Project description:The purpose of this study was to make a single comparison between Cqf genes expressed during the vegetative stages of infection on the telial host (oak leaf) versus the aecial host (pine stem). A large proportion of genes were expressed in both hosts and significantly differentially expressed genes were enriched for candidate fungal effectors (small secreted proteins). These results suggest that the Cqf rust fungus uses a largely common set of genes to create two very different infection phenotypes. This study was based on hybridizations to custom microarrays containing features representing 8692 gene models from a Cqf genome sequencing project midpoint assembly. Two Agilent 4 X 44K microarray slides were populated with 60-mer probes (1 to 5 per transcript), designed using AgilentM-bM-^@M-^Ys web-based eArray software. Labeled target cRNA (complementary RNA) was generated using AgilentM-bM-^@M-^Ys Low Input Quick Amp Labeling Kit, such that oak and pine samples were labeled with either cy3 or cy5 an equal number of times across the experiment. Each microarray was hybridized with labeled cRNA target derived from a single oak sample and labeled cRNA target derived from a single pine sample. There were a total of eight oak sample replications and eight pine sample replications. Target hybridization and scanning were performed by the University of FloridaM-bM-^@M-^Ys Interdisciplinary Center for Biotechnology Research using standard procedures and an Agilent G250B Scanner.
Project description:Bursaphelenchus xylophilus is known as the causative agent of pine wilt disease with complex life cycles. In this research, newly published Bursaphelenchus xylophilus genome data were employed to annotate its miRNAs based on deep sequencing technologies. Four small RNA libraries derived from different infection stages of pine wilt disease were constructed and sequenced. Consequently, we obtained hundreds of evolutionarily conserved miRNAs as well as novel miRNA candidates. The analysis of miRNA expression patterns showed that most miRNAs were expressed at extraordinarily high levels during the middle stage of pine wilt disease. Subsequent stem-loop RT-PCR experiments were carried out to validate our results. Functional analysis proved that expression levels of miR-73 and miR-239 were mutually exclusive with their target GH45 cellulase genes., genes known to be responsible for the degradation of the pine cell walls. In addition, another set of atypical miRNAs, termed mirtrons, were identified from B. xylophilus introns. This discovery has expanded the current knowledgebase of such splicing-derived miRNAs into B. xylophilus. Thus, our research has provided detailed characterization of B. xylophilus miRNAs expression patterns during the pathological process of pine wilt disease. The findings will contribute to more in-depth understanding of this devastating plant disease.
Project description:DNA methylation is a pivotal process that regulates gene expression and facilitates rapid adaptation to challenging environments. The pine wood nematode (PWN; Bursaphelenchus xylophilus), the causative agent of pine wilt disease, survives and spreads at low temperatures through third-stage dispersal larvae, making it a major pathogen for pine wood in Asia. To comprehend the impact of DNA methylation on the formation and environmental adaptation of third-stage dispersal larvae, we conducted whole-genome bisulfite sequencing and transcriptional sequencing on both the third-stage dispersal larvae and three other stages propagative larvae of PWN.
Project description:DNA methylation is a pivotal process that regulates gene expression and facilitates rapid adaptation to challenging environments. The pine wood nematode (PWN; Bursaphelenchus xylophilus), the causative agent of pine wilt disease, survives and spreads at low temperatures through third-stage dispersal larvae, making it a major pathogen for pine wood in Asia. To comprehend the impact of DNA methylation on the formation and environmental adaptation of third-stage dispersal larvae, we conducted whole-genome bisulfite sequencing and transcriptional sequencing on both the third-stage dispersal larvae and three other stages propagative larvae of PWN.