Pathogenic Candidate Research Articles

Proteomic analyses of Fusarium graminearum grown under mycotoxin‐inducing conditions

Non-gel-based quantitative proteomics technology was used to profile protein expression differences when Fusarium graminearum was induced to produce trichothecenes in vitro. As F. graminearum synthesizes and secretes trichothecenes early in the cereal host invasion process, we hypothesized that proteins contributing to infection would also be induced under conditions favouring mycotoxin synthesis. Protein samples were extracted from three biological replicates of a time course study and subjected to iTRAQ (isobaric tags for relative and absolute quantification) analysis. Statistical analysis of a filtered dataset of 435 proteins revealed 130 F. graminearum proteins that exhibited significant changes in expression, of which 72 were upregulated relative to their level at the initial phase of the time course. There was good agreement between upregulated proteins identified by 2-D PAGE/MS/MS and iTRAQ. RT-PCR and northern hybridization confirmed that genes encoding proteins which were upregulated based on iTRAQ were also transcriptionally active under mycotoxin producing conditions. Numerous candidate pathogenicity proteins were identified using this technique. These will provide leads in the search for mechanisms and markers of host invasion and novel antifungal targets.

A Glimpse of Streptococcal Toxic Shock Syndrome from Comparative Genomics of S. suis 2 Chinese Isolates

Background Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen, causing more than 200 cases of severe human infection worldwide, with the hallmarks of meningitis, septicemia, arthritis, etc. Very recently, SS2 has been recognized as an etiological agent for streptococcal toxic shock syndrome (STSS), which was originally associated with Streptococcus pyogenes (GAS) in Streptococci. However, the molecular mechanisms underlying STSS are poorly understood.Methods and FindingsTo elucidate the genetic determinants of STSS caused by SS2, whole genome sequencing of 3 different Chinese SS2 strains was undertaken. Comparative genomics accompanied by several lines of experiments, including experimental animal infection, PCR assay, and expression analysis, were utilized to further dissect a candidate pathogenicity island (PAI). Here we show, for the first time, a novel molecular insight into Chinese isolates of highly invasive SS2, which caused two large-scale human STSS outbreaks in China. A candidate PAI of ∼89 kb in length, which is designated 89K and specific for Chinese SS2 virulent isolates, was investigated at the genomic level. It shares the universal properties of PAIs such as distinct GC content, consistent with its pivotal role in STSS and high virulence.ConclusionsTo our knowledge, this is the first PAI candidate from S. suis worldwide. Our finding thus sheds light on STSS triggered by SS2 at the genomic level, facilitates further understanding of its pathogenesis and points to directions of development on some effective strategies to combat highly pathogenic SS2 infections.

A Functional Screen to Characterize the Secretomes of Eukaryotic Pathogens and Their Hosts In Planta

Complex suites of proteins that are secreted by plants and phytopathogens into the plant apoplast play crucial roles in surveillance, assault, defense, and counter-defense. High-throughput genome-scale strategies are being developed to better understand the nature of these "secretomes" and the identity of pathogen-derived effector proteins that subvert plant defenses and promote pathogenicity. Although combined bioinformatic and experimental approaches recently have provided comprehensive coverage of secreted proteins from bacterial phytopathogens, far less is known about the secretomes and batteries of effectors of eukaryotic phytopathogens; notably fungi and oomycetes. The yeast secretion trap (YST) represents a potentially valuable technique to simultaneously target pathogen and host secretomes in infected plant material. A YST screen, using a new vector system, was applied to study the interaction between tomato (Solanum lycopersicum) and the oomycete Phytophthora infestans, revealing sets of genes encoding secreted proteins from both pathogen and host. Most of those from the oomycete had no identifiable function and were detectable in planta only during pathogenesis, underlining the value of YST as a tool to identify new candidate effectors and pathogenicity factors. In addition, the majority of the P. infestans proteins had homologs in the genomes of the related oomycetes R. sojae and P. ramorum.

Genomic Structure and Phylogeny of the Plant Pathogen Ralstonia solanacearum Inferred from Gene Distribution Analysis

In the present study, we investigated the gene distribution among strains of the highly polymorphic plant pathogenic beta-proteobacterium Ralstonia solanacearum, paying particular attention to the status of known or candidate pathogenicity genes. Based on the use of comparative genomic hybridization on a pangenomic microarray for the GMI1000 reference strain, we have defined the conditions that allowed comparison of the repertoires of genes among a collection of 18 strains that are representative of the biodiversity of the R. solanacearum species. This identified a list of 2,690 core genes present in all tested strains. As a corollary, a list of 2,338 variable genes within the R. solanacearum species has been defined. The hierarchical clustering based on the distribution of variable genes is fully consistent with the phylotype classification that was previously defined from the nucleotide sequence analysis of four genes. The presence of numerous pathogenicity-related genes in the core genome indicates that R. solanacearum is an ancestral pathogen. The results establish the long coevolution of the two replicons that constitute the bacterial genome. We also demonstrate the clustering of variable genes in genomic islands. Most genomic islands are included in regions with an alternative codon usage, suggesting that they originate from acquisition of foreign genes through lateral gene transfers. Other genomic islands correspond to genes that have the same base composition as core genes, suggesting that they either might be ancestral genes lost by deletion in certain strains or might originate from horizontal gene transfers.

Book Review

Book Review

Transformation of Fungal Grapevine Trunk Disease Pathogens with the Green Fluorescent Protein Gene

Eutypa lata and Phaeomoniella chlamydospora are important fungal grapevine trunk disease pathogens. Studies of their epidemiology and disease progression are hampered by the slow growth rates of the fungi and a lack of molecular tools. A protoplast-based transformation system was developed using hygromycin resistance as a positive selectable marker. The system was used to introduce a green fluorescent protein reporter gene that was expressed in the hyphae of both species. The reporter strains will be useful tools for further epidemiological studies. Furthermore the transformation system will allow for genetic dissection of candidate pathogenicity or virulence genes by targeted replacement or disruption, in order to discern the role of these genes in the disease process.

The Cladosporium fulvum Bap1 gene: evidence for a novel class of Yap-related transcription factors with ankyrin repeats in phytopathogenic fungi

The Saccharomyces cerevisiae transcription factor gene YAP1 is a key regulatory gene conferring resistance against oxidative stress and various cytotoxic compounds. It would be of interest to isolate homologous genes in pathogenic fungi as candidate pathogenicity genes. We have developed a generally applicable hybridization method, using a single degenerate oligonucleotide probe designed on the basis of the conserved DNA-binding region of yAP1 homologs in yeast species, to clone a related gene, Bap1, from the tomato pathogen, Cladosporium fulvum. This gene has the capacity to code for a possible transcription factor consisting of an N-terminal, yAP1-related, basic DNA-binding domain. It lacks an obvious downstream leucine zipper but includes C-terminal ankyrin-like repeats. The Cochliobolus carbonum pathogenicity factor TOXEp shows a similar modular structure. BAP1 and TOXEp appear to represent a novel fungus-specific class of Yap basic DNA-binding domain-type transcription factors with ankyrin-like repeats (bANK proteins) in phytopathogenic fungi. A possible role of BAP1 in a membrane transporter-mediated detoxification or secretion process is discussed.