Intra species dissection of phytophthora capsici resistance in black pepper.

INTRODUCTION: Black pepper, a financially significant tropical crop, assumes a pivotal role in global agriculture for the major source of specie flavor. Nonetheless, the growth and productivity of black pepper face severe impediments due to the destructive pathogen Phytophthora capsici, ultimately resulting in black pepper blight. The dissecting for the genetic source of pathogen resistance for black pepper is beneficial for its global production. The genetic sources include the variations on gene coding sequences, transcription capabilities and epigenetic modifications, which exerts hierarchy of influences on plant defense against pathogen. However, the understanding of genetic source of disease resistance in black pepper remains limited. METHODS: The wild species Piper flaviflorum (P. flaviflorum, Pf) is known for blight resistance, while the cultivated species P. nigrum is susceptible. To dissecting the genetic sources of pathogen resistance for black pepper, the chromatin modification on H3K4me3 and transcriptome of black pepper species were profiled for genome wide comparative studies, applied with CUT&Tag and RNA sequencing technologies. RESULTS: The intraspecies difference between P. flaviflorum and P. nigrum on gene body region led to coding variations on 5137 genes, including 359 gene with biotic stress responses and regulation. P. flaviflorum exhibited a more comprehensive resistance response to Phytophthora capsica in terms of transcriptome features. The pathogen responsive transcribing was significant associated with histone modification mark of H3K4me3 in black pepper. The collective data on variations of sequence, transcription activity and chromatin structure lead to an exclusive jasmonic acid-responsive pathway for disease resistance in P. flaviflorum was revealed. This research provides a comprehensive frame work to identify the fine genetic source for pathogen resistance from wild species of black pepper.

Regulation of Glandular Size and Phytoalexin Biosynthesis by a Negative Feedback Loop in Cotton.

Plants have evolved diverse defense mechanisms encompassing physical and chemical barriers. Cotton pigment glands are known for containing various defense metabolites, but the precise regulation of gland size to modulate defense compound levels remains enigmatic. Here, it is discovered that the VQ domain-containing protein JAVL negatively regulates pigment gland size and the biosynthesis of defense compounds, while the MYC2-like transcription factor GoPGF has the opposite effect. Notably, GoPGF directly activates the expression of JAVL, whereas JAVL suppresses GoPGF transcription, establishing a negative feedback loop that maintains the expression homeostasis between GoPGF and JAVL. Furthermore, it is observed that JAVL negatively regulates jasmonate levels by inhibiting the expression of jasmonate biosynthetic genes and interacting with GoPGF to attenuate its activation effects, thereby maintaining homeostatic regulation of jasmonate levels. The increased expression ratio of GoPGF to JAVL leads to enlarged pigment glands and elevated jasmonates and defense compounds, enhancing insect and pathogen resistance in cotton. These findings unveil a new mechanism for regulating gland size and secondary metabolites biosynthesis, providing innovative strategies for strengthening plant defense.

Metabolomic Analysis Reveals Domestication-Driven Reshaping of Polyphenolic Antioxidants in Soybean Seeds.

Crop domestication has resulted in nutrient losses, so evaluating the reshaping of phytonutrients is crucial for improving nutrition. Soybean is an ideal model due to its abundant phytonutrients and wild relatives. In order to unravel the domestication consequence of phytonutrients, comparative and association analyses of metabolomes and antioxidant activities were performed on seeds of six wild (Glycine soja (Sieb. and Zucc.)) and six cultivated soybeans (Glycine max (L.) Merr.). Through ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), we observed a greater metabolic diversity in wild soybeans, which also displayed higher antioxidant activities. (-)-Epicatechin, a potent antioxidant, displayed a 1750-fold greater abundance in wild soybeans than in cultivated soybeans. Multiple polyphenols in the catechin biosynthesis pathway were significantly higher in wild soybeans, including phlorizin, taxifolin, quercetin 3-O-galactoside, cyanidin 3-O-glucoside, (+)-catechin, (-)-epiafzelechin, catechin-glucoside, and three proanthocyanidins. They showed significant positive correlations with each other and antioxidant activities, indicating their cooperative contribution to the high antioxidant activities of wild soybeans. Additionally, natural acylation related to functional properties was characterized in a diverse range of polyphenols. Our study reveals the comprehensive reprogramming of polyphenolic antioxidants during domestication, providing valuable insights for metabolism-assisted fortification of crop nutrition.

Approaches for Modes of Action Study of Long Non-Coding RNAs: From Single Verification to Genome-Wide Determination.

Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides (nt) that are not translated into known functional proteins. This broad definition covers a large collection of transcripts with diverse genomic origins, biogenesis, and modes of action. Thus, it is very important to choose appropriate research methodologies when investigating lncRNAs with biological significance. Multiple reviews to date have summarized the mechanisms of lncRNA biogenesis, their localization, their functions in gene regulation at multiple levels, and also their potential applications. However, little has been reviewed on the leading strategies for lncRNA research. Here, we generalize a basic and systemic mind map for lncRNA research and discuss the mechanisms and the application scenarios of 'up-to-date' techniques as applied to molecular function studies of lncRNAs. Taking advantage of documented lncRNA research paradigms as examples, we aim to provide an overview of the developing techniques for elucidating lncRNA interactions with genomic DNA, proteins, and other RNAs. In the end, we propose the future direction and potential technological challenges of lncRNA studies, focusing on techniques and applications.

Biotinylated Tn5 transposase-mediated CUT&Tag efficiently profiles transcription factor-DNA interactions in plants.

In contrast to CUT&Tag approaches for profiling bulk histone modifications, current CUT&Tag methods for analysing specific transcription factor (TF)-DNA interactions remain technically challenging due to TFs having relatively low abundance. Moreover, an efficient CUT&Tag strategy for plant TFs is not yet available. Here, we first applied biotinylated Tn5 transposase-mediated CUT&Tag (B-CUT&Tag) to produce high-quality libraries for interrogating TF-DNA interactions. B-CUT&Tag combines streptavidin-biotin-based DNA purification with routine CUT&Tag, optimizing the removal of large amounts of intact chromatin not targeted by specific TFs. The biotinylated chromatin fragments are then purified for construction of deep sequencing libraries or qPCR analysis. We applied B-CUT&Tag to probe genome-wide DNA targets of Squamosa promoter-binding-like protein 9 (SPL9), a well-established TF in Arabidopsis; the resulting profiles were efficient and consistent in demonstrating its well-established target genes in juvenile-adult transition/flowering, trichome development, flavonoid biosynthesis, wax synthesis and branching. Interestingly, our results indicate functions of AtSPL9 in modulating growth-defence trade-offs. In addition, we established a method for applying qPCR after CUT&Tag (B-CUT&Tag-qPCR) and successfully validated the binding of SPL9 in Arabidopsis and PHR2 in rice. Our study thus provides a convenient and highly efficient CUT&Tag strategy for profiling TF-chromatin interactions that is widely applicable to the annotation of cis-regulatory elements for crop improvement.

Physiological and transcriptomic evidence of antioxidative system and ion transport in chromium detoxification in germinating seedlings of soybean.

Chromium (Cr) toxicity impairs the productivity of crops and is a major threat to food security worldwide. However, the effect of Cr toxicity on seed germination and transcriptome of germinating seedlings of soybean crop has not been fully explored. In this study, two Cr-tolerant lines (J82, S125) and two Cr-sensitive ones (LD1, RL) were screened out of twenty-one soybean (Glycine max L.) genotypes based on seed germination rate, seed germinative energy, seed germination index, and growth of germinating seedlings under 50 mg L-1 Cr treatment. We found that Cr stress inhibits the growth of soybean seed germinating seedlings due to the Cr-induced overaccumulation of reactive oxygen species (ROS). Significantly different levels of element contents, antioxidant enzyme activities, malondialdehyde content were observed in the four soybean genotypes with contrasting Cr tolerance. Further, a total of 13,777 differentially expressed genes (DEGs) were identified in transcriptomic sequencing and 1298 DEGs in six gene modules were found highly correlated with the physiological traits by weighted correlation network analysis (WGCNA) analysis. The DEGs encoding antioxidant enzymes, transcription factors, and ion transporters are proposed to confer Cr tolerance in soybean germinating seedlings by reducing the uptake and translocation of Cr, decreasing the level of ROS, and keeping the osmotic balance in soybean germinating seedings. In conclusion, our study provided a molecular regulation network on soybean Cr tolerance at seed germinating stage and identified candidate genes for molecular breeding of low Cr accumulation soybean cultivars.

A novel strand-specific RNA-sequencing protocol using dU-adaptor-assembled Tn5.

Strand-specific RNA-seq is a powerful tool for the discovery of novel transcripts, annotation of genomes, and profiling of gene expression levels. Tn5 transposase has been successfully applied in massive-scale sequencing projects; in particular, Tn5 adaptor modification is used in epigenetics, genomic structure, and chromatin visualization. We developed a novel dU-adaptor-assembled Tn5-mediated strand-specific RNA-sequencing protocol and compared this method with the leading dUTP method in terms of experimental procedure and multiple quality metrics of the generated libraries. The results showed that the dU-Tn5 method is easy to operate and generates a strand-specific RNA-seq library of comparable quality considering library complexity, strand-specificity, evenness, and continuity of annotated transcript coverage. We also evaluated the performance of the dU-Tn5 method in identifying nitrogen-responsive protein-coding genes and long non-coding RNAs in soybean roots. The results indicated that ~62-70% of differentially expressed genes detected from conventional libraries were also detected in dU-Tn5 libraries, indicating good agreement of our method with the current standard; moreover, their fold-changes were highly correlated (R>0.9). Thus, our method provides a promising 'do-it-yourself' stranded RNA-seq procedure for gene expression profiling.

Transcriptome and Metabolome Analysis of a Late-Senescent Vegetable Soybean during Seed Development Provides New Insights into Degradation of Chlorophyll.

(1) Background: Senescence represents the final stage of plant growth and development, which transfers nutrients to growing seeds and directly affects the yield and quality of crops. However, little is known about chlorophyll degradation in developing and maturing seeds, in contrast to leaf senescence; (2) Methods: RNA-Seq was used to analyze the differentially expressed genes of different late-senescent germplasms. A widely untargeted metabolic analysis was used to analyze differential metabolites. In addition, qRT-PCR was conducted to detect gene expression levels; (3) Results: Transcriptome analysis revealed that ZX12 seeds have a higher expression level of the chlorophyll synthesis genes in the early stage of maturity, compared with ZX4, and have a lower expression level of chlorophyll degradation genes in the late stage of maturity. Flavonoids were the primary differential metabolites, and ZX12 contains the unique and highest expression of three types of metabolites, including farrerol-7-O-glucoside, cyanidin-3-o-(6'-o-feruloyl) glucoside, and kaempferide-3-o-(6'-malonyl) glucoside. Among them, farrerol-7-O-glucoside and cyanidin-3-o-(6'-o-feruloyl) glucoside are flavonoid derivatives containing mono and dihydroxy-B-ring chemical structures, respectively; and (4) Conclusions: It is speculated that the two metabolites can slow down the degradation process of chlorophyll by scavenging oxygen-free radicals in the chloroplast.

Comparative Transcriptome and Metabolome Analysis of Resistant and Susceptible Piper Species Upon Infection by the Oomycete Phytophthora Capsici.

Phytophthora capsici is a destructive oomycete pathogen that causes devastating disease in black pepper, resulting in a significant decline in yield and economic losses. Piper nigrum (black pepper) is documented as susceptible to P. capsici, whereas its close relative Piper flaviflorum is known to be resistant. However, the molecular mechanism underlying the resistance of P. flaviflorum remains obscure. In this study, we conducted a comparative transcriptome and metabolome analysis between P. flaviflorum and P. nigrum upon P. capsici infection and found substantial differences in their gene expression profiles, with altered genes being significantly enriched in terms relating to plant-pathogen interaction, phytohormone signal transduction, and secondary metabolic pathways, including phenylpropanoid biosynthesis. Further metabolome analysis revealed the resistant P. flaviflorum to have a high background endogenous ABA reservoir and time-course-dependent accumulation of ABA and SA upon P. capsici inoculation, while the susceptible P. nigrum had a high background endogenous IAA reservoir and time-course-dependent accumulation of JA-Ile, the active form of JA. Investigation of the phenylpropanoid biosynthesis metabolome further indicated the resistant P. flaviflorum to have more accumulation of lignin precursors than the susceptible P. nigrum, resulting in a higher accumulation after inoculation. This study provides an overall characterization of biologically important pathways underlying the resistance of P. flaviflorum, which theoretically explains the advantage of using this species as rootstock for the management of oomycete pathogen in black pepper production.

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation.

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Functional examination of lncRNAs in allotetraploid Gossypium hirsutum.

BACKGROUND: An evolutionary model using diploid and allotetraploid cotton species identified 80 % of non-coding transcripts in allotetraploid cotton as being uniquely activated in comparison with its diploid ancestors. The function of the lncRNAs activated in allotetraploid cotton remain largely unknown. RESULTS: We employed transcriptome analysis to examine the relationship between the lncRNAs and mRNAs of protein coding genes (PCGs) in cotton leaf tissue under abiotic stresses. LncRNA expression was preferentially associated with that of the flanking PCGs. Selected highly-expressed lncRNA candidates (n = 111) were subjected to a functional screening pilot test in which virus-induced gene silencing was integrated with abiotic stress treatment. From this low-throughput screen, we obtained candidate lncRNAs relating to plant height and tolerance to drought and other abiotic stresses. CONCLUSIONS: Low-throughput screen is an effective method to find functional lncRNA for further study. LncRNAs were more active in abiotic stresses than PCG expression, especially temperature stress. LncRNA XLOC107738 may take a cis-regulatory role in response to environmental stimuli. The degree to which lncRNAs are constitutively expressed may impact expression patterns and functions on the individual gene level rather than in genome-wide aggregate.

Absence of CG methylation alters the long noncoding transcriptome landscape in multiple species.

The noncoding regions throughout the genome are in large part comprised of transposable elements (TEs), some of which are functionalized with long intergenic noncoding RNAs (lincRNAs). DNA methylation is predominantly associated with TEs, but little is known about its contribution to the transcription of lincRNAs. Here, we examine the lincRNA profiles of DNA methylation-related mutants of five species, Arabidopsis, rice, tomato, maize, and mouse, to elucidate patterns in lincRNA regulation under altered DNA methylation status. Significant activation of lincRNAs was observed in the absence of CG DNA methylation rather than non-CG. Our study establishes a working model of the contribution of DNA methylation to regulation of the dynamic activity of lincRNA transcription.

Neofunctionalization of a polyploidization-activated cotton long intergenic non-coding RNA DAN1 during drought stress regulation.

The genomic shock of whole-genome duplication (WGD) and hybridization introduces great variation into transcriptomes, for both coding and noncoding genes. An altered transcriptome provides a molecular basis for improving adaptation during the evolution of new species. The allotetraploid cotton, together with the putative diploid ancestor species compose a fine model for study the rapid gene neofunctionalization over the genome shock. Here we report on Drought-Associated Non-coding gene 1 (DAN1), a long intergenic noncoding RNA (lincRNA) that arose from the cotton progenitor A-diploid genome after hybridization and WGD events during cotton evolution. DAN1 in allotetraploid upland cotton (Gossypium hirsutum) is a drought-responsive lincRNA predominantly expressed in the nucleoplasm. Chromatin isolation by RNA purification profiling and electrophoretic mobility shift assay analysis demonstrated that GhDAN1 RNA can bind with DNA fragments containing AAAG motifs, similar to DNA binding with one zinc finger transcription factor binding sequences. The suppression of GhDAN1 mainly regulates genes with AAAG motifs in auxin-response pathways, which are associated with drought stress regulation. As a result, GhDAN1-silenced plants exhibit improved tolerance to drought stress. This phenotype resembles the drought-tolerant phenotype of the A-diploid cotton ancestor species, which has an undetectable expression of DAN1. The role of DAN1 in cotton evolution and drought tolerance regulation suggests that the genomic shock of interspecific hybridization and WGD stimulated neofunctionalization of non-coding genes during the natural evolutionary process.

Functional analysis of the cotton CLE polypeptide signaling gene family in plant growth and development.

The CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (ESR)-RELATED (CLE) gene family encodes a large number of polypeptide signaling molecules involved in the regulation of shoot apical meristem division and root and vascular bundle development in a variety of plants. CLE family genes encode important short peptide hormones; however, the functions of these signaling polypeptides in cotton remain largely unknown. In the current work, we studied the effects of the CLE family genes on growth and development in cotton. Based on the presence of a conserved CLE motif of 13 amino acids, 93 genes were characterized as GhCLE gene family members, and these were subcategorized into 7 groups. A preliminary analysis of the cotton CLE gene family indicated that the activity of its members tends to be conserved in terms of both the 13-residue conserved domain at the C-terminus and their subcellular localization pattern. Among the 14 tested genes, the ectopic overexpression of GhCLE5::GFP partially mimicked the phenotype of the clv3 mutant in Arabidopsis. GhCLE5 could affect the endogenous CLV3 in binding to the receptor complex, comprised of CLV1, CLV2, and CRN, in the yeast two-hybrid assay and split-luciferase assay. Silencing GhCLE5 in cotton caused a short seedling phenotype. Therefore, we concluded that the cotton GhCLE gene family is functionally conserved in apical shoot development regulation. These results indicate that CLE also plays roles in cotton development as a short peptide hormone.

Pseudomonas species isolated via high-throughput screening significantly protect cotton plants against verticillium wilt.

Verticillium wilt (VW) caused by Verticillium dahliae is a devastating soil-borne disease that causes severe yield losses in cotton and other major crops worldwide. Here we conducted a high-throughput screening of isolates recovered from 886 plant rhizosphere samples taken from the three main cotton-producing areas of China. Fifteen isolates distributed in different genera of bacteria that showed inhibitory activity against V. dahliae were screened out. Of these, two Pseudomonas strains, P. protegens XY2F4 and P. donghuensis 22G5, showed significant inhibitory action against V. dahliae. Additional comparative genomic analyses and phenotypical assays confirmed that P. protegens XY2F4 and P. donghuensis 22G5 were the strains most efficient at protecting cotton plants against VW due to specific biological control products they produced. Importantly, we identified a significant efficacy of the natural tropolone compound 7-hydroxytropolone (7-HT) against VW. By phenotypical assay using the wild-type 22G5 and its mutant strain in 7-HT production, we revealed that the 7-HT produced by P. donghuensis is the major substance protecting cotton against VW. This study reveals that Pseudomonas specifically has gene clusters that allow the production of effective antipathogenic metabolites that can now be used as new agents in the biocontrol of VW.

Efficient chromatin profiling of H3K4me3 modification in cotton using CUT&Tag.

BACKGROUND: In 2019, Kaya-Okur et al. reported on the cleavage under targets and tagmentation (CUT&Tag) technology for efficient profiling of epigenetically modified DNA fragments. It was used mainly for cultured cell lines and was especially effective for small samples and single cells. This strategy generated high-resolution and low-background-noise chromatin profiling data for epigenomic analysis. CUT&Tag is well suited to be used in plant cells, especially in tissues from which small samples are taken, such as ovules, anthers, and fibers. RESULTS: Here, we present a CUT&Tag protocol step by step using plant nuclei. In this protocol, we quantified the nuclei that can be used in each CUT&Tag reaction, and compared the efficiency of CUT&Tag with chromatin immunoprecipitation with sequencing (ChIP-seq) in the leaves of cotton. A general workflow for the bioinformatic analysis of CUT&Tag is also provided. Results indicated that, compared with ChIP-seq, the CUT&Tag procedure was faster and showed a higher-resolution, lower-background signal than did ChIP. CONCLUSION: A CUT&Tag protocol has been refined for plant cells using intact nuclei that have been isolated.

Role of phasiRNAs from two distinct phasing frames of GhMYB2 loci in cis- gene regulation in the cotton genome.

BACKGROUND: Phased small interfering RNA (phasiRNA) is primarily derived from the 22-nt miRNA targeting loci. GhMYB2, a gene with potential roles in cotton fiber cell fate determination, is a target gene of miR828 and miR858 in the generation of phasiRNAs. RESULTS: In the presented work, through the evaluation of phasing scores and phasiRNA distribution pattern, we found that phasiRNAs from GhMYB2 were derived from the 3' cleavage fragments of 22-nt miR828 and 21-nt miR858 respectively. These two miRNA targeting sites initiated two phasing frames on transcripts of one locus. By means of RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we further demonstrated that phasiRNAs derived from the two phasing frames played a role in cis-regulation of GhMYB2. The phasiRNAs derived from GhMYB2 were expressed in the somatic tissues, especially in anther and hypocotyl. We further employed our previous small RNA sequencing data as well as the degradome data of cotton fiber bearing ovules, anthers, hypocotyls and embryogenic calli tissues published in public databases, to validate the expression, phasing pattern and functions of phasiRNAs. CONCLUSIONS: The presenting research provide insights of the molecular mechanism of phasiRNAs in regulation of GhMYB2 loci.

LncRNAs in polyploid cotton interspecific hybrids are derived from transposon neofunctionalization.

BACKGROUND: Interspecific hybridization and whole genome duplication are driving forces of genomic and organism diversification. But the effect of interspecific hybridization and whole genome duplication on the non-coding portion of the genome in particular remains largely unknown. In this study, we examine the profile of long non-coding RNAs (lncRNAs), comparing them with that of coding genes in allotetraploid cotton (Gossypium hirsutum), its putative diploid ancestors (G. arboreum; G. raimondii), and an F1 hybrid (G. arboreum × G. raimondii, AD). RESULTS: We find that most lncRNAs (80%) that were allelic expressed in the allotetraploid genome. Moreover, the genome shock of hybridization reprograms the non-coding transcriptome in the F1 hybrid. Interestingly, the activated lncRNAs are predominantly transcribed from demethylated TE regions, especially from long interspersed nuclear elements (LINEs). The DNA methylation dynamics in the interspecies hybridization are predominantly associated with the drastic expression variation of lncRNAs. Similar trends of lncRNA bursting are also observed in the progress of polyploidization. Additionally, we find that a representative novel lncRNA XLOC_409583 activated after polyploidization from a LINE in the A subgenome of allotetraploid cotton was involved in control of cotton seedling height. CONCLUSION: Our results reveal that the processes of hybridization and polyploidization enable the neofunctionalization of lncRNA transcripts, acting as important sources of increased plasticity for plants.

Cysteine protease enhances plant-mediated bollworm RNA interference.

Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665-673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.

Gossypol-enhanced P450 gene pool contributes to cotton bollworm tolerance to a pyrethroid insecticide.

Cotton plants accumulate phytotoxins, including gossypol and related sesquiterpene aldehydes, to resist insect herbivores and pathogens. To counteract these defensive plant secondary metabolites, cotton bollworms (Helicoverpa armigera) elevate their production of detoxification enzymes, including cytochrome P450 monooxygenases (P450s). Besides their tolerance to phytotoxin, cotton bollworms have quickly developed resistance to deltamethrin, a widely used pyrethroid insecticide in cotton field. However, the relationship between host plant secondary metabolites and bollworm insecticide resistance is poorly understood. Here, we show that exogenously expressed CYP6AE14, a gossypol-inducible P450 of cotton bollworm, has epoxidation activity towards aldrin, an organochlorine insecticide, indicating that gossypol-induced P450s participate in insecticide metabolism. Gossypol-ingested cotton bollworm larvae showed higher midgut P450 enzyme activities and exhibited enhanced tolerance to deltamethrin. The midgut transcripts of bollworm larvae administrated with different phytochemicals and deltamethrin were then compared by microarray analysis, which showed that gossypol and deltamethrin induced the most similar P450 expression profiles. Gossypol-induced P450s exhibited high divergence and at least five of them (CYP321A1, CYP9A12, CYP9A14, CYP6AE11 and CYP6B7) contributed to cotton bollworm tolerance to deltamethrin. Knocking down one of them, CYP9A14, by plant-mediated RNA interference (RNAi) rendered the larvae more sensitive to the insecticide. These data demonstrate that generalist insects can take advantage of secondary metabolites from their major host plants to elaborate defence systems against other toxic chemicals, and impairing this defence pathway by RNAi holds a potential for reducing the required dosages of agrochemicals in pest control.