Guangcun He

Identification and characterization of Bph14, a gene conferring resistance to brown planthopper in rice

Planthoppers are highly destructive pests in crop production worldwide. Brown planthopper (BPH) causes the most serious damage of the rice crop globally among all rice pests. Growing resistant varieties is the most effective and environment-friendly strategy for protecting the crop from BPH. More than 19 BPH-resistance genes have been reported and used to various extents in rice breeding and production. In this study, we cloned Bph14, a gene conferring resistance to BPH at seedling and maturity stages of the rice plant, using a map-base cloning approach. We show that Bph14 encodes a coiled-coil, nucleotide-binding, and leucine-rich repeat (CC-NB-LRR) protein. Sequence comparison indicates that Bph14 carries a unique LRR domain that might function in recognition of the BPH insect invasion and activating the defense response. Bph14 is predominantly expressed in vascular bundles, the site of BPH feeding. Expression of Bph14 activates the salicylic acid signaling pathway and induces callose deposition in phloem cells and trypsin inhibitor production after planthopper infestation, thus reducing the feeding, growth rate, and longevity of the BPH insects. Our work provides insights into the molecular mechanisms of rice defense against insects and facilitates the development of resistant varieties to control this devastating insect.

Knockdown of midgut genes by dsRNA-transgenic plant-mediated RNA interference in the Hemipteran insect Nilaparvata lugens

Background RNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders. Methodology/Principal Findings The Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed. Conclusions Our study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants.

Identification and mapping of two brown planthopper resistance genes in rice

Abstract The brown planthopper (BPH) is one of the most serious insect pests of rice. In this study, we conducted a molecular marker-based genetic analysis of the BPH resistance of ’B5’, a highly resistant line that derived its resistant genes from the wild rice Oryza officinalis. Insect resistance was evaluated using 250 F3 families from a cross between ’B5’ and ’Minghui 63’, based on which the resistance of each F2 plant was inferred. Two bulks were made by mixing, respectively, DNA samples from highly resistant plants and highly susceptible plants selected from the F2 population. The bulks were surveyed for restriction fragment length polymorphism using probes representing all 12 chromosomes at regular intervals. The survey revealed two genomic regions on chromosome 3 and chromosome 4 respectively that contained genes for BPH resistance. The existence of the two loci were further assessed by QTL (quantitative trait locus) analysis, which resolved these two loci to a 14.3-cM interval on chromosome 3 and a 0.4-cM interval on chromosome 4. Comparison of the chromosomal locations and reactions to BPH biotypes indicated that these two genes are different from at least nine of the ten previously identified BPH resistance genes. Both of the genes had large effects on BPH resistance and the two loci acted essentially independent of each other in determining t he resistance. These two genes may be a useful BPH resistance resource for rice breeding programs.

Rice UDP-Glucose Pyrophosphorylase1 Is Essential for Pollen Callose Deposition and Its Cosuppression Results in a New Type of Thermosensitive Genic Male Sterility

UDP-glucose pyrophosphorylase (UGPase) catalyzes the reversible production of glucose-1-phosphate and UTP to UDP-glucose and pyrophosphate. The rice (Oryza sativa) genome contains two homologous UGPase genes, Ugp1 and Ugp2. We report a functional characterization of rice Ugp1, which is expressed throughout the plant, with highest expression in florets, especially in pollen during anther development. Ugp1 silencing by RNA interference or cosuppression results in male sterility. Expressing a double-stranded RNA interference construct in Ugp1-RI plants resulted in complete suppression of both Ugp1 and Ugp2, together with various pleiotropic developmental abnormalities, suggesting that UGPase plays critical roles in plant growth and development. More importantly, Ugp1-cosuppressing plants contained unprocessed intron-containing primary transcripts derived from transcription of the overexpression construct. These aberrant transcripts undergo temperature-sensitive splicing in florets, leading to a novel thermosensitive genic male sterility. Pollen mother cells (PMCs) of Ugp1-silenced plants appeared normal before meiosis, but during meiosis, normal callose deposition was disrupted. Consequently, the PMCs began to degenerate at the early meiosis stage, eventually resulting in complete pollen collapse. In addition, the degeneration of the tapetum and middle layer was inhibited. These results demonstrate that rice Ugp1 is required for callose deposition during PMC meiosis and bridges the apoplastic unloading pathway and pollen development.

Understanding rice plant resistance to the Brown Planthopper (Nilaparvata lugens): A proteomic approach

Engineering and breeding resistant plant varieties are the most effective and environmentally friendly ways to control agricultural pests and improve crop performance. However, the mechanism of plant resistance to pests is poorly understood. Here we used a quantitative mass‐spectrometry‐based proteomic approach for comparative analysis of expression profiles of proteins in rice leaf sheaths in responses to infestation by the brown planthopper (Nilaparvata lugens Stål, BPH), which is a serious rice crop pest. Proteins involved in multiple pathways showed significant changes in expression in response to BPH feeding, including jasmonic acid synthesis proteins, oxidative stress response proteins, beta‐glucanases, protein; kinases, clathrin protein, glycine cleavage system protein, photosynthesis proteins and aquaporins. The corresponding genes of eight important proteins were further analyzed by quantitative RT‐PCR. Proteomic and transcript responses that were related to wounding, oxidative and pathogen stress overlapped considerably between BPH‐resistant (carrying the resistance gene BPH15) and susceptible rice lines. In contrast, proteins and genes related to callose metabolism remained unchanged and glycine cleavage system protein was up‐regulated in the BPH‐resistant lines, indicating that they have an efficient and specific defense mechanism. Our results provide new information about the interaction between rice and the BPH.

Towards Understanding of Molecular Interactions between Rice and the Brown Planthopper

The brown planthopper (BPH) is the most notorious pest of rice (Oryza sativa). Studies of rice-BPH interaction have contributed to development of new rice varieties, offering an effective means for long-lasting control of BPH. Here, we review the status of knowledge of the molecular basis of rice-BPH interaction, from the perspective of immunity. The BPH has complicated feeding behaviors on rice, which are mainly related to host resistance. Now, 24 resistance genes have been detected in rice, indicating gene-for-gene relationships with biotypes of the BPH. However, only one BPH resistance gene (Bph14) was identified and characterized using map-based cloning. Bph14 encodes an immune receptor of NB-LRR family, providing a means for studying the molecular mechanisms of rice resistance to BPH. Plant hormones (e.g. salicylic acid and jasmonate/ethylene), Ca(2+), mitogen-activated protein kinases (MAPKs), and OsRac1 play important roles in the immune response of rice to BPH. Signal transduction leads to modifying expression of defense-related genes and defense mechanisms against BPH, including sieve tube sealing, production of secondary metabolites, and induction of proteinase inhibitor. A model for the molecular interactions between rice and the BPH is proposed, although many details remain to be investigated that are valuable for molecular design of BPH-resistant rice varieties.

Differential gene expression in response to brown planthopper feeding in rice

Plant responses to herbivores are complex. 108 cDNA clones representing genes relating to plant responses to chewing insect-feeding, pathogen infection, wounding and other stresses were collected. Northern blot and cDNA array analysis were employed to investigate gene expression regulated by piercing-sucking insect, brown planthopper (BPH), Nilaparvata lugens (Homoptera: Dephacidae) on both the resistant and susceptible rice genotypes. After BPH feeding in rice for 72 h, the expression of most tested genes was affected. 14 genes in resistant rice variety B5 and 44 genes in susceptible MH63 were significantly up- or down-regulated. Most of the well-regulated genes were grouped in the categories of signaling pathways, oxidative stress/apoptosis, wound-response, drought-inducible and pathogen-related proteins. Those related to the flavonoid pathway, aromatic metabolidsm and the octadecanoid pathway were mostly kept unchanged or down-regulated. Our results indicate that BPH feeding induces plant responses which would take part in a jasmonic acid-independent pathway and crosstalk with those related to abiotic stress, pathogen invasion and phytohormone signaling pathways.

Identification of genes responsive to brown planthopper Nilaparvata lugens Stål (Homoptera: Delphacidae) feeding in rice

The brown planthopper (Nilaparvata lugens Stål) is a rice (Oryza sativa L.)-specific herbivore that ingests assimilates from the phloem, which leads to stunted growth and therefore poor yield of rice. In this study, 27 planthopper-responsive genes in rice plants were isolated by suppression subtractive hybridization (SSH), and confirmed by filter cDNA array hybridization and northern blot analysis. Among these transcripts, 25 were induced and 2 were suppressed by N. lugens feeding. The responsive genes are involved in photosynthesis, macromolecule degradation, signal transduction and defenses against stress and pathogens. Overall, genes involved in macromolecule degradation and plant defenses were up-regulated, whereas those involved in photosynthesis and cell growth were down-regulated. Our data suggest that leaf senescence is most likely activated in the susceptible rice plants damaged by N. lugens insects.

Bacterial Symbionts of the Brown Planthopper, Nilaparvata lugens (Homoptera: Delphacidae)

ABSTRACT The brown planthopper (Nilaparvata lugens Stål), the most destructive pest of rice, has been identified, including biotypes with high virulence towards previously resistant rice varieties. There have also been many reports of a yeast-like symbiont of N. lugens, but little is known about the bacterial microbes. In this study, we examined the bacterial microbes in N. lugens and identified a total of 18 operational taxonomic units (OTUs) representing four phyla (Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes) by sequencing and analyzing 16S rRNA gene libraries obtained from three populations of N. lugens, which were maintained on the rice varieties TN1, Mudgo, and ASD7. Several of the OTUs were similar to previously reported secondary symbionts of other insects, including an endosymbiont of the psyllid Glycapsis brimblecombei, an Asaia sp. found in the mosquito Anopheles stephensi, and Wolbachia, found in the mite Metaseiulus occidentalis. However, the species and numbers of the detected OTUs differed substantially among the N. lugens populations. Further, in situ hybridization analysis using digoxigenin-labeled probes indicated that OTU 1 was located in hypogastrium tissues near the ovipositor and ovary in biotype 1 insects, while OTU 2 was located in the front of the ovipositor sheath in biotype 2 insects. In addition, masses of bacterium-like organisms were observed in the tubes of salivary sheaths in rice plant tissues that the insects had fed upon. The results provide indications of the diversity of the bacterial microbes harbored by the brown planthopper and of possible associations between specific bacterial microbes and biotypes of N. lugens.

Identification of Quantitative Trait Loci Across Recombinant Inbred Lines and Testcross Populations for Traits of Agronomic Importance in Rice

This study was conducted to determine whether quantitative trait loci (QTL) controlling traits of agronomic importance detected in recombinant inbred lines (RILs) are also expressed in testcross (TC) hybrids of rice. A genetic map was constructed using an RIL population derived from a cross between B5 and Minghui 63, a parent of the most widely grown hybrid rice cultivar in China. Four TC hybrid populations were produced by crossing the RILs with three maintaining lines for the widely used cytoplasmic male-sterile (CMS) lines and the genic male-sterile line Peiai64s. The mean values of the RILs for the seven traits investigated were significantly correlated to those of the F1 hybrids in the four TC populations. Twenty-seven main-effect QTL were identified in the RILs. Of these, the QTL that had the strongest effect on each of the seven traits in the RILs was detected in two or more of the TC populations, and six other QTL were detected in one TC population. Epistatic analysis revealed that the effect of epistatic QTL was relatively weak and cross combination specific. Searching publicly available QTL data in rice revealed the positional convergence of the QTL with the strongest effect in a wide range of populations and under different environments. Since the main-effect QTL is expressed across different testers, and in different genetic backgrounds and environments, it is a valuable target for gene manipulation and for further application in rice breeding. When a restorer line that expresses main-effect QTL is bred, it could be used in a number of cross combinations.