OsmiR319-OsPCF5 modulate resistance to brown planthopper in rice through association with MYB proteins.

BACKGROUND: The brown planthopper (BPH) is a kind of piercing-sucking insect specific to rice, with the damage tops the list of pathogens and insects in recent years. microRNAs (miRNAs) are pivotal regulators of plant-environment interactions, while the mechanism underlying their function against insects is largely unknown. RESULTS: Here, we confirmed that OsmiR319, an ancient and conserved miRNA, negatively regulated resistance to BPHs, with overexpression of OsmiR319 susceptible to BPH, while suppression of OsmiR319 resistant to BPH in comparison with wild type. Meanwhile, we identified several targets of OsmiR319 that may mediate BPH resistance. Among them, OsPCF5 was the most obviously induced by BPH feeding, and over expression of OsPCF5 was resistance to BPH. In addition, various biochemical assays verified that OsPCF5 interacted with several MYB proteins, such as OsMYB22, OsMYB30, and OsMYB30C.Genetically, we revealed that both OsMYB22 and OsMYB30C positively regulated BPH resistance. Genetic interaction analyses confirmed that OsMYB22 and OsMYB30C both function in the same genetic pathway with OsmiR319b to mediate BPH resistance. CONCLUSIONS: Altogether, we revealed that OsPCF5 regulates BPH resistance via association with several MYB proteins downstream of OsmiR319, these MYB proteins might function as regulators of BPH resistance through regulating the phenylpropane synthesis.

InClust+: the deep generative framework with mask modules for multimodal data integration, imputation, and cross-modal generation.

BACKGROUND: With the development of single-cell technology, many cell traits can be measured. Furthermore, the multi-omics profiling technology could jointly measure two or more traits in a single cell simultaneously. In order to process the various data accumulated rapidly, computational methods for multimodal data integration are needed. RESULTS: Here, we present inClust+, a deep generative framework for the multi-omics. It's built on previous inClust that is specific for transcriptome data, and augmented with two mask modules designed for multimodal data processing: an input-mask module in front of the encoder and an output-mask module behind the decoder. InClust+ was first used to integrate scRNA-seq and MERFISH data from similar cell populations, and to impute MERFISH data based on scRNA-seq data. Then, inClust+ was shown to have the capability to integrate the multimodal data (e.g. tri-modal data with gene expression, chromatin accessibility and protein abundance) with batch effect. Finally, inClust+ was used to integrate an unlabeled monomodal scRNA-seq dataset and two labeled multimodal CITE-seq datasets, transfer labels from CITE-seq datasets to scRNA-seq dataset, and generate the missing modality of protein abundance in monomodal scRNA-seq data. In the above examples, the performance of inClust+ is better than or comparable to the most recent tools in the corresponding task. CONCLUSIONS: The inClust+ is a suitable framework for handling multimodal data. Meanwhile, the successful implementation of mask in inClust+ means that it can be applied to other deep learning methods with similar encoder-decoder architecture to broaden the application scope of these models.

A novel transcriptional repressor complex MYB22-TOPLESS-HDAC1 promotes rice resistance to brown planthopper by repressing F3'H expression.

The brown planthopper (BPH) is the most destructive pest of rice. The MYB transcription factors are vital for rice immunity, but most are activators. Although MYB22 positively regulates rice resistance to BPH and has an EAR motif associated with active repression, it remains unclear whether it is a transcriptional repressor affecting rice-BPH interaction. Genetic analyses revealed that MYB22 regulates rice resistance to BPH via its EAR motif. Several biochemical experiments (e.g. transient transcription assay, Y2H, LCA, and BiFC) indicated that MYB22 is a transcriptional repressor that interacts with the corepressor TOPLESS via its EAR motif and recruits HDAC1 to form a tripartite complex. Flavonoid-3'-hydroxylase (F3'H) is a flavonoid biosynthesis pathway-related gene that negatively regulates rice resistance to BPH. Based on a bioinformatics analysis and the results of EMSA and transient transcription assays, MYB22 can bind directly to the F3'H promoter and repress gene expression along with TOPLESS and HDAC1. We revealed a transcriptional regulatory mechanism influencing the rice-BPH interaction that differs from previously reported mechanisms. Specifically, MYB22-TOPLESS-HDAC1 is a novel transcriptional repressor complex with components that synergistically and positively regulate rice resistance to BPH through the transcriptional repression of F3'H.

OsmiR396/growth regulating factor modulate rice grain size through direct regulation of embryo-specific miR408.

microRNAs (miRNAs) are promising targets for crop improvement of complex agricultural traits. Coordinated activity between/among different miRNAs may fine-tune specific developmental processes in diverse organisms. Grain size is a main factor determining rice (Oryza sativa L.) crop yield, but the network of miRNAs influencing this trait remains uncharacterized. Here we show that sequestering OsmiR396 through target mimicry (MIM396) can substantially increase grain size in several japonica and indica rice subspecies and in plants with excessive tillers and a high panicle density. Thus, OsmiR396 has a major role related to the regulation of rice grain size. The grain shape of Growth Regulating Factor8 (OsGRF8)-overexpressing transgenic plants was most similar to that of MIM396 plants, suggesting OsGRF8 is a major mediator of OsmiR396 in grain size regulation. A miRNA microarray analysis revealed changes to the expression of many miRNAs, including OsmiR408, in the MIM396 plants. Analyses of gene expression patterns and functions indicated OsmiR408 is an embryo-specific miRNA that positively regulates grain size. Silencing OsmiR408 expression (miR408KO) using CRISPR technology resulted in small grains. Moreover, we revealed the direct regulatory effects of OsGRF8 on OsMIR408 expression. A genetic analysis further showed that the large-grain phenotype of MIM396 plants could be complemented by miR408KO. Also, several hormone signaling pathways might be involved in the OsmiR396/GRF-meditated grain size regulation. Our findings suggest that genetic regulatory networks comprising various miRNAs, such as OsmiR396 and OsmiR408, may be crucial for controlling rice grain size. Furthermore, the OsmiR396/GRF module may be important for breeding new high-yielding rice varieties.

Identification of the rice genes and metabolites involved in dual resistance against brown planthopper and rice blast fungus.

Brown planthopper (BPH) and blast disease jointly or individually cause big yield losses every year. To identify genes and metabolites with potential contributions to the dual resistance against both biotic-stress factors, we carried out a transcriptome and metabolome analysis for susceptible and resistant rice varieties after BPH and rice blast infestations. Coexpression network analysis identified a modular pattern that had the highest correlation coefficients (0.81) after the BPH and rice blast (-0.81) treatments. In total, 134 phenylpropanoid biosynthesis pathway-related genes were detected in this group. We found that the flavanone 3-hydroxylase gene (OsF3H) had opposite expression trends in response to BPH and rice blast infestations whereas the OsF3'H had similar expression patterns. Genetics analysis confirmed that the OsF3H gene knockdown lines demonstrated the opposite resistance phenotypes against BPH and rice blast, whereas the OsF3'H knockout lines enhanced rice resistance against both pests. Consistently, our metabolomics analysis identified the metabolite eriodictyol, one putative essential product of these two genes, that was more highly accumulated in the resistant rice variety of RHT than in the susceptible variety MDJ. This study highlights a useful strategy for identifying more genes and metabolites that have potential synergistic effects on rice against to multiple biotic stresses.

DSMNC: a database of somatic mutations in normal cells.

Numerous non-inherited somatic mutations, distinct from those of germ-line origin, occur in somatic cells during DNA replication per cell-division. The somatic mutations, recording the unique genetic cell-lineage 'history' of each proliferating normal cell, are important but remain to be investigated because of their ultra-low frequency hidden in the genetic background of heterogeneous cells. Luckily, the recent development of single-cell genomics biotechnologies enables the screening and collection of the somatic mutations, especial single nucleotide variations (SNVs), occurring in normal cells. Here, we established DSMNC: a database of somatic mutations in normal cells (http://dsmnc.big.ac.cn/), which provides most comprehensive catalogue of somatic SNVs in single cells from various normal tissues. In the current version, the database collected ∼0.8 million SNVs accumulated in ∼600 single normal cells (579 human cells and 39 mouse cells). The database interface supports the user-friendly capability of browsing and searching the SNVs and their annotation information. DSMNC, which serves as a timely and valuable collection of somatic mutations in individual normal cells, has made it possible to analyze the burdens and signatures of somatic mutations in various types of heterogeneous normal cells. Therefore, DSMNC will significantly improve our understanding of the characteristics of somatic mutations in normal cells.

Novel crosstalk between ethylene- and jasmonic acid-pathway responses to a piercing-sucking insect in rice.

Ethylene (ET) and jasmonic acid (JA) play important roles in plant defenses against biotic stresses. Crosstalk between JA and ET has been well studied in mediating pathogen resistance, but its roles in piercing-sucking insect resistance are unclear. The brown planthopper (BPH; Nilaparvata lugens) is the most notorious piercing-sucking insect specific to rice (Oryza sativa) that severely affects yield. A genetic analysis revealed that OsEBF1 and OsEIL1, which are in the ET signaling pathway, positively and negatively regulated BPH resistance, respectively. Molecular and biochemical analyses revealed direct interactions between OsEBF1 and OsEIL1. OsEBF1, an E3 ligase, mediated the degradation of OsEIL1 through the ubiquitination pathway, indicating the negative regulation of the ET-signaling pathway in response to BPH infestation. An RNA sequencing analysis revealed that a JA biosynthetic pathway-related gene, OsLOX9, was downregulated significantly in the oseil1 mutant. Biochemical analyses, including yeast one-hybrid, dual luciferase, and electrophoretic mobility shift assay, confirmed the direct regulation of OsLOX9 by OsEIL1. This study revealed the synergistic and negative regulation of JA and ET pathways in response to piercing-sucking insect attack. The synergistic mechanism was realized by transcriptional regulation of OsEIL1 on OsLOX9. OsEIL1-OsLOX9 is a novel crosstalk site in these two phytohormone signaling pathways.

Deficiency of mitochondrial outer membrane protein 64 confers rice resistance to both piercing-sucking and chewing insects in rice.

The brown planthopper (BPH) and striped stem borer (SSB) are the most devastating insect pests in rice (Oryza sativa) producing areas. Screening for endogenous resistant genes is the most practical strategy for rice insect-resistance breeding. Forty-five mutants showing high resistance against BPH were identified in a rice T-DNA insertion population (11,000 putative homozygous lines) after 4 years of large-scale field BPH-resistance phenotype screening. Detailed analysis showed that deficiency of rice mitochondrial outer membrane protein 64 (OM64) gene resulted in increased resistance to BPH. Mitochondrial outer membrane protein 64 protein is located in the outer mitochondrial membrane by subcellular localization and its deficiency constitutively activated hydrogen peroxide (H2 O2 ) signaling, which stimulated antibiosis and tolerance to BPH. The om64 mutant also showed enhanced resistance to SSB, a chewing insect, which was due to promotion of Jasmonic acid biosynthesis and related responses. Importantly, om64 plants presented no significant changes in rice yield-related characters. This study confirmed OM64 as a negative regulator of rice herbivore resistance through regulating H2 O2 production. Mitochondrial outer membrane protein 64 is a potentially efficient candidate to improve BPH and SSB resistance through gene deletion. Why the om64 mutant was resistant to both piercing-sucking and chewing insects via a gene deficiency in mitochondria is discussed.

A nuclease specific to lepidopteran insects suppresses RNAi.

More than 70% of all agricultural pests are insects in the order Lepidoptera, which, unlike other related insect orders, are not very sensitive to RNAi, limiting genetic studies of this insect group. However, the reason for this distinct lepidopteran characteristic is unknown. Previously, using transcriptome analysis of the Asian corn borer Ostrinia furnacalis, we identified a gene, termed up56, that is up-regulated in response to dsRNA. Here we report that this Lepidoptera-specific gene encodes a nuclease that contributes to RNAi insensitivity in this insect order. Its identity was experimentally validated, and sequence analysis indicated that up56 encodes a previously uncharacterized protein with homologous sequences in seven other lepidopteran species. Its computationally predicted three-dimensional structure revealed a high structural similarity to human exonuclease I. Exposure to dsRNA in O. furnacalis strongly up-regulated this gene's expression, and the protein could digest single-stranded RNA (ssRNA), dsRNA, and dsDNA both in vitro and in vivo Of note, we found that this up-regulation of up56 expression is faster than that of the gene encoding the key RNAi-associated nuclease Dicer. up56 knockdown in O. furnacalis significantly enhanced RNAi efficiency. Moreover, up56 overexpression in Drosophila melanogaster suppressed RNAi efficiency. Finally, up56 knockdown significantly increased the amount and diversity of small RNAs. Therefore, we renamed this protein RNAi efficiency-related nuclease (REase). In conclusion, we propose that REase may explain why lepidopterans are refractory to RNAi and that it represents a target for further research of RNAi efficiency in this insect order.

The OsmiR396-OsGRF8-OsF3H-flavonoid pathway mediates resistance to the brown planthopper in rice (Oryza sativa).

Multi-functional microRNAs (miRNAs) are emerging as key modulators of plant-pathogen interactions. Although the involvement of some miRNAs in plant-insect interactions has been revealed, the underlying mechanisms are still elusive. The brown planthopper (BPH) is the most notorious rice (Oryza sativa)-specific insect that causes severe yield losses each year and requires urgent biological control. To reveal the miRNAs involved in rice-BPH interactions, we performed miRNA sequencing and identified BPH-responsive OsmiR396. Sequestering OsmiR396 by overexpressing target mimicry (MIM396) in three genetic backgrounds indicated that OsmiR396 negatively regulated BPH resistance. Overexpression of one BPH-responsive target gene of OsmiR396, growth regulating factor 8 (OsGRF8), showed resistance to BPH. Furthermore, the flavonoid contents increased in both the OsmiR396-sequestered and the OsGRF8 overexpressing plants. By analysing 39 natural rice varieties, the elevated flavonoid contents were found to correlate with enhanced BPH resistance. Artificial applications of flavonoids to wild type (WT) plants also increased resistance to BPH. A BPH-responsive flavanone 3-hydroxylase (OsF3H) gene in the flavonoid biosynthetic pathway was proved to be directly regulated by OsGRF8. A genetic functional analysis of OsF3H revealed its positive role in mediating both the flavonoid contents and BPH resistance. And analysis of the genetic correlation between OsmiR396 and OsF3H showed that down-regulation of OsF3H complemented the BPH resistance characteristic and simultaneously decreased the flavonoid contents of the MIM396 plants. Thus, we revealed a new BPH resistance mechanism mediated by the OsmiR396-OsGRF8-OsF3H-flavonoid pathway. Our study suggests potential applications of miRNAs in BPH resistance breeding.

Modulation of plant architecture by the miR156f-OsSPL7-OsGH3.8 pathway in rice.

Tiller number and plant height are two of the main features of plant architecture that directly influence rice yield. Auxin and miR156, an extensively studied small RNA (smRNA), are both broadly involved in plant development and physiology, suggesting a possible relationship between the two. In this study, we identified a rice T-DNA insertion cluster and dwarf (cd) mutant that has an increased tiller number and reduced plant height. The T-DNA insertion was in close proximity to the miR156f gene and was associated with its up-regulation. Plants overexpressing miR156f resembled the cd mutant. In contrast, plants overexpressing an miR156f target mimic (MIM156fOE) had a reduced tiller number and increased height. Genetic analysis showed that OsSPL7 is a target of miR156f that regulates plant architecture. Plants overexpressing OsSPL7 had a reduced tiller number, while OsSPL7 RNAi plants had an increased tiller number and a reduced height. We also found that OsSPL7 binds directly to the OsGH3.8 promoter to regulate its transcription. Overexpression of OsGH3.8 and OsGH3.8 RNAi partially complemented the MIM156fOE and cd mutant phenotypes, respectively. Our combined data show that the miR156f-OsSPL7-OsGH3.8 pathway regulates tiller number and plant height in rice, and this pathway may allow crosstalk between miR156 and auxin.

OsEXPA10 mediates the balance between growth and resistance to biotic stress in rice.

KEY MESSAGE: OsEXPA10 gene coordinates the balance between rice development and biotic resistance. Expansins are proteins that can loosen the cell wall. Previous studies have indicated that expansin-encoding genes were involved in defense against abiotic stress, but little is known about the involvement of expansins in biotic stress. Brown planthopper (BPH) is one of the worst insect pests of rice in the Asia-Pacific planting area, and many efforts have been made to identify and clone BPH-resistance genes for use in breeding resistant cultivars. At the same time, rice blast caused by Magnaporthe grisea is one of the three major diseases that severely affect rice production worldwide. Here, we demonstrated that one rice expansin-encoding gene, OsEXPA10, functions in both rice growth and biotic resistance. Over expression of OsEXPA10 improved rice growth but also increased susceptibility to BPH infestation and blast attack, while knock-down OsEXPA10 gene expression resulted in reduced plant height and grain size, but also increased resistance to BPH and the blast pathogen. These results imply that OsEXPA10 mediates the balance between rice development and biotic resistance.

Acetoacetate Accelerates Muscle Regeneration and Ameliorates Muscular Dystrophy in Mice.

Acetoacetate (AA) is a ketone body and acts as a fuel to supply energy for cellular activity of various tissues. Here, we uncovered a novel function of AA in promoting muscle cell proliferation. Notably, the functional role of AA in regulating muscle cell function is further evidenced by its capability to accelerate muscle regeneration in normal mice, and it ameliorates muscular dystrophy in mdx mice. Mechanistically, our data from multiparameter analyses consistently support the notion that AA plays a non-metabolic role in regulating muscle cell function. Finally, we show that AA exerts its function through activation of the MEK1-ERK1/2-cyclin D1 pathway, revealing a novel mechanism in which AA serves as a signaling metabolite in mediating muscle cell function. Our findings highlight the profound functions of a small metabolite as signaling molecule in mammalian cells.

New insights into an RNAi approach for plant defence against piercing-sucking and stem-borer insect pests.

Insect double-stranded (ds)RNA expression in transgenic crops can increase plant resistance to biotic stress; however, creating transgenic crops to defend against every insect pest is impractical. Arabidopsis Mob1A is required for organ growth and reproduction. When Arabidopsis roots were soaked in dsMob1A, the root lengths and numbers were significantly suppressed and plants could not bolt or flower. Twenty-four hours after rice roots were immersed in fluorescent-labelled dsEYFP (enhanced yellow fluorescent protein), fluorescence was observed in the rice sheath and stem and in planthoppers feeding on the rice. The expression levels of Ago and Dicer in rice and planthoppers were induced by dsEYFP. When rice roots were soaked in dsActin, their growth was also significantly suppressed. When planthoppers or Asian corn borers fed on rice or maize that had been irrigated with a solution containing the dsRNA of an insect target gene, the insect's mortality rate increased significantly. Our results demonstrate that dsRNAs can be absorbed by crop roots, trigger plant and insect RNAi and enhance piercing-sucking and stem-borer insect mortality rates. We also confirmed that dsRNA was stable under outdoor conditions. These results indicate that the root dsRNA soaking can be used as a bioinsecticide strategy during crop irrigation.

Proteomic Analysis of Silkworm Antennae.

The silkworm Bombyx mori is an oligophagous insect that feeds mainly on mulberry leaves. The olfactory system of silkworm is a good model to study olfaction in Lepidoptera. Here, we carried out shotgun proteomic analysis and MS sequencing of the silkmoth antennae. A total of 364 proteins were detected, 77 were female specific, 143 were male specific, and 144 were expressed in both male and female antennae. Five odorant-binding proteins, two chemosensory proteins, and one olfactory receptor were identified. They may play a major role in the perception of odorants. An esterase and an aldehyde dehydrogenase were found only in male antennae. Glutathione S-transferases (GSTs) and cytochrome P450s, also found in silkworm antennae, may be involved in the degradation of xenobiotics. Additionally, antioxidation proteins and immunity proteins were identified. Juvenile hormone binding proteins (JHBP), juvenile hormone resistance protein II, and juvenile hormone episode hydrolase (JHEH) were found in the proteomic analysis, which suggests that the antennae are a target for juvenile hormone in the silkworm. Our results provide insight into the expression of proteins in the antennae of silkworm and will facilitate the future functional analysis of silkworm antennae.

Determination of genomic copy number alteration emphasizing a restriction site-based strategy of genome re-sequencing.

MOTIVATION: Copy number abbreviation (CNA) is one type of genomic aberration that is often induced by genome instability and is associated with diseases such as cancer. Determination of the genome-wide CNA profile is an important step in identifying the underlying mutation mechanisms. Genomic data based on next-generation sequencing technology are particularly suitable for determination of high-quality CNA profile. Now is an important time to reevaluate the use of sequencing techniques for CNA analysis, especially with the rapid growth of the different targeted genome and whole-genome sequencing strategies. RESULTS: In this study, we provide a comparison of resequencing strategies, with regard to their utility, applied to the same hepatocellular carcinoma sample for copy number determination. These strategies include whole-genome, exome and restriction site-associated DNA (RAD) sequencing. The last of these strategies is a targeted sequencing technique that involves cutting the genome with a restriction enzyme and isolating the targeted sequences. Our data demonstrate that RAD sequencing is an efficient and comprehensive strategy that allows the cost-effective determination of CNAs. Further investigation of RAD sequencing data led to the finding that a precise measurement of the allele frequency would be a helpful complement to the read depth for CNA analysis for two reasons. First, knowledge of the allele frequency helps to resolve refined calculations of allele-specific copy numbers, which, in turn, identify the functionally important CNAs that are under natural selection on the parental alleles. Second, this knowledge enables deconvolution of CNA patterns in complex genomic regions.

Bacteria-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management.

Since the discovery of the RNA interference (RNAi) mechanism, it has been widely used in the fields of gene function, biomedicine, and crop pest control. In the direction of agricultural application, this technology is highly expected, especially in the field of pest control, which is called "the third revolution in the history of pesticides". Currently, RNA biopesticides are developing rapidly all over the world. A genetically modified product (MON87411) has been approved for marketing, and a large number of agricultural companies are developing products based on direct spraying RNA biopesticides and submitting them for regulatory approval. The biggest problem that has emerged for spray RNA biopesticides is the technology for large-scale and low-cost production of dsRNA. At present, the bacterial fermentation production technology can realize large-scale dsRNA production with a yield of 4.23~182 mg/L bacterial solution. This article describes the experimental protocol for dsRNA production technology based on bacterial fermentation.

Identification of differential expression genes associated with host selection and adaptation between two sibling insect species by transcriptional profile analysis.

BACKGROUND: Cotton bollworm (Helicoverpa armigera) and oriental tobacco budworm (Helicoverpa assulta) are noctuid sibling species. Under artificial manipulation, they can mate and produce fertile offspring. As serious agricultural insect pests, cotton bollworms are euryphagous insects, but oriental tobacco budworms are oligophagous insects. To identify the differentially expressed genes that affect host recognition and host adaptation between the two species, we constructed digital gene expression tag profiles for four developmental stages of the two species. High-throughput sequencing results indicated that we have got more than 23 million 17nt clean tags from both species, respectively. The number of unique clean tags was nearly same in both species (approximately 357,000). RESULTS: According to the gene annotation results, we identified 83 and 68 olfaction related transcripts from H. armigera and H. assulta, respectively. At the same time, 1137 and 1138 transcripts of digestion enzymes were identified from the two species. Among the olfaction related transcripts, more odorant binding protein and G protein-coupled receptor were identified in H. armigera than in H. assulta. Among the digestion enzymes, there are more detoxification enzyme, e.g. P450, carboxypeptidase and ATPase in H. assulta than in H. armigera. These differences partially explain that because of the narrow host plant range of H. assulta, more detoxification enzymes would help them increase the food detoxification and utilization efficiency. CONCLUSIONS: This study supplied some differentially expressed genes affecting host selection and adaptation between the two sibling species. These genes will be useful information for studying on the evolution of host plant selection. It also provides some important target genes for insect species-specific control by RNAi technology.

Disruption of an N-acetyltransferase gene in the silkworm reveals a novel role in pigmentation.

The pigmentation of insects has served as an excellent model for the study of morphological trait evolution and developmental biology. The melanism (mln) mutant of the silkworm Bombyx mori is notable for its strong black coloration, phenotypic differences between larval and adult stages, and its widespread use in strain selection. Here, we report the genetic and molecular bases for the formation of the mln morphological trait. Fine mapping revealed that an arylalkylamine N-acetyltransferase (AANAT) gene co-segregates with the black coloration patterns. Coding sequence variations and expression profiles of AANAT are also associated with the melanic phenotypes. A 126 bp deletion in the mln genome causes two alternatively spliced transcripts with premature terminations. An enzymatic assay demonstrated the absolute loss of AANAT activity in the mutant proteins. We also performed RNA interference of AANAT in wild-type pupae and observed a significant proportion of adults with ectopic black coloration. These findings indicate that functional deletion of this AANAT gene accounts for the mln mutation in silkworm. AANAT is also involved in a parallel melanin synthesis pathway in which ebony plays a role, whereas no pigmentation defect has been reported in the Drosophila model or in other insects to date. To the best of our knowledge, the mln mutation is the first characterized mutant phenotype of insects with AANAT, and this result contributes to our understanding of dopamine metabolism and melanin pattern polymorphisms.

OsmiR159 Modulate BPH Resistance Through Regulating G-Protein γ Subunit GS3 Gene in Rice.

Brown planthopper (BPH) is the most destructive insect pest to rice that causes tremendous yield loss each year in rice planting Asia and South-East Asia areas. Compared with traditional chemical-based treatment, utilization of plant endogenous resistance is a more effective and environmental-friendly way for BPH control. Accordingly, quite a few quantitative trait loci (QTLs) for BPH resistance were cloned using forward genetics. However, BPH is apt to change quickly into new biotypes to overcome plant resistance, therefore, new resistance resources and genes are continuously needed. miRNAs are important regulators in both plant development and physiological regulation including immunity, and might be used as effective supplements for BPH resistance QTLs. miR159 is an ancient and conserved miRNA. In this study, we found that each OsMIR159 gene in rice responded to BPH feeding very obviously, and genetic function assay proved them to negatively regulate BPH resistance, with STTM159 showing resistance to BPH, and over expression of OsmiR159d susceptible to BPH. One target genes of OsmiR159, OsGAMYBL2, positively regulated BPH resistance. Further biochemical studies revealed that OsGAMYBL2 could directly bind to the promoter of G-protein γ subunit encoding GS3 gene and repress its expression. And genetically, GS3 responded to BPH feeding promptly and negatively regulated BPH resistance, GS3 over expression plants were susceptible to BPH, while GS3 knock-out plants were resistant to BPH. Thus, we identified new function of OsmiR159-OsGAMYBL2 in mediating BPH response, and revealed a new OsmiR159-G protein pathway that mediates BPH resistance in rice.