Rongzhi Chen

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.

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.

A rice β-1,3-glucanase gene Osg1 is required for callose degradation in pollen development

Plant β-1,3-glucanases are involved in plant defense and development. In rice (Oryza sativa), 14 genes encoding putative β-1,3-glucanases have been isolated and sequenced. However, only limited information is available on the function of these β-1,3-glucanase genes. In this study, we report a detailed functional characterization of one of these genes, Osg1. Osg1 encodes a glucanase carrying no C-terminal extension. Osg1 was found to be expressed throughout the plant and highly expressed in florets, leaf sheaths, and leaf blades. Investigations using real-time PCR, immunocytochemical analysis, and a GUS-reporter gene driven by the Osg1 promoter indicated that Osg1 was mainly expressed at the late meiosis, early microspore, and middle microspore stages in the florets. To elucidate the role of Osg1, we suppressed expression of the Osg1 gene by RNA interference in transgenic rice. The silencing of Osg1 resulted in male sterility. The pollen mother cells appeared to be normal in Osg1-RI plants, but callose degradation was disrupted around the microspores in the anther locules of the Osg1-RI plants at the early microspore stage. Consequently, the release of the young microspores into the anther locules was delayed, and the microspores began to degenerate later. These results provide evidence that Osg1 is essential for timely callose degradation in the process of tetrad dissolution.

Responses of Two Contrasting Genotypes of Rice to Brown Planthopper

Rice (Oryza sativa L.) and brown planthoppers (BPH) (Nilaparvata lugens Stål) provide an ideal system for studying molecular mechanisms involved in the interactions between plants and phloem-feeding insects. The phenotypic responses and changes in transcript profiles of seedlings representing two rice cultivars differing in resistance to the BPH were analyzed. In the BPH-compatible (susceptible) cv. MH63, BPH feeding reduced three examined plant growth parameters (leaf area expansion, height increases, and dry weight increases) and photosynthetic rates of the leaves. In the BPH-incompatible (resistant) cv. B5, BPH feeding caused slight reductions in protein and sucrose contents, but the plants maintained their photosynthetic activity and grew normally. A cDNA microarray containing 1,920 suppression subtractive hybridization clones was used to explore the transcript profiles differences in the two cultivars under control and BPH-feeding conditions. In total, 160 unique genes were detected as being significantly affected by BPH feeding in rice plants, covering a wide range of functional categories, and there were 38 genes that showed the similar transcript pattern in both genotypes. The physiological responses and transcript profiles of plants represented in both genotypes suggested that multiple pathways might be involved in reprogramming of BPH-infested rice plants. The differences in transcript levels between the compatible and incompatible interactions revealed in this study were not only the reaction of resistance and susceptibility but also reflections of different damage rates and genotypic backgrounds of the rice cultivars.

Allelic diversity in an NLR gene BPH9 enables rice to combat planthopper variation

Significance Insect pests represent a major constraint that reduces crop yield and quality globally. Host plant resistance is often used as a key tactic to control insect pests, but is frequently overcome by newly emerged insect populations. In nature, plants have developed various strategies for sustainable defense. In this work, we isolated a brown planthopper-resistance gene, BPH9, and show that alleles of this gene locus have been widely used in rice breeding and saved rice production from massive brown planthopper (BPH) damage. Allelic diversity in this gene locus has provided resistance to rice against different BPH populations. Manipulating allelic diversity of the gene may provide a strategy for developing resistant varieties to cope with evolving insect populations with new virulence variation. Brown planthopper (BPH), Nilaparvata lugens Stål, is one of the most devastating insect pests of rice (Oryza sativa L.). Currently, 30 BPH-resistance genes have been genetically defined, most of which are clustered on specific chromosome regions. Here, we describe molecular cloning and characterization of a BPH-resistance gene, BPH9, mapped on the long arm of rice chromosome 12 (12L). BPH9 encodes a rare type of nucleotide-binding and leucine-rich repeat (NLR)-containing protein that localizes to the endomembrane system and causes a cell death phenotype. BPH9 activates salicylic acid- and jasmonic acid-signaling pathways in rice plants and confers both antixenosis and antibiosis to BPH. We further demonstrated that the eight BPH-resistance genes that are clustered on chromosome 12L, including the widely used BPH1, are allelic with each other. To honor the priority in the literature, we thus designated this locus as BPH1/9. These eight genes can be classified into four allelotypes, BPH1/9-1, -2, -7, and -9. These allelotypes confer varying levels of resistance to different biotypes of BPH. The coding region of BPH1/9 shows a high level of diversity in rice germplasm. Homologous fragments of the nucleotide-binding (NB) and leucine-rich repeat (LRR) domains exist, which might have served as a repository for generating allele diversity. Our findings reveal a rice plant strategy for modifying the genetic information to gain the upper hand in the struggle against insect herbivores. Further exploration of natural allelic variation and artificial shuffling within this gene may allow breeding to be tailored to control emerging biotypes of BPH.

A rice lectin receptor-like kinase that is involved in innate immune responses also contributes to seed germination

Seed germination and innate immunity both have significant effects on plant life spans because they control the plants entry into the ecosystem and provide defenses against various external stresses, respectively. Much ecological evidence has shown that seeds with high vigor are generally more tolerant of various environmental stimuli in the field than those with low vigor. However, there is little genetic evidence linking germination and immunity in plants. Here, we show that the rice lectin receptor-like kinase OslecRK contributes to both seed germination and plant innate immunity. We demonstrate that knocking down the OslecRK gene depresses the expression of α–amylase genes, reducing seed viability and thereby decreasing the rate of seed germination. Moreover, it also inhibits the expression of defense genes, and so reduces the resistance of rice plants to fungal and bacterial pathogens as well as herbivorous insects. Yeast two-hybrid and co-immunoprecipitation experiments revealed that OslecRK interacts with an actin-depolymerizing factor (ADF) in vivo via its kinase domain. Moreover, the rice adf mutant exhibited a reduced seed germination rate due to the suppression of α–amylase gene expression. This mutant also exhibited depressed immune responses and reduced resistance to biotic stresses. Our results thus provide direct genetic evidence for a common physiological pathway connecting germination and immunity in plants. They also partially explain the common observation that high-vigor seeds often perform well in the field. The dual effects of OslecRK may be indicative of progressive adaptive evolution in rice.

Knockdown of GDCH gene reveals reactive oxygen species-induced leaf senescence in rice

Glycine decarboxylase complex (GDC) is a multi-protein complex, comprising P-, H-, T- and L-protein subunits, which plays a major role in photorespiration in plants. While structural analysis has demonstrated that the H subunit of GDC (GDCH) plays a pivotal role in GDC, research on the role of GDCH in biological processes in plants is seldom reported. Here, the function of GDCH, stresses resulting from GDCH-knockdown and the interactions of these stresses with other cellular processes were studied in rice plants. Under high CO(2), the OsGDCH RNA interference (OsGDCH-RNAi) plants grew normally, but under ambient CO(2), severely suppressed OsGDCH-RNAi plants (SSPs) were non-viable, which displayed a photorespiration-deficient phenotype. Under ambient CO(2), chlorophyll loss, protein degradation, lipid peroxidation and photosynthesis decline occurred in SSPs. Electron microscopy studies showed that chloroplast breakdown and autophagy took place in these plants. Reactive oxygen species (ROS), including O2(-) and H(2)O(2), accumulated and the antioxidant enzyme activities decreased in the leaves of SSPs under ambient CO(2). The expression of transcription factors and senescence-associated genes (SAGs), which was up-regulated in SSPs after transfer to ambient CO(2), was enhanced in wild-type plants treated with H(2)O(2). Evidences demonstrate ROS induce senescence in SSPs, and transcription factors OsWRKY72 may mediate the ROS-induced senescence.

Phloem-exudate proteome analysis of response to insect brown plant-hopper in rice

Brown plant-hopper (Nilaparvata lugens Stål, BPH), one of the most devastating agricultural insect pests of rice throughout Asia, ingests nutrients from rice sieve tubes and causes a dramatic yield loss. Planting resistant variety is an efficient and economical way to control this pest. Understanding the mechanisms of host resistance is extremely valuable for molecular design of resistant rice variety. Here, we used an iTRAQ-based quantitative proteomics approach to perform analysis of protein expression profiles in the phloem exudates of BPH-resistant and susceptible rice plants following BPH infestation. A total of 238 proteins were identified, most of which were previously described to be present in the phloem of rice and other plants. The expression of genes for selected proteins was confirmed using a laser capture micro-dissection method and RT-PCR. The mRNAs for three proteins, RGAP, TCTP, and TRXH, were further analyzed by using in situ mRNA hybridization and localized in the phloem cells. Our results showed that BPH feeding induced significant changes in the abundance of proteins in phloem sap of rice involved in multiple pathways, including defense signal transduction, redox regulation, and carbohydrate and protein metabolism, as well as cell structural proteins. The results presented provide new insights into rice resistance mechanisms and should facilitate the breeding of novel elite BPH-resistant rice varieties.