MicroRNA-encoded regulatory peptides modulate cadmium tolerance and accumulation in rice.

MicroRNAs (miRNAs) are small noncoding RNAs that play a vital role in plant responses to abiotic and biotic stresses. Recently, it has been discovered that some primary miRNAs (pri-miRNAs) encode regulatory short peptides called miPEPs. However, the presence of miPEPs in rice, and their functions in response to abiotic stresses, particularly stress induced by heavy metals, remain poorly understood. Here, we identified a functional small peptide (miPEP156e) encoded by pri-miR156e that regulates the expression of miR156 and its target SPL genes, thereby affecting miR156-mediated cadmium (Cd) tolerance in rice. Overexpression of miPEP156e led to decreased uptake and accumulation of Cd and reactive oxygen species (ROS) levels in plants under Cd stress, resulting in improved rice Cd tolerance, as observed in miR156-overexpressing lines. Conversely, miPEP156e mutants displayed sensitivity to Cd stress due to the elevated accumulation of Cd and ROS. Transcriptome analysis further revealed that miPEP156e improved rice Cd tolerance by modulating Cd transporter genes and ROS scavenging genes. Our study provides insights into the regulatory mechanism of miPEP156e in rice response to Cd stress and demonstrates the potential of miPEPs as an effective tool for improving crop abiotic stress tolerance.

Rice responds to Spodoptera frugiperda infestation via epigenetic regulation of H3K9ac in the jasmonic acid signaling and phenylpropanoid biosynthesis pathways.

KEY MESSAGE: This study provided important insights into the complex epigenetic regulatory of H3K9ac-modified genes involved in the jasmonic acid signaling and phenylpropanoid biosynthesis pathways of rice in response to Spodoptera frugiperda infestation. Physiological and molecular mechanisms underlying plant responses to insect herbivores have been well studied, while epigenetic modifications such as histone acetylation and their potential regulation at the genomic level of hidden genes remain largely unknown. Histone 3 lysine 9 acetylation (H3K9ac) is an epigenetic marker widely distributed in plants that can activate gene transcription. In this study, we provided the genome-wide profiles of H3K9ac in rice (Oryza sativa) infested by fall armyworm (Spodoptera frugiperda, FAW) using CUT&Tag-seq and RNA-seq. There were 3269 and 4609 up-regulated genes identified in plants infested by FAW larvae for 3 h and 12 h, respectively, which were mainly enriched in alpha-linolenic acid and phenylpropanoid pathways according to transcriptomic analysis. In addition, CUT&Tag-seq analysis revealed increased H3K9ac in FAW-infested plants, and there were 422 and 543 up-regulated genes enriched with H3K9ac observed at 3 h and 12 h after FAW feeding, respectively. Genes with increased H3K9ac were mainly enriched in the transcription start site (TSS), suggesting that H3K9ac is related to gene transcription. Integrative analysis of both RNA-seq and CUT&Tag-seq data showed that up-expressed genes with H3K9ac enrichment were mainly involved in the jasmonic acid (JA) and phenylpropanoid pathways. Particularly, two spermidine hydroxycinnamoyl transferase genes SHT1 and SHT2 involved in phenolamide biosynthesis were highly modified by H3K9ac in FAW-infested plants. Furthermore, the Ossht1 and Ossht2 transgenic lines exhibited decreased resistance against FAW larvae. Our findings suggest that rice responds to insect herbivory via H3K9ac epigenetic regulation in the JA signaling and phenolamide biosynthesis pathways.

High nitrogen application in maize enhances insecticide tolerance of the polyphagous herbivore Spodoptera litura by induction of detoxification enzymes and intensification of cuticle.

Nitrogen (N) is one of the most intensively used fertilizers in cropping system and could exert a variety of bottom-up effects on the ecological fitness of herbivores. However, the effects of increased N inputs on insect pesticide tolerance have not been comprehensively understood. Bioassays showed that high N (HN) applied to maize plants significantly increased larval tolerance of Spodoptera litura to multiple insecticides. Activities of detoxification enzymes were significantly higher in the larvae fed on maize plants supplied with HN. RNA-seq analysis showed that numerous GST and cuticle-related genes were induced in the larvae fed on HN maize. RT-qPCR analysis further confirmed four GST genes and larval-specific cuticle gene LCP167. Furthermore, when injected with dsRNA specific to GSTe1, GSTs5, and LCP167, the mortality of larvae treated with methomyl was about 3-fold higher than that of dsGFP-injected larvae. Electron microscope observation showed that cuticle of the larvae fed on HN maize was thicker than the medium level of N. These findings suggest that increased application of N fertilizer enhances insecticide tolerance of lepidopteran pests via induction of detoxification enzymes and intensification of cuticle. Thus, overuse of N fertilizer may increase pest insecticide tolerance and usage of chemical insecticides.

Study on Active Components and Mechanism of Lettuce Latex Against Spodoptera Litura.

Plant latex is a sticky emulsion exuded from laticifers once the plant is damaged. Latex is an essential component of plant defense against herbivores. Lettuce (Lactuca sativa L.) in the Compositae family has relatively fewer insect herbivores compared with other leaf vegetables. The larvae of a generalist lepidopteran pest Spodoptera litura (Fabricius) avoided feeding on living lettuce plants. However, the larvae rapidly damaged the excised leaves that were unable to produce latex. Six compounds were isolated from lettuce latex. They were identified as 2,5-dihydroxybenzaldehyde (1), 3ß-hydroxy-4,15-dehydrograndolide (2), annuolide D (3), lactucin (4), lactucopicrin (5), and hanphyllin (6). Bioassays showed that the inhibition rate of compound 1 (2,5-dihydroxybenzaldehyde) and 6 (hanphyllin, a sesquiterpene lactone) on the weight gain of S. litura were 52.4 % and 10 %, respectively, at the concentration of 100 µg/g. RNA-seq analyses showed that larval exposure to compound 1 down-regulated the genes associated with heterobiotic metabolism including drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, retinol metabolism, glutathione metabolism, and drug metabolism-other enzymes (mainly uridine diphosphate glucuronyltransferase, UGTs). RT-qPCR further confirmed that 33 genes in the family of carboxylesterase (CarE), P450s and UGTs were down-regulated by compound 1. The activities of CarE, P450s and UGTs in the larvae fed on diets containing compound 1 were significantly lower than those fed on control diets, with the inhibition for the three detoxification enzymes being 55.4 %, 53.9 %, and 52.9 %. These findings suggest that secondary metabolites including 2,5-dihydroxybenzaldehyde in the latex play a key role in protecting lettuce from insect herbivory.

Metabolomic Profiling Reveals the Anti-Herbivore Mechanisms of Rice (Oryza sativa).

The use of secondary metabolites of rice to control pests has become a research hotspot, but little is known about the mechanism of rice self-resistance. In this study, metabolomics analysis was performed on two groups of rice (T1, with insect pests; T2, without pests), indicating that fatty acids, alkaloids, and phenolic acids were significantly up-regulated in T1. The up-regulated metabolites (p-value < 0.1) were enriched in linoleic acid metabolism, terpene, piperidine, and pyridine alkaloid biosynthesis, α-linolenic acid metabolism, and tryptophan metabolism. Six significantly up-regulated differential metabolites in T1 were screened out: N-trans-feruloyl-3-methoxytyramine (1), N-trans-feruloyltyramine (2), N-trans-p-coumaroyltyramine (3), N-cis-feruloyltyramine (4), N-phenylacetyl-L-glutamine (5), and benzamide (6). The insect growth inhibitory activities of these six different metabolites were determined, and the results show that compound 1 had the highest activity, which significantly inhibited the growth of Chilo suppressalis by 59.63%. Compounds 2-4 also showed a good inhibitory effect on the growth of Chilo suppressalis, while the other compounds had no significant effect. RNA-seq analyses showed that larval exposure to compound 1 up-regulated the genes that were significantly enriched in ribosome biogenesis in eukaryotes, the cell cycle, ribosomes, and other pathways. The down-regulated genes were significantly enriched in metabolic pathways, oxidative phosphorylation, the citrate cycle (TCA cycle), and other pathways. Eighteen up-regulated genes and fifteen down-regulated genes from the above significantly enriched pathways were screened out and verified by real-time quantitative PCR. The activities of detoxification enzymes (glutathione S-transferase (GST); UDP-glucuronosyltransferase (UGT); and carboxylesterase (CarE)) under larval exposure to compound 1 were measured, which indicated that the activity of GST was significantly inhibited by compound 1, while the activities of the UGT and CarE enzymes did not significantly change. As determined by UPLC-MS, the contents of compound 1 in the T1 and T2 groups were 8.55 ng/g and 0.53 ng/g, respectively, which indicated that pest insects significantly induced the synthesis of compound 1. Compound 1 may enhance rice insect resistance by inhibiting the detoxification enzyme activity and metabolism of Chilo suppressalis, as well as promoting cell proliferation to affect its normal growth and development process. The chemical-ecological mechanism of the insect resistance of rice is preliminarily clarified in this paper.

Integration of transcriptome and metabolome analyses reveals the role of OsSPL10 in rice defense against brown planthopper.

KEY MESSAGE: OsSPL10 is a negative regulator of rice defense against BPH, knockout of OsSPL10 enhances BPH resistance through upregulation of defense-related genes and accumulation of secondary metabolites. Rice (Oryza sativa L.), one of the most important staple foods worldwide, is frequently attacked by various herbivores, including brown planthopper (BPH, Nilaparvata lugens). BPH is a typical monophagous, phloem-sucking herbivore that has been a substantial threat to rice production and global food security. Understanding the regulatory mechanism of defense responses to BPH is essential for improving BPH resistance in rice. In this study, a SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 10 (OsSPL10) transcription factor was found to play a negative role in the defenses of rice against BPH. To gain insights into the molecular and biochemical mechanisms of OsSPL10, we performed combined analyses of transcriptome and metabolome, and revealed that knockout of OsSPL10 gene improved rice resistance against BPH by enhancing the direct and indirect defenses. Genes involved in plant hormone signal transduction, MAPK signaling pathway, phenylpropanoid biosynthesis, and plant-pathogen interaction pathway were significantly upregulated in spl10 mutant. Moreover, spl10 mutant exhibited increased accumulation of defense-related secondary metabolites in the phenylpropanoid and terpenoid pathways. Our findings reveal a novel role for OsSPL10 gene in regulating the rice defense responses, which can be used as a potential target for genetic improvement of BPH resistance in rice.

Transcription factor OsbZIP49 controls tiller angle and plant architecture through the induction of indole-3-acetic acid-amido synthetases in rice.

Tiller angle is an important determinant of plant architecture in rice (Oryza sativa L.). Auxins play a critical role in determining plant architecture; however, the underlying metabolic and signaling mechanisms are still largely unknown. In this study, we have identified a member of the bZIP family of TGA class transcription factors, OsbZIP49, that participates in the regulation of plant architecture and is specifically expressed in gravity-sensing tissues, including the shoot base, nodes and lamina joints. Transgenic rice plants overexpressing OsbZIP49 displayed a tiller-spreading phenotype with reduced plant height and internode lengths. In contrast, CRISPR/Cas9-mediated knockout of OsbZIP49 resulted in a compact architecture. Follow-up studies indicated that the effects of OsbZIP49 on tiller angles are mediated through changes in shoot gravitropic responses. Additionally, we provide evidence that OsbZIP49 activates the expression of indole-3-acetic acid-amido synthetases OsGH3-2 and OsGH3-13 by directly binding to TGACG motifs located within the promoters of both genes. Increased GH3-catalyzed conjugation of indole-3-acetic acid (IAA) in rice transformants overexpressing OsbZIP49 resulted in the increased accumulation of IAA-Asp and IAA-Glu, and a reduction in local free auxin, tryptamine and IAA-Glc levels. Exogenous IAA or naphthylacetic acid (NAA) partially restored shoot gravitropic responses in OsbZIP49-overexpressing plants. Knockout of OsbZIP49 led to reduced expression of both OsGH3-2 and OsGH3-13 within the shoot base, and increased accumulation of IAA and increased OsIAA20 expression levels were observed in transformants following gravistimulation. Taken together, the present results reveal the role transcription factor OsbZIP49 plays in determining plant architecture, primarily due to its influence on local auxin homeostasis.

Enhanced anti-herbivore defense of tomato plants against Spodoptera litura by their rhizosphere bacteria.

BACKGROUND: The use of beneficial microorganisms as an alternative for pest control has gained increasing attention. The objective of this study was to screen beneficial rhizosphere bacteria with the ability to enhance tomato anti-herbivore resistance. RESULTS: Rhizosphere bacteria in tomato field from Fuqing, one of the four locations where rhizosphere bacteria were collected in Fujian, China, enhanced tomato resistance against the tobacco cutworm Spodoptera litura, an important polyphagous pest. Inoculation with the isolate T6-4 obtained from the rhizosphere of tomato field in Fuqing reduced leaf damage and weight gain of S. litura larvae fed on the leaves of inoculated tomato plants by 27% in relative to control. Analysis of 16S rRNA gene sequence identities indicated that the isolate T6-4 was closely related to Stenotrophomonas rhizophila supported with 99.37% sequence similarity. In the presence of S. litura infestation, inoculation with the bacterium led to increases by a 66.9% increase in protease inhibitor activity, 53% in peroxidase activity and 80% in polyphenol oxidase activity in the leaves of inoculated plants as compared to the un-inoculated control. Moreover, the expression levels of defense-related genes encoding allene oxide cyclase (AOC), allene oxide synthase (AOS), lipoxygenase D (LOXD) and proteinase inhibitor (PI-II) in tomato leaves were induced 2.2-, 1.7-, 1.4- and 2.7-fold, respectively by T6-4 inoculation. CONCLUSION: These results showed that the tomato rhizosphere soils harbor beneficial bacteria that can systemically induce jasmonate-dependent anti-herbivore resistance in tomato plants.

Cadmium transfer between maize and soybean plants via common mycorrhizal networks.

More than 80% terrestrial plants establish mutualistic symbiosis with soil-borne arbuscular mycorrhizal fungi (AMF). These fungi not only significantly improve plant nutrient acquisition and stress resistance, but also mitigate heavy metal phytotoxicity, Furthermore, the extraradical mycorrhizal mycelia can form common mycorrhizal networks (CMNs) that link roots of multiple plants in a community. Here we show that the networks mediate migration of heavy metal cadmium (Cd) from maize (Zea mays L.) to soybean (Glycine max (Linn.) Merr.) plants. CMNs between maize and soybean plants were established after inoculation of maize plants with AMF Funneliformis mosseae. Application of CdCl2 in maize plants led to 64.4% increase in the shoots and 48.2% increase in the roots in Cd content in CMNs-connected soybean plants compared to the control without Cd treatment in maize. Meanwhile, although the CMNs-connected soybean plants did not directly receive Cd supply, they upregulated transcriptional levels of Cd transport-related genes HATPase and RSTK 2.13- and 5.96-fold, respectively, induced activities of POD by 44.8% in the leaves, and increased MDA by 146.2% in the roots. Furthermore, Cd addition inhibited maize growth but mycorrhizal colonization improved plant performance in presence of Cd stress. This finding demonstrates that mycorrhizal networks mediate the transfer of Cd between plants of different species, suggesting a potential to use CMNs as a conduit to transfer toxic heavy metals from main food crops to heavy metal hyperaccumulators via intercropping.

Activation of the NR2E nuclear receptor HR83 leads to metabolic detoxification-mediated chlorpyrifos resistance in Nilaparvata lugens.

Increased production of detoxification enzymes appears to be the primary route for insecticide resistance in many crop pests. However, the mechanisms employed by resistant insects for overexpression of detoxification genes involved in insecticide resistance remain obscure. We report here that the NR2E nuclear receptor HR83 plays a critical role in chlorpyrifos resistance by regulating the expression of detoxification genes in the brown planthopper (BPH), Nilaparvata lugens. HR83 was highly expressed in the fat body and ovary of adult females in chlorpyrifos-resistant BPHs. Knockdown of HR83 by RNA interference showed no effect on female fecundity, whereas caused a decrease of resistance to chlorpyrifos. This treatment also led to a dramatic reduction in the expression of multiple detoxification genes, including four UDP-glycosyltransferases (UGTs), three cytochrome P450 monooxygenases (P450s) and four carboxylesterases (CarEs). Among these HR83-regulated genes, UGT-1-3, UGT-2B10, CYP6CW1, CYP4CE1, CarE and Esterase E4-1 were over-expressed both in the fat body and ovary of the resistant BPHs. Functional analyses revealed that UGT-2B10, CYP4CE1, CarE and Esterase E4-1 are essential for the resistance of BPH to chlorpyrifos. Generally, this study implicates HR83 in the metabolic detoxification-mediated chlorpyrifos resistance and suggests that the regulation of detoxification genes may be an ancestral function of the NR2E nuclear receptor subfamily.

Effect of hepatocyte nuclear factor 4 on the fecundity of Nilaparvata lugens: Insights from RNA interference combined with transcriptomic analysis.

Hepatocyte nuclear factor 4 (HNF4) plays essential roles in regulating lipid metabolism and glucose homeostasis in female insects. However, little is known about the role of HNF4 in insect fecundity. Here we demonstrate that HNF4 regulates female fecundity by affecting egg hatching in the brown planthopper (BPH) Nilaparvata lugens. HNF4 was highly expressed in the ovary and fat body of female adult. RNA interference-mediated HNF4 knockdown resulted in a dramatic reduction in egg hatchability and caused a severe block in embryonic development, while showed no significant effects on ovary development and egg laying. Transcriptome sequencing analysis showed that 72 genes encoding ribosome proteins were significantly down-regulated in the HNF4-silenced BPH and "ribosome" was the most-enriched pathway for the down-regulated genes. These results suggest that HNF4 controls the dynamics of egg structure, likely through its regulation of ribosome protein genes, which in turn affects the embryonic development and egg hatching.

Control of Panama disease of banana by intercropping with Chinese chive (Allium tuberosum Rottler): cultivar differences.

Panama disease (Fusarium wilt disease) caused by Fusarium oxysporum f. sp. cubense race 4 (FOC) severely threatens banana (Musa spp.) production worldwide. Intercropping of banana with Allium plants has shown a potential to reduce Panama disease. In this study, six cultivars of Chinese chive (Allium tuberosum Rottler) were selected to compare their differences in antifungal activity and active compounds. Three cultivars Duokang Fujiu 11, Fujiuhuang 2, and Duokang Sijiqing with higher levels of antifungal compounds were further used for intercropping with banana in the pots and field to compare their effects on growth and disease incidence of banana.The six cultivars showed significant differences in antifungal activity against FOC mycelia growth in both leaf volatiles and aqueous leachates. The aqueous leachates displayed stronger antifungal activity than the volatiles. FJH cultivar showed the best inhibitory effect among all six cultivars. Contents of three main antifungal compounds dipropyl trisulfide (DPT), dimethyl trisulfide (DMT), and 2-methyl-2-pentenal (MP) in volatiles and aqueous leachates varied considerably among cultivars. Pot and field experiments showed that intercropping with three selected Chinese chive cultivars significantly improved banana vegetative growth, increased photosynthetic characteristics and yield but decreased disease incidence of Panama disease.Our results indicate that intercropping with Chinese chive shows potential to reduce banana Panama disease and selection of appropriate cultivars is vital for effective disease control.

Priming and filtering of antiherbivore defences among Nicotiana attenuata plants connected by mycorrhizal networks.

Arbuscular mycorrhizal fungi (AMF) establish symbiotic associations with a majority of terrestrial plants to form underground common mycorrhizal networks (CMNs) that connect neighbouring plants. Because Nicotiana attenuata plants do not respond to herbivory-elicited volatiles from neighbours, we used this ecological model system to evaluate if CMNs function in interplant transmission of herbivory-elicited responses. A mesocosm system was designed to establish and remove CMNs linking N. attenuata plants to examine the herbivory-elicited metabolic and hormone responses in CMNs-connected "receiver" plants after the elicitation of "donor" plants by wounding (W) treated with Manduca sexta larval oral secretions (OS). AMF colonization increased constitutive jasmonate (JA and JA-Ile) levels in N. attenuata roots but did not affect well-characterized JAs-regulated defensive metabolites in systemic leaves. Interestingly, larger JAs bursts, and higher levels of several amino acids and particular sectors of hydroxygeranyllinalool diterpene glycoside metabolism were elevated in the leaves of W + OS-elicited "receivers" with CMN connections with "donors" that had been W + OS-elicited 6 hr previously. Our results demonstrate that AMF colonization alone does not enhance systemic defence responses but that sectors of systemic responses in leaves can be primed by CMNs, suggesting that CMNs can transmit and even filter defence signalling among connected plants.

The roles of jasmonate signalling in nitrogen uptake and allocation in rice (Oryza sativa L.).

Herbivore damage by chewing insects activates jasmonate (JA) signalling that can elicit systemic defense responses in rice. Few details are known, however, concerning the mechanism, whereby JA signalling modulates nutrient status in rice in response to herbivory. (15 NH4 )2 SO4 labelling experiments, proteomic surveys, and RT-qPCR analyses were used to identify the roles of JA signalling in nitrogen (N) uptake and allocation in rice plants. Exogenous applications of methyl jasmonate (MeJA) to rice seedlings led to significantly reduced N uptake in roots and reduced translocation of recently-absorbed 15 N from roots to leaves, likely occurring as a result of down-regulation of glutamine synthetase cytosolic isozyme 1-2 and ferredoxin-nitrite reductase. Shoot MeJA treatment resulted in a remobilization of endogenous unlabelled 14 N from leaves to roots, and root MeJA treatment also increased 14 N accumulation in roots but did not affect 14 N accumulation in leaves of rice. Additionally, proteomic and RT-qPCR experiments showed that JA-mediated plastid disassembly and dehydrogenases GDH2 up-regulation contribute to N release in leaves to support production of defensive proteins/compounds under N-limited condition. Collectively, our results indicate that JA signalling mediates large-scale systemic changes in N uptake and allocation in rice plants.

Plant allelochemicals affect tolerance of polyphagous lepidopteran pest Helicoverpa armigera (Hübner) against insecticides.

Cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), is a polyphagous lepidopteran pest distributed worldwide with a broad spectrum of host plants. However, the mechanism of H. armigera adaptation to various insecticides and defensive allelochemicals in its host plants is not fully understood. Therefore, this study examined the influence of consumption of plant allelochemicals on larval tolerance to methomyl and chlorpyrifos insecticides in H. armigera and its possible mechanism. Twelve plant allelochemicals were screened to evaluate their effects on larval sensitivity to methomyl. Of which flavone, coumarin, DIMBOA (2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one) and visnagin significantly reduced larval sensitivity to methomyl. Application of cytochrome P450 inhibitor piperonyl butoxide (PBO) significantly increased the mortality of methomyl-treated larvae. In contrast, PBO addition significantly decreased the mortality of chlorpyrifos-treated larvae. Moreover, allelochemical consumption enhanced the activities of glutathione S-transferase, carboxylesterase, cytochrome P450 and acetylcholinesterase in the midgut and fat body. The qRT-PCR analysis confirms that P450 genes, CYP6B2, CYP6B6 and CYP6B7 were induced by the four allelochemicals in the midguts and the fat bodies. In conclusion, the generalist H. armigera can take benefit of plant allelochemicals from its host plants to elaborate its defense against insecticides.

Identification of a cytochrome P450 CYP6AB60 gene associated with tolerance to multi-plant allelochemicals from a polyphagous caterpillar tobacco cutworm (Spodoptera litura).

Generalist phytophagous insects adapt to adventurous chemical environment in a wide variety of host plants by extraordinary detoxifying metabolic abilities. However, how polyphagous insect cope with the diversity of plant defenses remains largely unknown and only a few counter-defense genes detoxifying a wide range of toxic secondary metabolites have been well characterized. Here, we identify a cytochrome P450 gene (CYP6AB60) from tobacco cutworm (Spodoptera litura) in response to three different plant's defense metabolites. After being exposed to artificial diet supplemented with coumarin (COU), xanthotoxin (XAN) or tomatine (TOM), activities of P450 and CYP6AB60 transcript levels in both midgut and fat body tissues were significantly increased. Developmental expression analysis revealed that CYP6AB60 was expressed highly during the larval stages, and tissue distribution analysis showed that CYP6AB60 was expressed extremely high in the midgut, which correspond to the physiological role of CYP6AB60 from S. litura larvae in response to plant allelochemicals. Furthermore, when larvae are injected with double-stranded RNA (dsRNA) specific to CYP6AB60, levels of this transcript in the midgut and fatbody decrease and the negative effect of plant's defense metabolites on larval growth is magnified. These data demonstrate that the generalist insect S. litura might take advantage of an individual detoxificative gene CYP6AB60 to toxic secondary metabolites from different host plants. The CYP6AB60 can be a potential gene to carry out RNAi-mediated crop protection against the major polyphagous pest S. litura in the future.

Bioactivation of aflatoxin B1 by a cytochrome P450, CYP6AE19 induced by plant signaling methyl jasmonate in Helicoverpa armigra (Hübner).

Herbivore attack leads to enhanced production of defensive compounds to mount anti-herbivore defense in plants via activation of the jasmonate signaling pathway. On the other hand, some herbivores can eavesdrop on plants defense signaling and up-regulate their cytochrome P450 genes to increase detoxification of defensive compounds. However, the ecological risk of eavesdropping on plant defense signaling is largely unknown. In this study, we examined the induction of cytochrome P450s by methyl jasmonate (MeJA) and its consequence on the toxicity of aflatoxin B1 (AFB1) to Helicoverpa armigra larvae. The results show that MeJA applications either in a diet or volatile exposure enhanced the toxicity of AFB1 to the larvae. RNA sequences analysis revealed that cytochrome P450 CYP6AE19 was highly induced when MeJA was applied with AFB1. In addition, HaGST encoding glutathione-S-transferase that mainly transforms aflatoxin B1 exo-8,9-epoxide to aflatoxin B1 exo-8,9-glutathione was also induced. RNA interference of CYP6AE19 via injecting a double-stranded RNA decreased mortality of larvae exposed to AFB1; while injecting a double-stranded RNA of HaGST increased larval mortality. Furthermore, a protein model was generated and a subsequent docking simulation for AFB1 suggests the bioactivation as a major mechanism of AFB1. This study provides evidence that MeJA increased larval mortality of H. armigera via induction of CYP6AE19 that can bioactivate AFB1.

Copper-induced H2O2 accumulation confers larval tolerance to xanthotoxin by modulating CYP6B50 expression in Spodoptera litura.

In the plant-insect arms race, plants synthesize toxic compounds to defend against herbivorous insects, whereas insects employ cytochrome P450 monooxygenases (P450s) to detoxify these phytotoxins. As ubiquitous environmental contaminants, heavy metals can be easily absorbed by plants and further accumulated in herbivorous insects through the food chains, resulting in tangible consequences for plant-insect interactions. However, whether heavy metals can influence P450 activities and thereby cause further effects on larval tolerance to phytotoxins remains unknown. In this study, we shown that prior exposure to copper (Cu) enhanced larval tolerance to xanthotoxin in Spodoptera litura, a major polyphagous pest of agriculture. P450 activities were induced in larvae exposed to Cu or xanthotoxin, and a midgut specific expressed P450 gene, CYP6B50 was cross-induced after exposure to these two toxic xenobiotics. Knocking down CYP6B50 by RNA interference (RNAi) rendered the larvae more sensitive to xanthotoxin. As defense against oxidative stress following metal exposure has been demonstrated to affect insecticide resistance, the reactive oxygen species (ROS) generation and antioxidant enzyme activities were assessed. Cu exposure caused the accumulation of hydrogen peroxide (H2O2) and enhanced the activities of superoxide dismutase (SOD) and peroxidase (POD) in larval midgut. In addition, two antioxidant response elements (AREs) were identified from the CYP6B50 promoter, indicating that Cu-induced CYP6B50 expression may be related to the ROS burst. Application of ROS scavenger N-acetylcysteine (NAC) effectively suppressed CYP6B50 expression, inhibited P450 activities and impaired larval tolerance to xanthotoxin that had been induced by Cu. These results indicate that the increase in CYP6B50 expression regulated by Cu-induced H2O2 generation contributed to the enhancement of larval tolerance to xanthotoxin in S. litura. Ingestion of heavy metals from their host plants can inadvertently boost the counter-defense system of herbivorous insects to protect themselves against plant defensive toxins.

Copper exposure enhances Spodoptera litura larval tolerance to ß-cypermethrin.

Environmental xenobiotics can influence the tolerance of insects to chemical insecticides. Heavy metals are widespread distributed, can be easily bio-accumulated in plants and subsequently within phytophagous insects via the food chains. However, less attention has been paid to the effect of heavy metal exposure on their insecticide tolerance. In this study, pre-exposure of copper (Cu, 25-100 mg kg-1) significantly enhanced the subsequent tolerance of Spodoptera litura to ß-cypermethrin, a widely used pyrethroid insecticide in crop field. Cytochrome P450 monooxygenases (CYPs) activities were cross-induced in larvae exposed to Cu and ß-cypermethrin, while the activities of glutathione S-transferase (GST) and carboxylesterase (CarE) were not affected. Application of piperonyl butoxide (PBO), a P450 synergist, effectively impaired the tolerance to ß-cypermethrin in Cu-exposed S. litura larvae with a synergistic ratio of 1.72, indicating that P450s contribute to larval tolerance to ß-cypermethrin induced by Cu exposure. Among the four CYP6AB family genes examined, only larval midgut-specific CYP6AB12 was found to be cross-induced by Cu and ß-cypermethrin. RNA interference (RNAi)-mediated silencing of CYP6AB12 effectively decreased the mRNA levels of the target gene, and significantly reduced the larval tolerance to ß-cypermethrin following exposure to Cu. These results showed that pre-exposure of heavy metal Cu enhanced larval tolerance to ß-cypermethrin in S. litura, possibly through the cross-induction of P450s. Our findings provide new insights on the relationship between heavy metals and chemical insecticides that may benefit both the risk evaluation of heavy metal contamination and development of pest management strategies.

Albocycline-type Macrolides with Antibacterial Activities from Streptomyces sp. 4205.

The actinomycete genus Streptomyces is characterized by producing bioactive secondary metabolites, including antibiotics. In this study, chemical and biological investigations were carried out on Streptomyces strain 4205 isolated from the paddy soil, leading to the identification and characterization of 10 albocycline-type macrolides, among which 4 compounds were new, namely albocyclines A-D (1-4). The structures of 1-10 were identified according to the 1D- and 2D-NMR spectroscopic data. Furthermore, compounds 1-10 were evaluated for antimicrobial activity. Compounds 5-7 displayed antimicrobial activities against Candidaalbicans ATCC 90028 with the same MIC value of 10.0 mg/mL and the IC50 values of 1.5, 1.0, and 1.0 mg/mL, respectively. Thus, the research on Streptomyces sp. is of vital significance for developing new antibiotic agents.