The specificity of the double-stranded RNA (dsRNA) used in the RNA interference (RNAi) technique is crucial for the success of sequence-specific gene silencing.  Currently, RNAi-mediated insect control is a trending research topic.  However, the off-target effects of the dsRNA in RNAi are a major concern.  In this study, the dsHvβ´COPI (coat protein complex I, β´ subunit)-treated and untreated transcriptomes of the 28-spotted potato lady beetle (Henosepilachna vigintioctopunctata) were compared to understand its off-target gene silencing effects.  The RNA-seq results revealed that 63 and 44 differentially expressed genes (DEGs) were upregulated and downregulated, respectively, in the dsHvβ´COPI treated group as compared with the control.  Validation of the differential expressions of some selected DEGs via reverse transcription-quantitative PCR (RT-qPCR) analysis confirmed the reliability of the transcriptome analysis results.  Further downstream analysis revealed that there were no genes homologous with Hvβ´COPI in H. vigintioctopunctata.  Additionally, no genes with a >11 bp continuous match with dsHvβ´COPI were found in the H. vigintioctopunctata transcriptome.  Six genes (Hvcitron, Hvhelicase, Hvtransposase, Hvserine, Hvdynein, and HvE3 ubiquitin) were selected to examine the off-target activity of dsHvβ´COPI based on their potential involvement in various H. vigintioctopunctata metabolic pathways.  The severity of silencing these six off-target genes was evaluated by employing RNAi.  The RNAi results confirmed the downregulation of the expression of all six genes, although there was no significant lethality.  The findings of this study will be helpful in the risk analysis of future RNAi-mediated pest control experiments.

A number of plant pathogenic species of Phytophthora are known to produce different classes of secretory proteins during interactions with their hosts.  Although several small cysteine-rich (SCR) secretory proteins, conserved in oomycete pathogens, have been identified in Phytophthora, their specific involvement in these interactions remains unknown.  In this study, an SCR effector encoded by Pnscr82 in P. nicotianae was identified and shown to have similarities to P. cactorum phytotoxic protein, PcF (Phytophthora cactorum Fragaria).  Agroinfection with potato virus X vector, PnSCR82, was capable of inducing plant hypersensitive cell death in Nicotiana benthamiana and Solanum lycopersicum.  Real-time PCR results indicated that transiently expressed PnSCR82 in N. benthamiana leaves activated the jasmonate, salicylic acid and ethylene signaling pathways.  Transient expression of PnSCR82 enhanced plant resisitance to P. capsici.  In summary, our results demonstrated that P. nicotianae PnSCR82 elicits defensive responses in N. benthamiana and may potentially play a significant role in future crop protection programs.

A recent breakthrough in agricultural biotechnology is the introduction of RNAi-mediated strategies in pest control.  However, the off-target effects of RNAi pest control are still not fully understood.  Here, we studied the off-target effects of two insecticidal siRNAs in both target and non-target insects.  The results revealed that off-target effects of insecticidal siRNAs occur widely in both target and non-target insects.  We classified the expression-changed genes according to their homology to the siRNA-targeted gene, related KEGG pathways with the siRNA-targeted gene and continuous matches with siRNAs.  Surprisingly, the unintended significant changes in gene expression levels did not strictly match with the number of contiguous nucleotides in the siRNAs.  As expected, the expression of small portions of the homologous and KEGG-related genes were significantly changed.  We calculated the Shannon entropy of the transcriptome profile of the insects after injecting them with insecticidal siRNAs.  Though hundreds of genes were affected in their expression levels post siRNA-treatment, the Shannon entropy of the transcriptome remained unchanged, suggesting that the transcriptome expression was balanced.  Our results provide evidence that siRNAs cross-reacted with individual genes in non-target species, but did not have significant effects on the integrity of the transcriptome profiles in either target or non-target species on a genomic scale.  The metric we proposed can be used to estimate the off-target effects of insecticidal siRNAs, which might be useful for evaluating the safety of RNAi in pest control.  

Methylation of the N6 position of adenine, termed N6-methyladenine, protects DNA from restriction endonucleases via the host-specific restriction-modification system. N6-methyladenine was discovered and has been well studied in bacteria. N6-adenine-specific DNA methyltransferase (N6AMT) is the main enzyme catalyzing the methylation of the adenine base and knowledge of this enzyme was mainly derived from work in prokaryotic models. However, large-scale gene discovery at the genome level in many model organisms indicated that the N6AMT gene also exists in eukaryotes, such as humans, mice, fruit flies and plants. Here, we cloned a N6AMT gene from Nilaparvata lugens (Nlu-N6AMT) and amplified its fulllength transcript. Then, we carried out a systematic investigation of N6AMT in 33 publically available insect genomes, indicating that all studied insects had N6AMT. Genomic structure analysis showed that insect N6AMT has short introns compared with the mammalian homologs. Domain and phylogenetic analysis indicated that insect N6AMT had a conserved N6-adenineMlase domain that is specific to catalyze the adenine methylation. Nlu-N6AMT was highly expressed in the adult female. We knocked down Nlu-N6AMT by feeding dsRNA from the second instar nymph to adult female, inducing retard development of adult female. In all, we provide the first genome-wide analysis of N6AMT in insects and presented the experimental evidence that N6AMT might have important functions in reproductive development and ovary maturation.

The normalization of quantitative real-time PCR (qPCR) is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions. qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, five new housekeeping genes (LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9) in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable than β-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantification. These results were further confirmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantification by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 or β-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replace β-actin as the reference genes for qPCR in L. striatellus.

In order to determine the potential for haploid induction via in vitro gynogenesis in tomato, the ovules and protoplasts of embryo sacs from the hybrids Zhongza 101 and Zhongza 105 were cultured. An efficient method of ovule isolation was established in this study. Using this method, 100-150 ovules could be isolated from one ovary. Isolated ovules were cultured on three induction media to induce gynogenesis in vitro. During culture, ovules were enlarged markedly, with opaque white color. When observed microscopically, there were cell divisions and cell clumps in embryo sacs. Subsequently, the cell clumps in embryo sacs ceased growth, likely because the integument grew faster than embryo sacs did and hindered the further development of embryo sacs. Therefore, subsequent callus morphogenesis might be originated from the integument. Thousands of calli from the two tomato varieties were obtained. Five diploid plants were regenerated after 15 months of subculturing. To eliminate the hindering effect of integument on embryo sac cells, the protoplasts of embryo sacs were prepared and cultured. After 48 hours of culture, the protoplasts of embryo sacs doubled in size and gradually formed clusters of cells. These results suggested that gynogenesis might be a potential way for haploid induction in tomato.