH-terminal hTCF-4 isoform, which contains two binding domains for c-terminal binding protein, a protein implicated in the repression of the TCF family transcriptional activity. Thus, loss of the TCF-4 capacity to interact with COOH-terminal binding protein could be an important event during colorectal carcinogenesis by modifying Wnt signaling.

BAT-26, an indicator of the replication error phenotype in colorectal cancers and cell lines.

Instability of microsatellites is a hallmark of the DNA replication error phenotype (RER+) due to the inactivation of mismatch repair genes. In humans, microsatellite instability has first been described in colorectal tumors developing in either hereditary nonpolyposis colorectal cancer or sporadic patients. Colorectal tumorigenesis in RER+ and RER- tumors is probably due to distinct mechanisms, and RER+ tumors have a better prognosis than RER- tumors. The study of the RER status of a tumor may thus be important in the future to determine biological prognosis factors and investigate therapeutical strategies. The RER status of 134 primary tumors and 26 cell lines derived from colorectal cancers was established by PCR amplification and analysis of a minimum of 32 microsatellite loci. This characterization allowed us to unambiguously classify 35 primary tumors and 7 cell lines as RER+. Typing of a single poly(A) tract, BAT-26, was sufficient to confirm the RER status of 159 of these 160 tumors and cell lines. Moreover, in DNA from unaffected individuals, normal tissues of a subset of the RER+ patients, and all RER- tumors or cell lines, BAT-26 was quasi-monomorphic, showing only minor size variations. BAT-26 alleles showing shortening from 4 to 15 bp were observed in all but 1 of the RER+ tumors and cell lines. The size difference between the range of normal large alleles and unstable small alleles was sufficient to be detected by electrophoresis on conventional polyacrylamide gels stained with ethidium bromide. We thus propose a simple, low-cost, and rapid method to screen for the RER status of colorectal cancer primary tumors and cell lines, even in the absence of matching normal DNA and, in most cases, without the need for radioactivity. This method could easily be set up in routine laboratories.

Frequent frameshift mutations of the TCF-4 gene in colorectal cancers with microsatellite instability.

About 15% of sporadic colorectal cancers show microsatellite instability (MSI) due to the inactivation of mismatch repair genes and are termed MSI-H tumors. In these tumors, frameshift mutations in coding repeats have been found within the TGFbeta-RII, BAX, and IGFRII genes that are probably involved in their progression. In the present work, we report frequent mutations in TCF-4, another target gene for instability. TCF-4 codes for a transcription factor that is a crucial member of the adenomatous polyposis coil (APC)/beta-catenin/T-cell factor (TCF) pathway. Fifty percent (4 of 8) of human MSI-H colorectal cell lines and 39% (19 of 49) of MSI-H colorectal primary tumors were found to have a 1-bp deletion in an (A)9 repeat within the coding region of this gene. In contrast, a frameshift mutation was found in only 1 of 56 non-MSI colorectal tumors and in none of 16 non-MSI colorectal cancer cell lines. These results suggest that TCF-4 frameshift mutations are selected for and play a role in colorectal MSI-H tumorigenesis. Depending on different reading frames due to alternatively spliced TCF-4 mRNA, the (A)9 repeat normally codes for several isoforms that could serve as modulators of TCF-4 transcriptional activity. The deletion of one nucleotide in this repeat could change TCF-4 transactivating properties by modifying the respective proportions of the different isoforms.

Microsatellite instability occurs in distinct subtypes of pediatric but not adult central nervous system tumors.

Length alterations in microsatellite repeats, termed microsatellite instability (MSI), are found in 10-15% of sporadic colon, endometrial, and gastric cancers harboring defects in DNA mismatch repair (MMR) genes We used the microsatellite markers Big Adenine Tract (BAT) 26 and BAT-25 from the reference panel of five markers recommended by the National Cancer Institute to evaluate the incidence of MSI in 206 central nervous system tumors. We screened 102 pediatric and 104 adult cases representing 165 astrocytic and 41 nonastrocytic tumors. The overall incidence of MSI was 8% (16 of 206). All 16 tumors with MSI were found in pediatric rather than adult patients. MSI was associated with two distinct subtypes of pediatric tumors occurring in 27% (12 of 45) of WHO grade III and grade IV astrocytomas and 24% (4 of 17) of gangliogliomas We evaluated the difference in clinicopathological and genetic features among 45 high-grade pediatric astrocytomas by MSI status. The median survival for pediatric patients with MSI (n = 12) was 8 months compared with 15 months for those patients without MSI (n = 33; P = 0.18). The frequency of p53 gene mutations was 13% for pediatric patients with MSI (n = 8) compared with 47% for those patients without MSI (n = 19; P = 0.19). These results revealed a trend between MSI status and prog nosis and MSI status and frequency of p53 gene mutations. Our data suggest that pediatric high-grade astrocytomas can be attributed to two different genetic pathways: a MMR-deficient pathway and a MMR proficient pathway.