Expression Analysis Reveals Differentially Expressed Genes in BPH and WBPH Associated with Resistance in Rice RILs Derived from a Cross between RP2068 and TN1.

BPH (brown planthopper) and WBPH (white backed planthopper) are significant rice pests that often co-occur as sympatric species and cause substantial yield loss. Despite their genetic similarities, different host-resistance genes confer resistance against these two hoppers. The defense mechanisms in rice against these pests are complex, and the molecular processes regulating their responses remain largely unknown. This study used specific recombinant inbred lines (RILs) derived from a cross between rice varieties RP2068-18-3-5 (BPH- and WBPH-resistant) and TN1 (BPH- and WBPH-susceptible) to investigate the mechanisms of interaction between these planthoppers and their rice hosts. WBPH and BPH were allowed to feed on specific RILs, and RNA-Seq was carried out on WBPH insects. Transcriptome profiling and qRT-PCR results revealed differential expression of genes involved in detoxification, digestion, transportation, cuticle formation, splicing, and RNA processing. A higher expression of sugar transporters was observed in both hoppers feeding on rice with resistance against either hopper. This is the first comparative analysis of gene expressions in these insects fed on genetically similar hosts but with differential resistance to BPH and WBPH. These results complement our earlier findings on the differential gene expression of the same RILs (BPH- or WBPH-infested) utilized in this study. Moreover, identifying insect genes and pathways responsible for countering host defense would augment our understanding of BPH and WBPH interaction with their rice hosts and enable us to develop lasting strategies to control these significant pests.

Infestation of Rice by Gall Midge Influences Density and Diversity of Pseudomonas and Wolbachia in the Host Plant Microbiome.

Background: The virulence of phytophagous insects is predominantly determined by their ability to evade or suppress host defense for their survival. The rice gall midge (GM, Orseolia oryzae), a monophagous pest of rice, elicits a host defense similar to the one elicited upon pathogen attack. This could be due to the GM feeding behaviour, wherein the GM endosymbionts are transferred to the host plant via oral secretions, and as a result, the host mounts an appropriate defense response(s) (i.e., up-regulation of the salicylic acid pathway) against these endosymbionts. Methods: The current study aimed to analyze the microbiome present at the feeding site of GM maggots to determine the exchange of bacterial species between GM and its host and to elucidate their role in rice-GM interaction using a next-generation sequencing approach. Results: Our results revealed differential representation of the phylum Proteobacteria in the GM-infested and -uninfested rice tissues. Furthermore, analysis of the species diversity of Pseudomonas and Wolbachia supergroups at the feeding sites indicated the exchange of bacterial species between GM and its host upon infestation. Conclusion: As rice-GM microbial associations remain relatively unstudied, these findings not only add to our current understanding of microbe-assisted insect-plant interactions but also provide valuable insights into how these bacteria drive insect-plant coevolution. Moreover, to the best of our knowledge, this is the first report analyzing the microbiome of a host plant (rice) at the feeding site of its insect pest (GM).

Geographic and Research Center Origins of Rice Resistance to Asian Planthoppers and Leafhoppers: Implications for Rice Breeding and Gene Deployment.

This study examines aspects of virulence to resistant rice varieties among planthoppers and leafhoppers. Using a series of resistant varieties, brown planthopper, Nilaparvata lugens, virulence was assessed in seedlings and early-tillering plants at seven research centers in South and East Asia. Virulence of the whitebacked planthopper, Sogatella furcifera, in Taiwan and the Philippines was also assessed. Phylogenetic analysis of the varieties using single-nucleotide polymorphisms (SNPs) indicated a clade of highly resistant varieties from South Asia with two further South Asian clades of moderate resistance. Greenhouse bioassays indicated that planthoppers can develop virulence against multiple resistance genes including genes introgressed from wild rice species. Nilaparvata lugens populations from Punjab (India) and the Mekong Delta (Vietnam) were highly virulent to a range of key resistance donors irrespective of variety origin. Sogatella furcifera populations were less virulent to donors than N. lugens; however, several genes for resistance to S. furcifera are now ineffective in East Asia. A clade of International Rice Research Institute (IRRI)-bred varieties and breeding lines, without identified leafhopper-resistance genes, were highly resistant to the green leafhopper, Nephotettix virescens. Routine phenotyping during breeding programs likely maintains high levels of quantitative resistance to leafhoppers. We discuss these results in the light of breeding and deploying resistant rice in Asia.

Metabolic and transcriptomic changes induced in host during hypersensitive response mediated resistance in rice against the Asian rice gall midge.

BACKGROUND: An incompatible interaction between rice (Oryza sativa) and the Asian rice gall midge (AGM, Orseolia oryzae Wood-Mason), that is usually manifested through a hypersensitive response (HR), represents an intricate relationship between the resistant host and its avirulent pest. We investigated changes in the transcriptome and metabolome of the host (indica rice variety: RP2068-18-3-5, RP), showing HR when attacked by an avirulent gall midge biotype (GMB1), to deduce molecular and biochemical bases of such a complex interaction. Till now, such an integrated analysis of host transcriptome and metabolome has not been reported for any rice-insect interaction. RESULTS: Transcript and metabolic profiling data revealed more than 7000 differentially expressed genes and 80 differentially accumulated metabolites, respectively, in the resistant host. Microarray data revealed deregulation of carbon (C) and nitrogen (N) metabolism causing a C/N shift; up-regulation of tetrapyrrole synthesis and down-regulation of chlorophyll synthesis and photosynthesis. Integrated results revealed that genes involved in lipid peroxidation (LPO) were up-regulated and a marker metabolite for LPO (azelaic acid) accumulated during HR. This coincided with a greater accumulation of GABA (neurotransmitter and an insect antifeedant) at the feeding site. Validation of microarray results by semi-quantitative RT-PCR revealed temporal variation in gene expression profiles. CONCLUSIONS: The study revealed extensive reprogramming of the transcriptome and metabolome of RP upon GMB1 infestation leading to an HR that was induced by the generation and release of reactive oxygen species i.e. singlet oxygen and resulted in LPO-mediated cell death. RP thus used HR as a means to limit nutrient supply to the feeding maggots and simultaneously accumulated GABA, strategies that could have led to maggot mortality. The integrated results of transcript and metabolic profiling, for the first time, provided insights into an HR+ type of resistance in rice against gall midge.

Rice-gall midge interactions: Battle for survival.

Gall midges are insects specialized in maneuvering plant growth, metabolic and defense pathways for their benefit. The Asian rice gall midge and rice share such an intimate relationship that there is a constant battle for survival by either partner. Diverse responses by the rice host against the midge include necrotic hypersensitive resistance reaction, non-hypersensitive resistance reaction and gall-forming compatible interaction. Genetic studies have revealed that major R (resistance) genes confer resistance to gall midge in rice. Eleven gall midge R genes have been characterized so far in different rice varieties in India. In addition, no single R gene confers resistance against all the seven biotypes of the Asian rice gall midge, and none of the biotypes is virulent against all the resistance genes. Further, the interaction of the plant resistance gene with the insect avirulence gene is on a gene-for-gene basis. Our recent investigations involving suppressive subtraction hybridization cDNA libraries, microarray analyses, gene expression assays and metabolic profiling have revealed several molecular mechanisms, metabolite markers and pathways that are induced, down-regulated or altered in the rice host during incompatible or compatible interactions with the pest. This is also true for some of the pathways studied in the gall midge. Next generation sequencing technology, gene expression studies and conventional screening of gall midge cDNA libraries highlighted molecular approaches adopted by the insect to feed, survive and reproduce. This constant struggle by the midge to overcome the host defenses and the host to resist the pest has provided us with an opportunity to observe this battle for survival at the molecular level.

A putative candidate for the recessive gall midge resistance gene gm3 in rice identified and validated.

KEY MESSAGE: We report here tagging and fine-mapping of gm3 gene, development of a functional marker for it and its use in marker-assisted selection. The recessive rice gall midge resistance gene, gm3 identified in the rice breeding line RP2068-18-3-5 confers resistance against five of the seven Indian biotypes of the Asian rice gall midge Orseolia oryzae. We report here tagging and fine-mapping of gm3 gene, development of a functional marker for it and demonstrated its use in marker-assisted selection (MAS). A mapping population consisting of 302 F10 recombinant inbred lines derived from the cross TN1 (susceptible)/RP2068-18-3-5, was screened against gall midge biotype 4 (GMB4) and analyzed with a set of 89 polymorphic SSR markers distributed uniformly across the rice genome. Two SSR markers, RM17480 and gm3SSR4, located on chromosome 4L displayed high degree of co-segregation with the trait phenotype and flanked the gene. In silico analysis of the genomic region spanning these two markers contained 62 putatively expressed genes, including a gene encoding an NB-ARC (NBS-LRR) domain containing protein. A fragment of this gene was amplified with the designed marker, NBcloning 0.9 Kb from the two susceptible TN1, Improved Samba Mahsuri (B95-1) and two resistant cultivars, RP 2068-18-3-5 and Phalguna (with Gm2 gene). The amplicons were observed to be polymorphic between the susceptible and resistant genotypes and hence were cloned and sequenced. A new primer, gm3del3, which was designed based on sequence polymorphism, amplified fragments with distinct size polymorphism among RP2068-18-3-5, Phalguna and TN1 and B95-1 and displayed no recombination in the entire mapping population. Expression of the candidate NB-ARC gene in the susceptible TN1 and the resistant RP2068-18-3-5 plants following infestation with GMB4 was analyzed, through real-time reverse transcription PCR. Results showed twofold enhanced expression in RP2068-18-3-5 plants, but not in TN1 plants, 120 h after infestation. Amino acid sequence and structure analysis of the proteins coded by different alleles of gm3 gene showed deletion of eight amino acids due to an early stop codon in RP2068-18-3-5 resulting in a change in the functional domain of the protein. The gm3del3 was used as a functional marker for introgression of gm3 gene into the genetic background of the elite bacterial blight resistant cultivar Improved Samba Mahsuri (B95-1) through MAS.

Suppressive subtraction hybridization reveals that rice gall midge attack elicits plant-pathogen-like responses in rice.

The Asian rice gall midge, Orseolia oryzae (Diptera: Cecidomyiidae), is the third most destructive insect pest of rice (Oryza sativa L.). Till date, 11 gall midge resistance gene loci have been characterized in different rice varieties. To elucidate molecular basis of incompatible (hypersensitive response plus [HR+] type) and compatible rice-gall midge interactions, two suppressive subtraction hybridization cDNA libraries were constructed. These were enriched for differentially expressed transcripts after gall midge infestation in two rice varieties (resistant Suraksha and susceptible TN1). In total, 2784 ESTs were generated and sequenced from the two libraries, of which 1536 were from the resistant Suraksha and 1248 were from the susceptible TN1. Majority (80%) of the ESTs was non-redundant sequences with known functions and was classified into three principal gene ontology (GO) categories and 12 groups. Upregulation of NBS-LRR, Cytochrome P450, heat shock proteins, phenylalanine ammonia lyase and OsPR10α genes from the Suraksha library, as revealed by real-time PCR, indicated that R gene mediated, salicylic acid related defense pathway is likely to be involved in gall midge resistance. Present study suggested that resistance in Suraksha against gall midge is similar in nature to the resistance observed in plants against pathogens. However, in TN1, genes related to primary metabolism and redox were induced abundantly. Results suggested that genes encoding translationally controlled tumor protein and NAC domain proteins are likely to be involved in the gall midge susceptibility.

Pyrosequencing-based transcriptome analysis of the asian rice gall midge reveals differential response during compatible and incompatible interaction.

The Asian rice gall midge (Orseolia oryzae) is a major pest responsible for immense loss in rice productivity. Currently, very little knowledge exists with regard to this insect at the molecular level. The present study was initiated with the aim of developing molecular resources as well as identifying alterations at the transcriptome level in the gall midge maggots that are in a compatible (SH) or in an incompatible interaction (RH) with their rice host. Roche 454 pyrosequencing strategy was used to develop both transcriptomics and genomics resources that led to the identification of 79,028 and 85,395 EST sequences from gall midge biotype 4 (GMB4) maggots feeding on a susceptible and resistant rice variety, TN1 (SH) and Suraksha (RH), respectively. Comparative transcriptome analysis of the maggots in SH and RH revealed over-representation of transcripts from proteolysis and protein phosphorylation in maggots from RH. In contrast, over-representation of transcripts for translation, regulation of transcription and transcripts involved in electron transport chain were observed in maggots from SH. This investigation, besides unveiling various mechanisms underlying insect-plant interactions, will also lead to a better understanding of strategies adopted by insects in general, and the Asian rice gall midge in particular, to overcome host defense.

A novel mechanism of gall midge resistance in the rice variety Kavya revealed by microarray analysis.

The Asian rice gall midge [Orseolia oryzae (Wood-Mason)] is an important rice pest causing an annual average yield loss of about US $80 million in India. Rice varieties possess several discrete resistance (R) genes conferring resistance against the pest in two distinct ways, i.e., with (HR+ type) or without (HR- type) the expression of hypersensitive reaction (HR). The aim of the present work is to understand the molecular basis of compatible and incompatible (HR- type) rice gall midge interactions between the rice variety Kavya and the two gall midge biotypes: the virulent GMB4M and the avirulent GMB1 using transcriptional microarray gene expression analysis. A large number of differentially expressed genes (602genes in incompatible interaction and 1,330 genes in compatible interaction with at least twofold changes, p value <0.05) was obtained from the microarray analysis that could be grouped into six clusters based on their induction during both or either of the interactions. MapMan software was used for functional characterization of these genes into 13 categories (BINs). Real-time polymerase chain reaction validation of 26 genes selected through the analysis revealed four genes viz. NADPH oxidase, AtrbohF, cinnamoyl-CoA reductase, and von Willebrand factor type A domain containing protein coding genes to be significantly upregulated during the incompatible interaction. But most of the signature genes related to HR+ type resistance like salicylic acid pathway-related genes and disease resistance protein coding genes were downregulated. On the other hand, during the compatible interaction, genes related to primary metabolism and nutrient transport were upregulated and genes for defense and signaling were downregulated. We propose a hypothesis that HR- type of resistance in the rice variety Kavya against gall midge could be due to the constitutive expression of an R gene and a case of extreme resistance which is devoid of cell death. Compatible interaction, however, modulated a large number of differentially expressed transcripts to reprogram cell organization, cell remodeling, and relocation of nutrients through transport to support insect growth.

Differential gene expression in gall midge susceptible rice genotypes revealed by suppressive subtraction hybridization (SSH) cDNA libraries and microarray analysis.

BACKGROUND: A major pest of rice, the Asian rice gall midge (Orseolia oryzae Wood-Mason), causes significant yield losses in the rice growing regions throughout Asia. Feeding by the larvae induces susceptible plants to produce nutritive tissue to support growth and development. In order to identify molecular signatures during compatible interactions, genome wide transcriptional profiling was performed using SSH library and microarray technology. RESULTS: Results revealed up-regulation of genes related to primary metabolism, nutrient relocation, cell organization and DNA synthesis. Concomitantly, defense, secondary metabolism and signaling genes were suppressed. Further, real-time PCR validation of a selected set of 20 genes, in three susceptible rice varieties (TN1, Kavya and Suraksha) during the interaction with the respective virulent gall midge biotypes, also revealed variation in gene expression in Kavya as compared to TN1 and Suraksha. CONCLUSIONS: These studies showed that virulent insects induced the plants to step up metabolism and transport nutrients to their feeding site and suppressed defense responses. But Kavya rice mounted an elevated defense response during early hours of virulent gall midge infestation, which was over-powered later, resulting in host plant susceptibility.

Serine proteases-like genes in the asian rice gall midge show differential expression in compatible and incompatible interactions with rice.

The Asian rice gall midge, Orseolia oryzae (Wood-Mason), is a serious pest of rice. Investigations into the gall midge-rice interaction will unveil the underlying molecular mechanisms which, in turn, can be used as a tool to assist in developing suitable integrated pest management strategies. The insect gut is known to be involved in various physiological and biological processes including digestion, detoxification and interaction with the host. We have cloned and identified two genes, OoprotI and OoprotII, homologous to serine proteases with the conserved His(87), Asp(136) and Ser(241) residues. OoProtI shared 52.26% identity with mosquito-type trypsin from Hessian fly whereas OoProtII showed 52.49% identity to complement component activated C1s from the Hessian fly. Quantitative real time PCR analysis revealed that both the genes were significantly upregulated in larvae feeding on resistant cultivar than in those feeding on susceptible cultivar. These results provide an opportunity to understand the gut physiology of the insect under compatible or incompatible interactions with the host. Phylogenetic analysis grouped these genes in the clade containing proteases of phytophagous insects away from hematophagous insects.

Isolation and characterization of microsatellite loci in the Asian rice gall midge (Orseolia oryzae) (Diptera: Cecidomyiidae).

Microsatellite loci were isolated from the genomic DNA of the Asian rice gall midge, Orseolia oryzae (Wood-Mason) using a hybridization capture approach. A total of 90 non-redundant primer pairs, representing unique loci, were designed. These simple sequence repeat (SSR) markers represented di (72%), tri (15.3%), and complex repeats (12.7%). Three biotypes of gall midge (20 individuals for each biotype) were screened using these SSRs. The results revealed that 15 loci were hyper variable and showed polymorphism among different biotypes of this pest. The number of alleles ranged from two to 11 and expected heterozygosity was above 0.5. Inheritance studies with three markers (observed to be polymorphic between sexes) revealed sex linked inheritance of two SSRs (Oosat55 and Oosat59) and autosomal inheritance of one marker (Oosat43). These markers will prove to be a useful tool to devise strategies for integrated pest management and in the study of biotype evolution in this important rice pest.