The presence of GC-AG introns in Neurospora crassa and other euascomycetes determined from analyses of complete genomes: implications for automated gene prediction

AbstractA combination of experimental and computational approaches was employed to identify introns with noncanonical GC-AG splice sites (GC-AG introns) within euascomycete genomes. Evaluation of 2335 cDNA-confirmed introns from Neurospora crassa revealed 27 such introns (1.2%). A similar frequency (1.0%) of GC-AG introns was identified in Fusarium graminearum, in which 3 of 292 cDNA-confirmed introns contained GC-AG splice sites. Computational analyses of the N. crassa genome using a GC-AG intron consensus sequence identified an additional 20 probable GC-AG introns in this fungus. For 8 of the 47 GC-AG introns identified in N. crassa a GC donor site is also present in a homolog from Magnaporthe grisea, F. graminearum, or Aspergillus nidulans. In most cases, however, homologs in these fungi contain a GT-AG intron or no intron at the corresponding position. These findings have important implications for fungal genome annotation, as the automated annotations of euascomycete genomes incorrectly identified intron boundaries for all of the confirmed and probable GC-AG introns reported here

Characterization of a Fusarium graminearum Salicylate Hydroxylase

Salicylic acid (SA) plays an important role in regulating plant defense responses against pathogens. However, pathogens have evolved ways to manipulate plant SA-mediated defense signaling. Fusarium graminearum causes Fusarium head blight (FHB) and reduces crop yields and quality by producing various mycotoxins. In this study, we aimed to identify the salicylate hydroxylase in F. graminearum and determine its role in wheat head blight development. We initially identified a gene in F. graminearum strain NRRL 46422 that encodes a putative salicylate hydroxylase (designated FgShyC). However, the FgShyC deletion mutant showed a similar ability to degrade SA as wild-type strain 46422; nor did overexpression of FgShyC in E. coli convert SA to catechol. The results indicate that FgShyC is not involved in SA degradation. Further genome sequence analyses resulted in the identification of eight salicylate hydroxylase candidates. Upon addition of 1 mM SA, FGSG_03657 (designated FgShy1), was induced approximately 400-fold. Heterologous expression of FgShy1 in E. coli converted SA to catechol, confirming that FgShy1 is a salicylate hydroxylase. Deletion mutants of FgShy1 were greatly impaired but not completely blocked in SA degradation. Expression analyses of infected tissue showed that FgShy1 was induced during infection, but virulence assays revealed that deletion of FgShy1 alone was not sufficient to affect FHB severity. Although the Fgshy1 deletion mutant did not reduce pathogenicity, we cannot rule out that additional salicylate hydroxylases are present in F. graminearum and characterization of these enzymes will be necessary to fully understand the role of SA-degradation in FHB pathogenesis

A Targeted Multilocus Genotyping Assay for Lineage, Serogroup, and Epidemic Clone Typing of Listeria monocytogenes▿ †

A 30-probe assay was developed for simultaneous classification of Listeria monocytogenes isolates by lineage (I to IV), major serogroup (4b, 1/2b, 1/2a, and 1/2c), and epidemic clone (EC) type (ECI, ECIa, ECII, and ECIII). The assay was designed to facilitate rapid strain characterization and the integration of subtype data into risk-based inspection programs

Multilocus Genotyping Assays for Single Nucleotide Polymorphism-Based Subtyping of Listeria monocytogenes Isolates▿ †

Listeria monocytogenes is responsible for serious invasive illness associated with consumption of contaminated food and places a significant burden on public health and the agricultural economy. We recently developed a multilocus genotyping (MLGT) assay for high-throughput subtype determination of L. monocytogenes lineage I isolates based on interrogation of single nucleotide polymorphisms (SNPs) via multiplexed primer extension reactions. Here we report the development and validation of two additional MLGT assays that address the need for comprehensive DNA sequence-based subtyping of L. monocytogenes. The first of these novel MLGT assays targeted variation segregating within lineage II, while the second assay combined probes for lineage III strains with probes for strains representing a recently characterized fourth evolutionary lineage (IV) of L. monocytogenes. These assays were based on nucleotide variation identified in >3.8 Mb of comparative DNA sequence and consisted of 115 total probes that differentiated 93% of the 100 haplotypes defined by the multilocus sequence data. MLGT reproducibly typed the 173 isolates used in SNP discovery, and the 10,448 genotypes derived from MLGT analysis of these isolates were consistent with DNA sequence data. Application of the MLGT assays to assess subtype prevalence among isolates from ready-to-eat foods and food-processing facilities indicated a low frequency (6.3%) of epidemic clone subtypes and a substantial population of isolates (>30%) harboring mutations in inlA associated with attenuated virulence in cell culture and animal models. These mutations were restricted to serogroup 1/2 isolates, which may explain the overrepresentation of serotype 4b isolates in human listeriosis cases

A Single-Nucleotide-Polymorphism-Based Multilocus Genotyping Assay for Subtyping Lineage I Isolates of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations