Oxylipins produced by the 13-lipoxygenase (LOX) have been reported to play an important role in plant defense responses to herbivores. Yet, the role of oxylipins produced by the 9-LOX pathway in this process remains largely unknown. Here we cloned a gene encoding a chloroplast-localized 9-LOX, Osr9-LOX1, from rice. Transcriptional analysis revealed that herbivore infestation, mechanical wounding and jasmonic acid (JA) treatment either repressed or did not enhance the level of Osr9-LOX1 transcripts at early stages but did at later stages, whereas salicylic acid (SA) treatment quickly increased the transcript level of Osr9-LOX1. Antisense expression of Osr9-lox1 (as-r9lox1) decreased the amount of wound-induced (Z)-3-hexenal but increased levels of striped stem borer (SSB)-induced linolenic acid, JA, SA and trypsin protease inhibitors. These changes were associated with increased resistance in rice to the larvae of the SSB Chilo suppressalis. In contrast, although no significant differences were observed in the duration of the nymph stage or the number of eggs laid by female adults between the brown planthopper (BPH) Nilaparvata lugens that fed on as-r9lox1 lines and BPH that fed on wild-type (WT) rice plants, the survival rate of BPH nymphs that fed on as-r9lox1 lines was higher than that of nymphs that fed on WT plants, possibly because of a higher JA level. The results demonstrate that Osr9-LOX1 plays an important role in regulating an herbivore-induced JA burst and cross-talk between JA and SA, and in controlling resistance in rice to chewing and phloem-feeding herbivores.
NADPH-cytochrome P450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. It catalyzes electron transfer from NADPH to all known P450s, thus plays central roles not only in the metabolism of exogenous xenobiotics but also in the regulation of endogenous hormones in insects. In this study, a full-length cDNA encoding of a CPR (named CsCPR) was isolated from the Asiatic rice striped stem borer, Chilo suppressalis, by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA contains a 2061 bp open reading frame, which encodes an enzyme of 686 amino acid residues, with a calculated molecular mass of 77.6 kDa. The deduced peptide has hallmarks of typical CPR, including an N-terminal membrane anchor and the FMN, FAD and NADPH binding domains. The N-terminal-truncated protein fused with a 6 × His·tag was heterologously expressed in Escherichia coli Rosetta (DE3) cells and purified, specific activity and the Km values of the recombinant enzyme were determined. Tissue- and developmental stage-dependent expression of CsCPR mRNA was investigated by real-time quantitative PCR. The CsCPR mRNA was noticeably expressed in the digestive, metabolic, and olfactory organs of the larvae and adults of C. suppressalis. Our initial results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems.
Glutathione S-transferase (GST) genes control crucial traits for the metabolism of various toxins encountered by insects in host plants and the wider environment, including insecticides. The planthoppers Nilaparvata lugens and Sogatella furcifera are serious specialist pests of rice throughout eastern Asia. Their capacity to rapidly adapt to resistant rice varieties and to develop resistance to various insecticides has led to severe outbreaks over the last decade.
Transgenic rice expressing Bacillus thuringiensis (Bt) CrylAb protein is expected to be commercialized in China in the near future. The use of Bt rice for controlling insect pests sparks intensive debates regarding its biosafety. Folsomia candida is an euedaphic species and is often used as a "standard" test organism in assessing effects of environmental pollutants on soil organisms. In this study, growth, development, reproduction, and superoxide dismutase activity (SOD) of F. candida were investigated in the laboratory for populations reared on leaf tissue or leaf-soil mixtures of two CrylAb rice lines and a non-Bt rice isoline. Two independent tests were performed: 1) a 35-d test using petri dishes containing yeast diet (positive control) or fresh rice leaf tissue, and 2) a 28-d test in soil-litter microcosms containing yeast or a mixture of soil and rice leaf tissue. Biological parameters measured in both tests were number of progeny production, population growth rate, and SOD activity. For the petri dish test, data measured also included insect body length and number of exuviation. There were no significant differences between the populations reared on Bt and non-Bt rice leaf tissue in all measured parameters in both tests and for both Bt rice lines, suggesting no significant effects of the CrylAb protein in Bt rice on F. candida in the laboratory studies. Results of this study should add additional biosafety proofs for use of Bt rice to manage rice pests in China.
The oxylipin pathway is of central importance for plant defensive responses. Yet, the first step of the pathway, the liberation of linolenic acid following induction, is poorly understood. Phospholipases D (PLDs) have been hypothesized to mediate this process, but data from Arabidopsis (Arabidopsis thaliana) regarding the role of PLDs in plant resistance have remained controversial. Here, we cloned two chloroplast-localized PLD genes from rice (Oryza sativa), OsPLD?4 and OsPLD?5, both of which were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis, mechanical wounding, and treatment with jasmonic acid (JA). Antisense expression of OsPLD?4 and -?5 (as-pld), which resulted in a 50% reduction of the expression of the two genes, reduced elicited levels of linolenic acid, JA, green leaf volatiles, and ethylene and attenuated the SSB-induced expression of a mitogen-activated protein kinase (OsMPK3), a lipoxygenase (OsHI-LOX), a hydroperoxide lyase (OsHPL3), as well as a 1-aminocyclopropane-1-carboxylic acid synthase (OsACS2). The impaired oxylipin and ethylene signaling in as-pld plants decreased the levels of herbivore-induced trypsin protease inhibitors and volatiles, improved the performance of SSB and the rice brown planthopper Nilaparvata lugens, and reduced the attractiveness of plants to a larval parasitoid of SSB, Apanteles chilonis. The production of trypsin protease inhibitors in as-pld plants could be partially restored by JA, while the resistance to rice brown planthopper and SSB was restored by green leaf volatile application. Our results show that phospholipases function as important components of herbivore-induced direct and indirect defenses in rice.
How rice defends itself against pathogen infection is well documented, but little is known about how it defends itself against herbivore attack. We measured changes in the transcriptome and chemical profile of rice when the plant is infested by the striped stem borer (SSB) Chilo suppressalis. Infestation by SSBs resulted in changes in the expression levels of 4545 rice genes; this number accounts for about 8% of the genome and is made up of 18 functional groups with broad functions. The largest group comprised genes involved in metabolism, followed by cellular transport, transcription and cellular signaling. Infestation by SSBs modulated many genes responsible for the biosynthesis of plant hormones and plant signaling. Jasmonic acid (JA), salicylic acid (SA) and ethylene were the major hormones that shaped the SSB-induced defence responses of rice. Many secondary signal transduction components, such as those involved in Ca²? signaling and G-protein signaling, receptor and non-receptor protein kinases, and transcription factors were involved in the SSB-induced responses of rice. Photosynthesis and ATP synthesis from photophosphorylation were restricted by SSB feeding. In addition, SSB infestation induced the accumulation of defence compounds, including trypsin proteinase inhibitors (TrypPIs) and volatile organic compounds. These results demonstrate that SSB-induced defences required rice to reconfigure a wide variety of its metabolic, physiological and biochemical processes.
Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C(6), C(12), C(16), C(18:1), and C(24:1)-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe(2+). These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.
The spread of invasive species is a complex ecological process that is affected by both the biology of the species and the spatial structure of a landscape. The rice water weevil (Lissorhoptrus oryzophilus Kuschel), a notorious crop pest found in many parts of the world, is one of the most devastating invasive species in China, and has caused enormous economic losses and ecological damage. Little is known, however, as to how habitat and landscape features affect the spatial spread of this pest. Thus, the main goal of this study was to investigate the relationship between the observed spread pattern of L. oryzophilus and landscape structural factors in Zhejiang Province, China between 1993 and 2001. We quantified the invasive spread of the weevil in terms of both the proportion of infected area and spread distance each year as well as landscape structure and connectivity of rice paddies with landscape metrics. Our results showed that the spread of L. oryzophilus took place primarily in the southwest-northeast direction along coastal areas at a speed of about 36 km per year. The composition and spatial arrangement of landscape elements were key determinants of this unique spread pattern. In particular, the connectivity of early rice paddies was crucial for the invasive spread while other factors such as meteorological and geographical conditions may also have been relevant. To control the spread of the pest, we propose four management measures: (1) to implement a landscape-level planning scheme of cropping systems to minimize habitat area and connectivity for the pest, (2) to reduce the source populations at a local scale using integrated control methods, (3) to monitor and report invasive spread in a timely manner, and (4) to strengthen the quarantine system. To be most effective, all four management measures need to be implemented together through an integrated, multi-scaled approach.
Ceramidases catalyze the hydrolysis of ceramides to generate sphingosine (SPH) and fatty acids, and ceramide metabolism is implicated in various biological responses in Drosophila melanogaster. Here we report the cloning, biochemical characterization, and functional analysis of a Drosophila alkaline ceramidase (Dacer). Dacer, a membrane-bound protein of 284 amino acids, shares homology with yeast and mammalian alkaline ceramidases. Overexpression of Dacer in High Five insect cells increases ceramidase activity in the alkaline pH range, indicating that Dacer is a bona fide alkaline ceramidase. Dacer mRNA is highly expressed in the midgut and at the pupal stage. An inactivation of Dacer by insertional mutagenesis increases the levels of ceramides in both Drosophila pupae and adult flies. Dacer inactivation increases Drosophila pre-adult development time, lifespan, and anti-oxidative stress capacity. Collectively, these results suggest that Dacer plays an important role in the Drosophila development and longevity by controlling the metabolism of ceramides.
The jasmonic acid (JA) pathway plays a central role in plant defense responses against insects. Some phloem-feeding insects also induce the salicylic acid (SA) pathway, thereby suppressing the plants JA response. These phenomena have been well studied in dicotyledonous plants, but little is known about them in monocotyledons. We cloned a chloroplast-localized type 2 13-lipoxygenase gene of rice, OsHI-LOX, whose transcripts were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis and the rice brown planthopper (BPH) Niaparvata lugens, as well as by mechanical wounding and treatment with JA. Antisense expression of OsHI-LOX (as-lox) reduced SSB- or BPH-induced JA and trypsin protease inhibitor (TrypPI) levels, improved the larval performance of SBB as well as that of the rice leaf folder (LF) Cnaphalocrocis medinalis, and increased the damage caused by SSB and LF larvae. In contrast, BPH, a phloem-feeding herbivore, showed a preference for settling and ovipositing on WT plants, on which they consumed more and survived better than on as-lox plants. The enhanced resistance of as-lox plants to BPH infestation correlated with higher levels of BPH-induced H(2)O(2) and SA, as well as with increased hypersensitive response-like cell death. These results imply that OsHI-LOX is involved in herbivore-induced JA biosynthesis, and plays contrasting roles in controlling rice resistance to chewing and phloem-feeding herbivores. The observation that suppression of JA activity results in increased resistance to an insect indicates that revision of the generalized plant defense models in monocotyledons is required, and may help develop novel strategies to protect rice against insect pests.
Sensory neuron membrane proteins (SNMPs), which are located on the dendritic membrane of olfactory neurons, were considered as important components involved in pheromone reception in insects. In Drosophila melanogaster, mutants without SNMP are unable to evoke neuronal activities in the presence of pheromone cis-vaccenyl acetate (cVA). So deeply understanding the SNMPs functions may help to develop pheromone-mediated insect pest management tactics. The present study reports the identification and characterization of CmedSNMP1 and CmedSNMP2, two candidate SNMPs in the rice leaffolder, Cnaphalocrocis medinalis, one of the serious rice insect pests in Asia. The comparison of amino acid sequences shows that CmedSNMP1 and CmedSNMP2 are very similar to the previously reported SNMPs isolated from moths such as Ostrinia nubilalis and O. furnacalis, respectively, but the two CmedSNMPs share low identity with each other. The distribution patterns of two CmedSNMPs in different tissues of adult moths were examined using RT-PCR and quantitative real-time PCR. Although the two genes are expressed not only in antennae but also in nonolfactory tissues such as wings, legs, and body; the relative transcription level shows both CmedSNMP1 and CmedSNMP2 are highly enriched in antennae. The dN/dS ratios of the two CmedSNMPs indicate that the two genes are all subject to purifying selection and evolved to be functional genes. This work presents for the first time a study on the SNMPs of C. medinalis, which may help in providing guidance to future functional research of moth SNMPs.
Glutathione S-transferases (GSTs) have received considerable attention in insects for their roles in insecticide resistance. Laodelphax striatellus (Fallén) is a serious rice pest. L. striatellus outbreaks occur frequently throughout eastern Asia. A key problem in controlling this pest is its rapid adaptation to numerous insecticides. In this research, nine cDNAs encoding GSTs in L. striatellus were cloned and characterised.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.