Culex quinquefasciatus Late Trypsin Biosynthesis Is Translationally Regulated by Trypsin Modulating Oostatic Factor

Trypsin is a serine protease that is synthesized by the gut epithelial cells of female mosquitoes; it is the enzyme that digests the blood meal. To study its molecular regulation, Culex quinquefasciatus late trypsin was purified by diethylaminoethyl (DEAE), affinity, and C18 reverse-phase high performance liquid chromatography (HPLC) steps, and the N-terminal amino acid sequence was determined for molecular cloning. Five overlapping segments of the late trypsin cDNA were amplified by PCR, cloned, and the full sequence (855 bp) was characterized. Three-dimensional models of the pro-trypsin and activated trypsin were built and compared with other trypsin models. Trypsin modulating oostatic factor (TMOF) concentrations in the hemolymph were determined by ELISA and compared with trypsin activity in the gut after the blood meal. The results showed that there was an increase in TMOF concentrations circulating in the hemolymph which has correlated to the reduction of trypsin activity in the mosquito gut. Northern blot analysis of the trypsin transcripts after the blood meal indicated that trypsin activity also followed the increase and decrease of the trypsin transcript. Injections of different amounts of TMOF (0.025 to 50 μg) decreased the amounts of trypsin in the gut. However, Northern blot analysis showed that TMOF injections did not cause a decrease in trypsin transcript abundance, indicating that TMOF probably affected trypsin translation.

Novel small RNAs expressed by Bartonella bacilliformis under multiple conditions reveal potential mechanisms for persistence in the sand fly vector and human host

Bartonella bacilliformis, the etiological agent of Carrión’s disease, is a Gram-negative, facultative intracellular alphaproteobacterium. Carrión’s disease is an emerging but neglected tropical illness endemic to Peru, Colombia, and Ecuador. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant and repeated environmental shifts during its life cycle, including changes in pH and temperature. In most bacteria, small non-coding RNAs (sRNAs) serve as effectors that may post-transcriptionally regulate the stress response to such changes. However, sRNAs have not been characterized in B. bacilliformis, to date. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. sRNAs included the highly-conserved tmRNA, 6S RNA, RNase P RNA component, SRP RNA component, ffH leader RNA, and the alphaproteobacterial sRNAs αr45 and speF leader RNA. In addition, 153 other potential sRNAs of unknown function were discovered. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonella bacilliformis group I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we demonstrated the molecular targeting of Bartonella bacilliformis small RNA 9 (BbsR9) to transcripts of the ftsH, nuoF, and gcvT genes, in vitro.

Overexpression of transcription factor SlWRKY28 improved the tolerance of Populus davidiana × P. bolleana to alkaline salt stress

Background WRKY transcription factors (TFs) have been suggested to play crucial roles in the response to biotic and abiotic stresses. This study is the first to report the alkaline salt regulation of the WRKY gene. Results In this study, we cloned a WRKY gene (SlWRKY28) from the Salix linearistipularis and then transferred to the Populus davidiana × P. bolleana for expression. Sequence analysis on the transcriptome of Salix linearistipular showed the significant up-regulation of WRKY gene expression in response to salt-alkali stress in seedlings. Our data showed that SlWRKY28 localized to the nucleus. Furthermore, the expression of the SlWRKY28 from female plants increased with saline-alkali stress according to the northern blot analysis results. The results of 3,3′-Diaminobenzidine (DAB) staining showed that hydrogen peroxide (H2O2) concentration was lower under stress, but ascorbate peroxidase (APX) enzyme activity was significantly higher in the overexpressed plants than that in non-transgenic (NT) plants. Conclusions We found out the SlWRKY28 induced regulation of the enzyme gene in the reactive oxygen species (ROS) scavenging pathway is a potential mechanism for transgenic lines to improve their resistance to alkaline salt. This study shows theoretical and practical significance in determining SlWRKY28 transcription factors involved in the regulation of alkaline salt tolerance.

Silencing Dicer-Like Genes Reduces Virulence and sRNA Generation in Penicillium italicum, the Cause of Citrus Blue Mold

The Dicer protein is one of the most important components of RNAi machinery because it regulates the production of small RNAs (sRNAs) in eukaryotes. Here, Dicer1-like gene (Pit-DCL1) and Dicer2-like gene (Pit-DCL2) RNAi transformants were generated via pSilent-1 in Penicillium italicum (Pit), which is the causal agent of citrus blue mold. Neither transformant showed a change in mycelial growth or sporulation ability, but the pathogenicity of the Pit-DCL2 RNAi transformant to citrus fruits was severely impaired, compared to that of the Pit-DCL1 RNAi transformant and the wild type. We further developed a citrus wound-mediated RNAi approach with a double-stranded fragment of Pit-DCL2 generated in vitro, which achieved an efficiency in reducing Pi-Dcl2 expression and virulence that was similar to that of protoplast-mediated RNAi in P. italicum, suggesting that this approach is promising in the exogenous application of dsRNA to control pathogens on the surface of citrus fruits. In addition, sRNA sequencing revealed a total of 69.88 million potential sRNAs and 12 novel microRNA-like small RNAs (milRNAs), four of which have been predicated on target innate immunity or biotic stress-related genes in Valencia orange. These data suggest that both the Pit-DCL1 and Pit-DCL2 RNAi transformants severely disrupted the biogenesis of the potential milRNAs, which was further confirmed for some milRNAs by qRT-PCR or Northern blot analysis. These data suggest the sRNAs in P. italicum that may be involved in a molecular virulence mechanism termed cross-kingdom RNAi (ck-RNAi) by trafficking sRNA from P. italicum to citrus fruits.

Development of nematode resistance in Arabidopsis by HD-RNAi-mediated silencing of the effector gene Mi-msp2

Root-knot nematodes (RKNs) are devastating parasites that infect thousands of plants. As RKN infection is facilitated by oesophageal gland effector genes, one such effector gene, Mi-msp2, was selected for a detailed characterization. Based on domain analysis, the Mi-MSP2 protein contains an ShKT domain, which is likely involved in blocking K+ channels and may help in evading the plant defence response. Expression of the Mi-msp2 gene was higher in juveniles (parasitic stage of RKNs) than in eggs and adults. Stable homozygous transgenic Arabidopsis lines expressing Mi-msp2 dsRNA were generated, and the numbers of galls, females and egg masses were reduced by 52–54%, 60–66% and 84–95%, respectively, in two independent RNAi lines compared with control plants. Furthermore, expression analysis revealed a significant reduction in Mi-msp2 mRNA abundance (up to 88%) in female nematodes feeding on transgenic plants expressing dsRNA, and northern blot analysis confirmed expression of the Mi-msp2 siRNA in the transgenic plants. Interestingly, a significant reduction in the reproduction factor was observed (nearly 40-fold). These data suggest that the Mi-msp2 gene can be used as a potential target for RKN management in crops of economic importance.

Northern Blot Analysis

The whole workflow for the quantification of specific transcripts by Northern blot analysis is described in detail, including RNA isolation, probe generation via labelling with Dig-dUTP, hybridization, signal visualization and quantification.

Molecular Switch Controlling Expression of the Mannose-Specific Adhesin, Msa, inLactobacillus plantarum

ABSTRACTSome lactic acid bacteria, especiallyLactobacillusspp., possess adhesive properties enabling colonization of the human gastrointestinal tract. Two probioticLactobacillus plantarumstrains, WCSF1 and 299v, display highly different mannose-specific adhesion, withL. plantarum299v being superior toL. plantarumWCFS1 based on a yeast agglutination assay. A straightforward correlation between the mannose adhesion capacity and domain composition of the mannose-specific adhesin (Msa) in the two strains has not been demonstrated previously. In this study, we analyzed the promoter regions upstream of themsagene encoding a mannose-specific adhesin in these two strains. The promoter region was mapped by primer extension and DNA sequence analysis, and only a single nucleotide change was identified between the two strains. However, Northern blot analysis showed a strongermsatranscript band in 299v than in WCFS1 correlating with the different adhesion capacities. During the establishment of a high-throughput yeast agglutination assay, we isolated variants of WCFS1 that displayed a very strong mannose-specific adhesion phenotype. The region upstream of themsagene in these variants showed an inversion of a 104-bp fragment located between two perfectly inverted repeats present in the untranslated leader region. The inversion disrupts a strong hairpin structure that otherwise most likely would terminate themsatranscript. In addition, the ribosome binding site upstream of themsagene, which is also masked within this hairpin structure, becomes accessible upon inversion, thereby increasing the frequency of translation initiation in the variant strains. Furthermore, Northern blot analysis showed a higher abundance of themsatranscript in the variants than in the wild type, correlating with a strong-Msa phenotype.IMPORTANCEProbiotic strains possess adhesive properties enabling colonization of the human intestinal tract through interactions between molecules present on the probiotic bacteria and components of the epithelial surface. InLactobacillus plantarum, interaction is mediated through bacterial surface proteins like Msa, which binds to mannose residues present on the intestinal cells. Such interactions are believed to be important for the health-promoting effects of probiotics, including displacement of pathogens, immunomodulation, and protective effects on the intestinal barrier function. In this study, we have identified a new molecular switch controlling expression of themsagene inL. plantarumstrain WCFS1. Strains with increasedmsaexpression could be valuable in the development and manufacture of improved probiotic products.

Comparative analysis of mitochondrial genome between UG93A and UG93B reveal common feature of 5’-end heterogeneity in mitochondrial genes of kenaf

Plant cytoplasmic male sterility (CMS) being maternal phenomenon trait that result from pollen abortion and closely linked with mitochondrial DNA rearrangement in many crops including kenaf. However, the molecular mechanism in kenaf is poorly known. In present work, we described the mitochondrial genome in isonuclear CMS line UG93A and its maintainer line UG93B. Findings of the current study revealed that a total of 398 SNPs and 230 InDels were identified in UG93A mtDNA. Total of 26 SNPs variations and three InDels were identified in the coding region of atp6, indicating its active role in mitochondrial genome re-arrangement. Northern blot analysis showed that the transcripts of atp1, atp4, atp6, cox3 and sdh4 in F1 were consistent with UG93A but different for UG93B. The transcript of atp9 was found similar between UG93B and F1 while different for UG93A, which depict that atp9 may be regulated by nuclear genes in F1 hybrid. The expression of atp9 in UG93A was substantially lower compared with UG93B, suggesting its key role for energy supplying in microspore development of kenaf. Circularized RNA (CR)-RT-PCR revealed that mitochondrial RNAs with heterogeneous 5’-ends but uniform 3’ - ends are common feature in kenaf mitochondrial genes, and the promoter architecture analysis showed that the promoter sequences in kenaf mitochondrial genome are highly diverged in comparison to those in other plants. Our data highlight that the translation of mitochondrial genes in kenaf is closely associated with heterogeneity of the 5’-end of plant mRNA. The present result provides the basic information in understanding the transcription of kenaf mitochondrial genome and can be used as reference in other plants.