Analysis of the A-U rich hairpin from the intergenic region of tospovirus S RNA as target and inducer of RNA silencing

Earlier work indicated that Tomato spotted wilt virus (TSWV) messenger transcripts, and not the (anti)genomic RNAs, are targeted by the RNA silencing machinery. Here, the predicted AU-rich hairpin (HP) structure encoded by the intergenic region (IGR) of the TSWV S RNA, and present at the 3' end of viral mRNAs, was analyzed as a target and inducer for RNA silencing. Virus-derived siRNAs (vsiRNAs) purified from virus infected plants were found to derive from all three genomic RNA segments but predominantly the ambisense M and S RNAs. Further profiling on the S RNA sequence revealed that vsiRNAs were found from almost the entire S RNA sequence, except the IGR from where hardly any vsiRNAs were found. Similar profiles were observed with the distantly related Tomato yellow ring tospovirus (TYRV). Dicer cleavage assays using Drosophila melanogaster (Dm) embryo extracts showed that synthetic transcripts of the IGR-HP region were recognized as substrate for Dicer. Transient agroinfiltration assays of a GFP-sensor construct containing the IGR-HP sequence at its 3' UTR (GFP-HP) did not show more rapid/strong silencing and profiling of the corresponding siRNAs, generated outside the context of a viral infection, still revealed relatively low levels of IGR-HP-derived siRNAs. These data support the idea that the IGR-HP is a weak inducer of RNA silencing and only plays a minor role in the amplification of a strong antiviral RNAi response

Symbiodinium Transcriptomes: Genome Insights into the Dinoflagellate Symbionts of Reef-Building Corals

Dinoflagellates are unicellular algae that are ubiquitously abundant in aquatic environments. Species of the genus Symbiodinium form symbiotic relationships with reef-building corals and other marine invertebrates. Despite their ecologic importance, little is known about the genetics of dinoflagellates in general and Symbiodinium in particular. Here, we used 454 sequencing to generate transcriptome data from two Symbiodinium species from different clades (clade A and clade B). With more than 56,000 assembled sequences per species, these data represent the largest transcriptomic resource for dinoflagellates to date. Our results corroborate previous observations that dinoflagellates possess the complete nucleosome machinery. We found a complete set of core histones as well as several H3 variants and H2A.Z in one species. Furthermore, transcriptome analysis points toward a low number of transcription factors in Symbiodinium spp. that also differ in the distribution of DNA-binding domains relative to other eukaryotes. In particular the cold shock domain was predominant among transcription factors. Additionally, we found a high number of antioxidative genes in comparison to non-symbiotic but evolutionary related organisms. These findings might be of relevance in the context of the role that Symbiodinium spp. play as coral symbionts

Transcriptomic response of the red tide dinoflagellate, Karenia brevis, to nitrogen and phosphorus depletion and addition

<p>Abstract</p> <p>Background</p> <p>The role of coastal nutrient sources in the persistence of <it>Karenia brevis </it>red tides in coastal waters of Florida is a contentious issue that warrants investigation into the regulation of nutrient responses in this dinoflagellate. In other phytoplankton studied, nutrient status is reflected by the expression levels of N- and P-responsive gene transcripts. In dinoflagellates, however, many processes are regulated post-transcriptionally. All nuclear encoded gene transcripts studied to date possess a 5' <it>trans</it>-spliced leader (SL) sequence suggestive, based on the trypanosome model, of post-transcriptional regulation. The current study therefore sought to determine if the transcriptome of <it>K. brevis </it>is responsive to nitrogen and phosphorus and is informative of nutrient status.</p> <p>Results</p> <p>Microarray analysis of N-depleted <it>K. brevis </it>cultures revealed an increase in the expression of transcripts involved in N-assimilation (nitrate and ammonium transporters, glutamine synthetases) relative to nutrient replete cells. In contrast, a transcriptional signal of P-starvation was not apparent despite evidence of P-starvation based on their rapid growth response to P-addition. To study transcriptome responses to nutrient addition, the limiting nutrient was added to depleted cells and changes in global gene expression were assessed over the first 48 hours following nutrient addition. Both N- and P-addition resulted in significant changes in approximately 4% of genes on the microarray, using a significance cutoff of 1.7-fold and p ≤ 10^-4. By far, the earliest responding genes were dominated in both nutrient treatments by pentatricopeptide repeat (PPR) proteins, which increased in expression up to 3-fold by 1 h following nutrient addition. PPR proteins are nuclear encoded proteins involved in chloroplast and mitochondria RNA processing. Correspondingly, other functions enriched in response to both nutrients were photosystem and ribosomal genes.</p> <p>Conclusions</p> <p>Microarray analysis provided transcriptomic evidence for N- but not P-limitation in <it>K. brevis</it>. Transcriptomic responses to the addition of either N or P suggest a concerted program leading to the reactivation of chloroplast functions. Even the earliest responding PPR protein transcripts possess a 5' SL sequence that suggests post-transcriptional control. Given the current state of knowledge of dinoflagellate gene regulation, it is currently unclear how these rapid changes in such transcript levels are achieved.</p

Proteomic Analyses of Host and Pathogen Responses during Bovine Mastitis

The pursuit of biomarkers for use as clinical screening tools, measures for early detection, disease monitoring, and as a means for assessing therapeutic responses has steadily evolved in human and veterinary medicine over the past two decades. Concurrently, advances in mass spectrometry have markedly expanded proteomic capabilities for biomarker discovery. While initial mass spectrometric biomarker discovery endeavors focused primarily on the detection of modulated proteins in human tissues and fluids, recent efforts have shifted to include proteomic analyses of biological samples from food animal species. Mastitis continues to garner attention in veterinary research due mainly to affiliated financial losses and food safety concerns over antimicrobial use, but also because there are only a limited number of efficacious mastitis treatment options. Accordingly, comparative proteomic analyses of bovine milk have emerged in recent years. Efforts to prevent agricultural-related food-borne illness have likewise fueled an interest in the proteomic evaluation of several prominent strains of bacteria, including common mastitis pathogens. The interest in establishing biomarkers of the host and pathogen responses during bovine mastitis stems largely from the need to better characterize mechanisms of the disease, to identify reliable biomarkers for use as measures of early detection and drug efficacy, and to uncover potentially novel targets for the development of alternative therapeutics. The following review focuses primarily on comparative proteomic analyses conducted on healthy versus mastitic bovine milk. However, a comparison of the host defense proteome of human and bovine milk and the proteomic analysis of common veterinary pathogens are likewise introduced

Epithelial TGFβ engages growth-factor signalling to circumvent apoptosis and drive intestinal tumourigenesis with aggressive features

The pro-tumourigenic role of epithelial TGFβ signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFβ signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFβ signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFβ signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFβ signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC’s with born to be bad traits