Protein lysine acetylation is a reversible posttranslational modification and plays a pivotal role in a broad array of physiological functions. In our study, a strategy combining immunoaffinity enrichment of acetylated peptides based on anti-acetyllysine antibody with high-resolution tandem mass spectrometry was employed for a systemic survey of acetylation sites in a polyphagous pest insect Thrips tabaci. In total, 597 acetylated proteins containing 995 lysine acetylation sites were identified in T. tabaci. Interestingly, functional enrichment analysis showed that acetylated proteins are implicated in the regulation of diverse KEGG pathways, including carbohydrate metabolism, energy metabolism, amino acid metabolism, and translational process. In particular, a large fraction of metabolic enzymes, including multiple rate-limiting enzymes, was also found to be acetylated. Comparative analysis indicated that a proportion of euNOG entries was shared by three insects. Furthermore, motif analysis showed that the sequence flanking acetylation sites exhibited subcellular compartment-specific patterns. Protein-protein interaction network analysis demonstrated that acetylated proteins formed several densely connected sub-networks tightly associated with ribosome, fatty acid metabolism, oxidative phosphorylation and purine metabolism, thus strengthening the functional enrichment result. Overall, our study provides a comprehensive view of acetylation sites, facilitating an in-depth investigation of functional roles of acetylation in the future. SIGNIFICANCE: Onion thrips is a polyphagous agricultural pest insect. Insecticide resistance has been frequently reported due to the intensive use of chemical pesticides. Lysine acetylation is a ubiquitous posttranslational modification and plays important roles in gene regulation. An in-depth understanding of transcriptional regulation is crucial for designing novel and highly efficient pesticides. With high-resolution mass spectrometry based proteomics method, we systematically explored the acetylome in this insect. In total, 595 proteins containing 995 acetylation sites were identified in this study. Bioinformatic analysis revealed that acetylated proteins are implicated in regulating diverse biological processes, including carbohydrate metabolism, energy metabolism, amino acid metabolism, and translational process. Furthermore, protein-protein interaction network analysis showed that ribosome, fatty acid metabolism, oxidative metabolism and purine metabolism are significantly enriched for acetylated proteins. Our results provide insights into the targets of acetylation in onion thrips and facilitate elucidation of transcriptional regulation and design of novel control strategies against this insect.

Over the past decade, second-generation sequencing (SGS) has been widely used to elucidate the transcriptome across many organisms. However, the full-length (FL) transcripts and alternative splice (AS) isoforms could not be confidently and accurately defined with SGS. Pacific biosciences (PacBio) single-molecule real-time sequencing was conducted to obtain FL transcriptome data in the codling moth. In total, 25,940 high-quality FL isoforms were obtained and clustered to 14,099 nonredundant clusters. Interestingly, nearly 90% of nonredundant PacBio transcripts were novel compared to reference genes. Among them, 3389 transcripts potentially represented novel genes. Additionally, a large number of AS events were discovered, and most of the splice junctions in the PacBio isoforms could be supported by short reads in public datasets. Furthermore, 952 FL lncRNAs and 81 fusion transcripts were identified and validated using RT-PCR analysis. Overall, an atlas of FL transcripts was obtained in the codling moth, which will help provide further insights into the complexity of the transcriptome and facilitate improving genome annotations and functional studies in this insect.

Longan (Dimocarpus longan) is a subtropical fruit best known for its nutritious fruit and regarded as a precious tonic and traditional medicine since ancient times. High-quality chromosome-scale genome assembly is valuable for functional genomic study and genetic improvement of longan. Here, we report a chromosome-level reference genome sequence for longan cultivar JDB with an assembled genome of 455.5 Mb in size anchored to fifteen chromosomes, representing a significant improvement of contiguity (contig N50 = 12.1 Mb, scaffold N50 = 29.5 Mb) over a previous draft assembly. A total of 40 420 protein-coding genes were predicted in D. longan genome. Synteny analysis suggests longan shares the widespread gamma event with core eudicots, but has no other whole genome duplications. Comparative genomics showed that D. longan genome experienced significant expansions of gene families related to phenylpropanoid biosynthesis and UDP-glucosyltransferase. Deep genome sequencing analysis of longan cultivars identified longan biogeography as a major contributing factor for genetic diversity, and revealed a clear population admixture and introgression among cultivars of different geographic origins, postulating a likely migration trajectory of longan overall confirmed by existing historical records. Finally, genome-wide association studies (GWAS) of longan cultivars identified quantitative trait loci (QTL) for six different fruit quality traits and revealed a shared QTL containing three genes for total soluble solid and seed weight. The chromosome-level reference genome assembly, annotation and population genetic resource for D. longan will facilitate the molecular studies and breeding of desirable longan cultivars in the future.

The beet armyworm Spodoptera exigua is a polyphagous caterpillar that causes serious damage to many species of crops and vegetables. To gain insight into how this polyphagous insect differs from less harmful oligophagous species, we generated a chromosome-level assembly and compared it to closely related species with the same or different feeding habits.

Insect olfaction is vital for foraging, mating, host-seeking, and avoidance of predators/pathogens. In insects, odorant binding proteins (OBPs) are involved in transporting hydrophobic odor molecules from the external environment to receptor neurons. The codling moth, Cydia pomonella, one of the most destructive insect fruit pests, causes enormous economic losses. However, little is known about the number, variety, gains and losses, and evolution of OBP genes in C. pomonella. Here we report the identification of 40 OBPs in C. pomonella, most (75%) of which are classic OBPs, using genomic and transcriptomic analyses. Two OBP genes were lost in C. pomonella relative to possible distant ancestor in Lepidoptera lineage based on an analysis of gene gains and losses. The phylogenetic tree and chromosome location showed that the expansion of OBP genes mainly resulted from tandem duplications, as the CpomGOBP2 gene was duplicated twice along with loss of CpomPBPB. Two positive selection sites of the CpomGOBP1 gene were identified while other OBP genes evolved under purifying selection. Our results provide fundamental knowledge of OBP genes allowing further study of their function in C. pomonella.

Miniature inverted-repeat transposable elements (MITEs) have a broad impact on genome structure and function. Although MITEs are found associated to genes, little is known about their effect on gene regulation. We have identified a novel MITE family, named Triton, whilst analyzing two independent trichosanthin (TCS) gene promoters (TP9 and TP12) cloned from Trichosanthes kirilowii Maximowicz. Triton1 and Triton2 are nested in TP9, and Triton3 (with 93% sequence similarity to Triton2) is in TP12. To assess the effect of MITE insertion on TCS promoters, we excised Triton1 from TP9 and inserted it into TP12. GUS activity analysis revealed that nested Triton1 is required for effective repression of promoter activity. Detailed analyses of a series of 5'-truncated promoters concerning Triton1 showed that a dark-specific repressor and some constitutive elements endow Triton1 with ability to response to light conditions. These results suggest that Triton1 MITE, which contains cis-regulatory elements, could mediate gene expression.

The rapid wide-scale spread of fall armyworm (Spodoptera frugiperda) has caused serious crop losses globally. However, differences in the genetic background of subpopulations and the mechanisms of rapid adaptation behind the invasion are still not well understood. Here we report the assembly of a 390.38-Mb chromosome-level genome of fall armyworm derived from south-central Africa using Pacific Bioscience (PacBio) and Hi-C sequencing technologies, with scaffold N50 of 12.9 Mb and containing 22,260 annotated protein-coding genes. Genome-wide resequencing of 103 samples and strain identification were conducted to reveal the genetic background of fall armyworm populations in China. Analysis of genes related to pesticide- and Bacillus thuringiensis (Bt) resistance showed that the risk of fall armyworm developing resistance to conventional pesticides is very high. Laboratory bioassay results showed that insects invading China carry resistance to organophosphate and pyrethroid pesticides, but are sensitive to genetically modified maize expressing the Bt toxin Cry1Ab in field experiments. Additionally, two mitochondrial fragments were found to be inserted into the nuclear genome, with the insertion event occurring after the differentiation of the two strains. This study represents a valuable advance toward improving management strategies for fall armyworm.

Due to the monocultural basis of agricultural crops, mutated plant microbes with increased pathogenicity can easily spread in the field and lead to serious yield losses. As a major threat to a wide range of crop plants, oomycete pathogens continuously undergo adaptive evolution to overcome plant defense barriers. However, the genetic basis of their evolution at the molecular level remains largely unknown. Here, we investigated the nature variation and the population genomics of the soybean pathogen Phytophthora sojae by high-throughput genome re-sequencing. Genomic variation analysis revealed uneven "two-speed" evolutionary pattern with genes in gene-sparse regions (GSRs) showing higher rates of structural polymorphisms and positive selection. GSRs are enriched in effector genes and transposase-related genes. Our results also suggested that the NADH oxidase and MIP transporter gene families undergo rapid and diversifying selection. Furthermore, we demonstrated that P. sojae isolates possess varying numbers of RxLR effectors with diverse sequences, totaling 471 members. Among them, 42 core RxLR effectors are assumed to be important for infection. Finally, we observed that Avr genes exhibit abundant sequence variation in P. sojae isolates. Several novel variants lead to the evading of host resistance, including a complete deletion in Avr3c and amino acid mutations in Avr1a. Taken together, our results provide an adaptive landscape of P. sojae at single-nucleotide resolution, as well as resources for further resistance breeding and disease prevention against this important plant pathogen.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.

The mycotoxin fumonisin B1 (FB1) is a strong inducer of programmed cell death (PCD) in plants, but its underlying mechanism remains unclear. Here, we describe two ubiquitin ligases, RING DOMAIN LIGASE3 (RGLG3) and RGLG4, which control FB1-triggered PCD by modulating the jasmonate (JA) signalling pathway in Arabidopsis thaliana. RGLG3 and RGLG4 transcription was sensitive to FB1. Arabidopsis FB1 sensitivity was suppressed by loss of function of RGLG3 and RGLG4 and was increased by their overexpression. Thus RGLG3 and RGLG4 have coordinated and positive roles in FB1-elicited PCD. Mutated JA perception by coi1 disrupted the RGLG3- and RGLG4-related response to FB1 and interfered with their roles in cell death. Although FB1 induced JA-responsive defence genes, it repressed growth-related, as well as JA biosynthesis-related, genes. Consistently, FB1 application reduced JA content in wild-type plants. Furthermore, exogenously applied salicylic acid additively suppressed JA signalling with FB1 treatment, suggesting that FB1-induced salicylic acid inhibits the JA pathway during this process. All of these effects were attenuated in rglg3 rglg4 plants. Altogether, these data suggest that the JA pathway is hijacked by the toxin FB1 to elicit PCD, which is coordinated by Arabidopsis RGLG3 and RGLG4.

Mikania micrantha is one of the top 100 worst invasive species that can cause serious damage to natural ecosystems and substantial economic losses. Here, we present its 1.79 Gb chromosome-scale reference genome. Half of the genome is composed of long terminal repeat retrotransposons, 80% of which have been derived from a significant expansion in the past one million years. We identify a whole genome duplication event and recent segmental duplications, which may be responsible for its rapid environmental adaptation. Additionally, we show that M. micrantha achieves higher photosynthetic capacity by CO2 absorption at night to supplement the carbon fixation during the day, as well as enhanced stem photosynthesis efficiency. Furthermore, the metabolites of M. micrantha can increase the availability of nitrogen by enriching the microbes that participate in nitrogen cycling pathways. These findings collectively provide insights into the rapid growth and invasive adaptation.

Mikania micrantha is a noxious invasive plant causing enormous economic losses and ecological damage. Soil microbiome plays an important role in the invasion process of M. micrantha, while little is known about its rhizosphere microbiome composition and function. In this study, we identified the distinct rhizosphere microbial communities of M. micrantha, by comparing them with those of two coexisting native plants (Polygonum chinense and Paederia scandens) and the bulk soils, using metagenomics data from field sampling and pot experiment. As a result, the enrichment of phosphorus-solubilizing bacteria Pseudomonas and Enterobacter was consistent with the increased soil available phosphorus in M. micrantha rhizosphere. Furthermore, the pathogens of Fusarium oxysporum and Ralstonia solanacearum and pathogenic genes of type III secretion system (T3SS) were observed to be less abundant in M. micrantha rhizosphere, which might be attributed to the enrichment of biocontrol bacteria Catenulispora, Pseudomonas, and Candidatus Entotheonella and polyketide synthase (PKS) genes involved in synthesizing antibiotics and polyketides to inhibit pathogens. These findings collectively suggested that the enrichment of microbes involved in nutrient acquisition and pathogen suppression in the rhizosphere of M. micrantha largely enhances its adaptation and invasion to various environments.

Mikania micrantha is one of the worst invasive species globally and can cause significant negative impacts on agricultural and forestry economics, particularly in Asia and the Pacific region. The rust Puccinia spegazzinii has been used successfully as a biological control agent in several countries to help manage M. micrantha. However, the response mechanisms of M. micrantha to P. spegazzinii infection have never been studied. To investigate the response of M. micrantha to infection by P. spegazzinii, an integrated analysis of metabolomics and transcriptomics was performed. The levels of 74 metabolites, including organic acids, amino acids, and secondary metabolites in M. micrantha infected with P. spegazzinii, were significantly different compared to those in plants that were not infected. After P. spegazzinii infection, the expression of the TCA cycle gene was significantly induced to participate in energy biosynthesis and produce more ATP. The content of most amino acids, such as L-isoleucine, L-tryptophan and L-citrulline, increased. In addition, phytoalexins, such as maackiain, nobiletin, vasicin, arachidonic acid, and JA-Ile, accumulated in M. micrantha. A total of 4978 differentially expressed genes were identified in M. micrantha infected by P. spegazzinii. Many key genes of M. micrantha in the PTI (pattern-triggered immunity) and ETI (effector-triggered immunity) pathways showed significantly higher expression under P. spegazzinii infection. Through these reactions, M. micrantha is able to resist the infection of P. spegazzinii and maintain its growth. These results are helpful for us to understand the changes in metabolites and gene expression in M. micrantha after being infected by P. spegazzinii. Our results can provide a theoretical basis for weakening the defense response of M. micrantha to P. spegazzinii, and for P. spegazzinii as a long-term biological control agent of M. micrantha.

Magnaporthe oryzae, the ascomycete fungus that causes rice blast disease, initiates conidiation in response to light when grown on Prune-Agar medium containing both carbon and nitrogen sources. Macroautophagy/autophagy was shown to be essential for M. oryzae conidiation and induced specifically upon exposure to light but is undetectable in the dark. Therefore, it is inferred that autophagy is naturally induced by light, rather than by starvation during M. oryzae conidiation. However, the signaling pathway(s) involved in such phototropic induction of autophagy remains unknown. We identified an M. oryzae ortholog of GCN5 (MGG_03677), encoding a histone acetyltransferase (HAT) that negatively regulates light- and nitrogen-starvation-induced autophagy, by acetylating the autophagy protein Atg7. Furthermore, we unveiled novel regulatory mechanisms on Gcn5 at both transcriptional and post-translational levels, governing its function associated with the unique phototropic response of autophagy in this pathogenic fungus. Thus, our study depicts a signaling network and regulatory mechanism underlying the autophagy induction by important environmental clues such as light and nutrients.

The golden apple snail (Pomacea canaliculata) is a freshwater snail listed among the top 100 worst invasive species worldwide and a noted agricultural and quarantine pest that causes great economic losses. It is characterized by fast growth, strong stress tolerance, a high reproduction rate, and adaptation to a broad range of environments.

The spotted-wing Drosophila (Drosophila suzukii Matsumura) is native to eastern Asia, but has become a global threat to fruit production. In recent years, CRISPR/Cas9 targeting was established in this species allowing for functional genomic and genetic control studies. Here, we report the generation and characterization of Cas9-expressing strains of D. suzukii. Five independent transgenic lines were generated using a piggyBac construct containing the EGFP fluorescent marker gene and the Cas9 gene under the control of the D. melanogaster heat shock protein 70 promoter and 3'UTR. Heat-shock (HS) treated embryos were analyzed by reverse transcriptase PCR, revealing strong heat inducibility of the transgenic Cas9 expression. By injecting gRNA targeting EGFP into one selected line, 50.0% of G0 flies showed mosaic loss-of-fluorescence phenotype, and 45.5% of G0 flies produced G1 mutants without HS. Such somatic and germline mutagenesis rates were increased to 95.4% and 85.7%, respectively, by applying a HS. Parental flies receiving HS resulted in high inheritance of the mutation (92%) in their progeny. Additionally, targeting the endogenous gene yellow led to the lack of pigmentation and male lethality. We discuss the potential use of these efficient and temperature-dependent Cas9-expressing strains for the genetic studies in D. suzukii.

Long noncoding RNAs (lncRNAs) have emerged as an important class of transcriptional regulators in cellular processes. The past decades have witnessed great progress in lncRNA studies in a variety of organisms. The codling moth (Cydia pomonella L.) is an important invasive insect in China. However, the functional impact of lncRNAs in this insect remains unclear. In this study, an atlas of codling moth lncRNAs was constructed based on publicly available RNA-seq datasets.

The Cydia pomonella granulovirus (CpGV) has been used as a biological control agent of codling moth (Cydia pomonella), a severe global pest on pome fruit. Despite the economic importance, our knowledge of its molecular biology is still limited and a detailed picture of its gene expression is still missing. Here, we sequenced the transcriptome of codling moth larvae infected with the Mexican isolate CpGV-M and analyzed the expression of viral genes at 12, 48, and 96 h post infection (hpi). The results showed that two genes (p6.9 and pp31/39K) related to DNA binding of virus production, were highly expressed at 48 and 96 hpi. From 48 to 96 hpi, the expression of genes associated with virus replication and dissemination decreased, whereas the expression of genes related to infectious virion production and per os infectivity increased. This study provides a comprehensive view of CpGV gene expression patterns in host larvae.

Mikania micrantha Kunth is a fast-growing global invasive weed species that causes severe damage to natural ecosystems and very large economic losses of forest and crop production. Although Puccinia spegazzinii can effectively inhibit the growth of M. micrantha and is used as a biological control strain in many countries, the mechanism of inhibiting the growth of M. micrantha is not clear. Here, we used a combination of phenotypic, enzyme activity, transcriptomic, and metabolomic approaches to study the response of M. micrantha after infection by P. spegazzinii. In the early stages of rust infection, jasmonic acid (JA), jasmonoyl-isoleucine (JA-Ile), and salicylic acid (SA) levels in infected leaves were significantly lower than those in uninfected leaves. In teliospore initial and developed stages of P. spegazzinii, JA and JA-Ile levels substantially increased by more than 6 times, which resulted in a significant decrease in the accumulation of defense hormone SA in infected leaves of M. micrantha. The contents of plant growth-promoting hormones were significantly reduced in the infected plants as a result of substantial downregulation of the expression of key genes related to hormone biosynthesis. Furthermore, rust infection led to high levels of reactive oxygen species in chloroplasts and the destruction of chlorophyll structure, which also led to decreased photosynthetic gene expression, net photosynthetic rate, activity of Rubisco, and levels of important organic acids in the Calvin cycle. We hypothesized that after P. spegazzinii infection, JA or JA-Ile accumulation not only inhibited SA levels to promote rust infection and development, but also impeded the rapid growth of M. micrantha by affecting plant growth hormones, carbon, and nitrogen metabolic pathways.