Sex specific expression and distribution of small RNAs in papaya

Background

Regulatory function of small non-coding RNAs (sRNA) in response to environmental and developmental cues has been established. Additionally, sRNA, also plays an important role in maintaining the heterochromatin and centromere structures of the chromosome. Papaya, a trioecious species with recently evolved sex chromosomes, has emerged as an excellent model system to study sex determination and sex chromosome evolution in plants. However, role of small RNA in papaya sex determination is yet to be explored.

Results

We analyzed the high throughput sRNAs reads in the Illumina libraries prepared from male, female, and hermaphrodite flowers of papaya. Using the sRNA reads, we identified 29 miRNAs that were not previously reported from papaya. Including this and two previous studies, a total of 90 miRNAs has been identified in papaya. We analyzed the expression of these miRNAs in each sex types. A total of 65 miRNAs, including 31 conserved and 34 novel mirNA, were detected in at least one library. Fourteen of the 65 miRNAs were differentially expressed among different sex types. Most of the miRNA expressed higher in male flowers were related to the auxin signaling pathways, whereas the miRNAs expressed higher in female flowers were the potential regulators of the apical meristem identity genes. Aligning the sRNA reads identified the sRNA hotspots adjacent to the gaps of the X and Y chromosomes. The X and Y chromosomes sRNA hotspots has a 7.8 and 4.4 folds higher expression of sRNA, respectively, relative to the chromosome wide average. Approximately 75% of the reads aligned to the X chromosome hotspot was identical to that of the Y chromosome hotspot.

Conclusion

By analyzing the large-scale sRNA sequences from three sex types, we identified the sRNA hotspots flanking the gaps of papaya X, Y, and Y^h chromosome. The sRNAs expression patterns in these regions were reminiscent of the pericentromeric region indicating that the only remaining gap in each of these chromosomes is likely the centromere. We also identified 14 differentially expressed miRNAs in male, female and hermaphrodite flowers of papaya. Our results provide valuable information toward understanding the papaya sex determination.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-20) contains supplementary material, which is available to authorized users.     

Activation of ethylene‐related genes in response to aphid feeding on resistant and susceptible melon and tomato plants

The simple gaseous compound ethylene (ET) has long been recognized as a common component of plant responses to insect feeding and pathogen attack. However, it is presently uncertain whether it plays a role in host–plant resistance to piercing–sucking insects such as aphids. In these experiments, we investigated the expression of key ET‐associated genes in resistant and susceptible interactions in two model systems: the tomato‐Mi‐Macrosiphum euphorbiae (Thomas) (Hemiptera: Aphididae: Macrosiphini) system and the melon‐virus aphid transmission gene (Vat)‐Aphis gossypii Glover (Hemiptera: Aphididiae: Aphidini) system. We examined expression patterns of genes associated with ET synthesis, perception, signal transduction, and downstream response. When compared with control plants, plants infested with aphids showed marked differences in gene expression. In particular, ET signaling pathway genes and downstream response genes were highly upregulated in the resistant interaction between A. gossypii and Vat⁺, indicating ET may play a role in Vat‐mediated host–plant resistance. A key integrator between the ET and jasmonic acid pathways (Cm‐ERF1) showed the strongest response.     

The diversity of small non-coding RNAs in the diatom Phaeodactylum tricornutum

Background

Marine diatoms constitute a major component of eukaryotic phytoplankton and stand at the crossroads of several evolutionary lineages. These microalgae possess peculiar genomic features and novel combinations of genes acquired from bacterial, animal and plant ancestors. Furthermore, they display both DNA methylation and gene silencing activities. Yet, the biogenesis and regulatory function of small RNAs (sRNAs) remain ill defined in diatoms.

Results

Here we report the first comprehensive characterization of the sRNA landscape and its correlation with genomic and epigenomic information in Phaeodactylum tricornutum. The majority of sRNAs is 25 to 30 nt-long and maps to repetitive and silenced Transposable Elements marked by DNA methylation. A subset of this population also targets DNA methylated protein-coding genes, suggesting that gene body methylation might be sRNA-driven in diatoms. Remarkably, 25-30 nt sRNAs display a well-defined and unprecedented 180 nt-long periodic distribution at several highly methylated regions that awaits characterization. While canonical miRNAs are not detectable, other 21-25 nt sRNAs of unknown origin are highly expressed. Besides, non-coding RNAs with well-described function, namely tRNAs and U2 snRNA, constitute a major source of 21-25 nt sRNAs and likely play important roles under stressful environmental conditions.

Conclusions

P. tricornutum has evolved diversified sRNA pathways, likely implicated in the regulation of largely still uncharacterized genetic and epigenetic processes. These results uncover an unexpected complexity of diatom sRNA population and previously unappreciated features, providing new insights into the diversification of sRNA-based processes in eukaryotes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-698) contains supplementary material, which is available to authorized users.     

An integrated approach for finding overlooked genes in Shigella

Background

The completion of numerous genome sequences introduced an era of whole-genome study. However, many genes are missed during genome annotation, including small RNAs (sRNAs) and small open reading frames (sORFs). In order to improve genome annotation, we aimed to identify novel sRNAs and sORFs in Shigella, the principal etiologic agents of bacillary dysentery.

Methodology/Principal Findings

We identified 64 sRNAs in Shigella, which were experimentally validated in other bacteria based on sequence conservation. We employed computer-based and tiling array-based methods to search for sRNAs, followed by RT-PCR and northern blots, to identify nine sRNAs in Shigella flexneri strain 301 (Sf301) and 256 regions containing possible sRNA genes. We found 29 candidate sORFs using bioinformatic prediction, array hybridization and RT-PCR verification. We experimentally validated 557 (57.9%) DOOR operon predictions in the chromosomes of Sf301 and 46 (76.7%) in virulence plasmid.We found 40 additional co-expressed gene pairs that were not predicted by DOOR.

Conclusions/Significance

We provide an updated and comprehensive annotation of the Shigella genome. Our study increased the expected numbers of sORFs and sRNAs, which will impact on future functional genomics and proteomics studies. Our method can be used for large scale reannotation of sRNAs and sORFs in any microbe with a known genome sequence.     

Identification and Comparative Analysis of the Tegillarca granosa Haemocytes MicroRNA Transcriptome in Response to Cd Using a Deep Sequencing Approach

Background

MicroRNAs (miRNAs) are endogenous non-coding small RNAs (sRNAs) that can base pair with their target mRNAs, which represses their translation or induces their degradation in various biological processes. To identify miRNAs regulated by heavy metal stress, we constructed two sRNA libraries for the blood clam Tegillarca granosa: one for organisms exposed to toxic levels of cadmium (Cd) and one for a control group.

Results

Sequencing of the two libraries and subsequent analysis revealed 215 conserved and 39 new miRNAs. Most of the new miRNAs in T. granosa were up- or down-regulated in response to Cd exposure. There were significant differences in expression between the Cd and control groups for 16 miRNAs. Of these, five miRNAs were significantly up-regulated and 11 were significantly down-regulated in the Cd stress library. Potential targets were predicted for the 16 differential miRNAs in pre-miRNAs identified according to sequence homology. Some of the predicted miRNA targets are associated with regulation of the response to stress induced by heavy metals. Five differentially expressed miRNAs (Tgr-nmiR-8, Tgr-nmiR-21, Tgr-miR-2a, Tgr-miR-10a-5p, and Tgr-miR-184b) were validated by qRT-PCR.

Conclusion

Our study is the first large-scale identification of miRNAs in T. granosa haemocytes. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in the responses of this species to environmental heavy metal stresses.     

sTarPicker: a method for efficient prediction of bacterial sRNA targets based on a two-step model for hybridization

Background

Bacterial sRNAs are a class of small regulatory RNAs involved in regulation of expression of a variety of genes. Most sRNAs act in trans via base-pairing with target mRNAs, leading to repression or activation of translation or mRNA degradation. To date, more than 1,000 sRNAs have been identified. However, direct targets have been identified for only approximately 50 of these sRNAs. Computational predictions can provide candidates for target validation, thereby increasing the speed of sRNA target identification. Although several methods have been developed, target prediction for bacterial sRNAs remains challenging.

Results

Here, we propose a novel method for sRNA target prediction, termed sTarPicker, which was based on a two-step model for hybridization between an sRNA and an mRNA target. This method first selects stable duplexes after screening all possible duplexes between the sRNA and the potential mRNA target. Next, hybridization between the sRNA and the target is extended to span the entire binding site. Finally, quantitative predictions are produced with an ensemble classifier generated using machine-learning methods. In calculations to determine the hybridization energies of seed regions and binding regions, both thermodynamic stability and site accessibility of the sRNAs and targets were considered. Comparisons with the existing methods showed that sTarPicker performed best in both performance of target prediction and accuracy of the predicted binding sites.

Conclusions

sTarPicker can predict bacterial sRNA targets with higher efficiency and determine the exact locations of the interactions with a higher accuracy than competing programs. sTarPicker is available at http://ccb.bmi.ac.cn/starpicker/.     

Characterization of Small RNAs and Their Targets from <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> Infected and Noninfected Cotton Root Tissues

Genes for host-plant resistant do exist in cotton (Gossypium spp.) but improvement of cotton cultivars with resistance is difficult due to intensive breeding. Identifying molecular-genetic mechanisms associated with disease resistance can offer a new way to combat a serious threat such as Fusarium oxysporum f. sp. vasinfectum (FOV). Here, we captured and annotated “top-layer” of abundantly and specifically expressed cotton root small RNA (sRNA) including microRNA (miR) sequences during FOV pathogenesis using size-directed and adenylated linker-based sRNA cloning strategy. A total of 4116 candidate sRNA sequences with 16 to 30 nucleotide (nt) length were identified from four complementary DNA (cDNA) libraries of noninfected and FOV race 3-infected roots of susceptible (“11970”) versus resistant (“Mebane B-1”) cotton genotypes (G. hirsutum L.). The highest numbers of sRNA signatures were those with 19–24 nt long in all libraries, and interestingly, the number of sRNAs substantially increased during FOV infection in a resistant genotype, while it sharply decreased in a susceptible genotype. In BLAST analysis, more than 73 % of sRNAs matched Gossypium (G. arboretum L., G. hirsutum, and G. barbadense L.) ESTs. A small percentage of sRNAs matched A. thaliana (1.68 %), T. cacao (1.26 %), fungal (2 %), and other organism (21.33 %) ESTs. mirBase comparisons showed that 4 % of sRNAs were homologous to previously reported plant miRs, among which we predicted novel cotton Ghr-miR-160 that was not registered in the cotton miR database. These major representative sRNA signatures targeted proteins associated with the key biological processes and molecular functions, explaining the molecular mechanisms of the host defense response during the FOV pathogenesis in cotton.     

Oral Delivery Mediated RNA Interference of a Carboxylesterase Gene Results in Reduced Resistance to Organophosphorus Insecticides in the Cotton Aphid,Aphis gossypii Glover

Background

RNA interference (RNAi) is an effective tool to examine the function of individual genes. Carboxylesterases (CarE, EC 3.1.1.1) are known to play significant roles in the metabolism of xenobiotic compounds in many insect species. Previous studies in our laboratory found that CarE expression was up-regulated in Aphis gossypii (Glover) (Hemiptera: Aphididae) adults of both omethoate and malathion resistant strains, indicating the potential involvement of CarE in organophosphorus (OP) insecticide resistance. Functional analysis (RNAi) is therefore warranted to investigate the role of CarE in A. gossypii to OPs resistance.

Result

CarE expression in omethoate resistant individuals of Aphis gossypii was dramatically suppressed following ingestion of dsRNA-CarE. The highest knockdown efficiency (33%) was observed at 72 h after feeding when dsRNA-CarE concentration was 100 ng/µL. The CarE activities from the CarE knockdown aphids were consistent with the correspondingly significant reduction in CarE expression. The CarE activity in the individuals of control aphids was concentrated in the range of 650–900 mOD/per/min, while in the individuals of dsRNA-CarE-fed aphids, the CarE activity was concentrated in the range of 500–800 mOD/per/min. In vitro inhibition experiments also demonstrated that total CarE activity in the CarE knockdown aphids decreased significantly as compared to control aphids. Bioassay results of aphids fed dsRNA-CarE indicated that suppression of CarE expression increased susceptibility to omethoate in individuals of the resistant aphid strains.

Conclusion

The results of this study not only suggest that ingestion of dsRNA through artificial diet could be exploited for functional genomic studies in cotton aphids, but also indicate that CarE can be considered as a major target of organophosphorus insecticide (OPs) resistance in A. gossypii. Further, our results suggest that the CarE would be a propitious target for OPs resistant aphid control, and insect-resistant transgenic plants may be obtained through plant RNAi-mediated silencing of insect CarE expression.     

Genome-wide identification of vegetative phase transition-associated microRNAs and target predictions using degradome sequencing in Malus hupehensis

Background

A long juvenile period between germination and flowering is a common characteristic among fruit trees, including Malus hupehensis (Pamp.) Rehd., which is an apple rootstock widely used in China. microRNAs (miRNAs) play an important role in the regulation of phase transition and reproductive growth processes.

Results

M. hupehensis RNA libraries, one adult and one juvenile phase, were constructed using tree leaves and underwent high-throughput sequencing. We identified 42 known miRNA families and 172 novel miRNAs. We also identified 127 targets for 25 known miRNA families and 168 targets for 35 unique novel miRNAs using degradome sequencing. The identified miRNA targets were categorized into 58 biological processes, and the 123 targets of known miRNAs were associated with phase transition processes. The KEGG analysis revealed that these targets were involved in starch and sucrose metabolism, and plant hormone signal transduction. Expression profiling of miRNAs and their targets indicated multiple regulatory functions in the phase transition. The higher expression level of mdm-miR156 and lower expression level of mdm-miR172 in the juvenile phase leaves implied that these two small miRNAs regulated the phase transition. mdm-miR160 and miRNA393, which regulate genes involved in auxin signal transduction, could also be involved in controlling this process. The identification of known and novel miRNAs and their targets provides new information on this regulatory process in M. hupehensis, which will contribute to the understanding of miRNA functions during growth, phase transition and reproduction in woody fruit trees.

Conclusions

The combination of sRNA and degradome sequencing can be used to better illustrate the profiling of hormone-regulated miRNAs and miRNA targets involving complex regulatory networks, which will contribute to the understanding of miRNA functions during growth, phase transition and reproductive growth in perennial woody fruit trees.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1125) contains supplementary material, which is available to authorized users.     

Association between Aphis gossypii genotype and phenotype on melon accessions

Development of molecular markers has allowed the characterization of several host–aphid interactions. We investigated the usefulness of microsatellite markers to characterize the plant resistance interaction in the model Aphis gossypii/Cucumis melo. Six aphid clones, collected in different localities and years and belonging to two multilocus genotypes (MLGs) based on eight microsatellite markers, were phenotyped on a set of 33 melon accessions, some of them known to carry the Vat gene. Three parameters were used: acceptance of plant, ability to colonize the plant and resistance to virus when inoculated by aphids. Concordance and correlation analyses showed that aphid clones sharing a same MLG exhibited a very agreeable phenotype on the set of accessions for acceptance of plant and resistance to virus when inoculated by aphids. From host point of view, melon accessions were grouped into four clear categories, resistant to aphids of both MLGs, only resistant to the NM1 MLG, only resistant to the C9 MLG, susceptible to both MLGs and another group of unclear characteristics. The four categories revealed different patterns of virulence for NM1 and C9 MLGs that are likely controlled by a single avirulence gene in accordance with a gene for gene interaction. In contrast, the ability to colonize the plant appeared slightly variable among clones sharing a same MLG. We hypothesize it is due to the putative polygenic control of this aphid trait. Because the phenotypic variability of A. gossypii matched the genetic variability revealed by eight microsatellite markers, these markers could be used to infer the frequency of biotypes in field experiments and help to elucidate the allele diversity of melon resistance genes.