The Draft Genome of Yellow Stem Borer, an Agriculturally Important Pest, Provides Molecular Insights into Its Biology, Development and Specificity Towards Rice for Infestation.

Yellow stem borer (YSB), Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae), a major monophagous insect pest of rice, causes significant yield losses. The rice-YSB interaction is very dynamic, making it difficult for management. The development of resistant lines has been unsuccessful as there are no effective resistant sources in the germplasm. Genome information is necessary for a better understanding of interaction with rice in terms of its recognition, response, and infestation mechanism. The draft genome of YSB is predicted to have 46,057 genes with an estimated size of 308 Mb, being correlated with the flow cytometry analysis. The existence of complex metabolic mechanisms and genes related to specific behavior was identified, being conditioned by a higher level of regulation. We deciphered the possible visual, olfactory, and gustatory mechanisms responsible for its evolution as a monophagous pest. Comparative genomic analysis revealed that YSB is unique in the way it has evolved. The obvious presence of high-immunity-related genes, well-developed RNAi machinery, and diverse effectors provides a means for developing genomic tools for its management. The identified 21,696 SSR markers can be utilized for diversity analysis of populations across the rice-growing regions. We present the first draft genome of YSB. The information emanated paves a way for biologists to design novel pest management strategies as well as for the industry to design new classes of safer and specific insecticide molecules.

Marker Assisted Forward Breeding to Combine Multiple Biotic-Abiotic Stress Resistance/Tolerance in Rice.

BACKGROUND: Unfavorable climatic changes have led to an increased threat of several biotic and abiotic stresses over the past few years. Looking at the massive damage caused by these stresses, we undertook a study to develop high yielding climate-resilient rice, using genes conferring resistance against blast (Pi9), bacterial leaf blight (BLB) (Xa4, xa5, xa13, Xa21), brown planthopper (BPH) (Bph3, Bph17), gall midge (GM) (Gm4, Gm8) and QTLs for drought tolerance (qDTY1.1 and qDTY3.1) through marker-assisted forward breeding (MAFB) approach. RESULT: Seven introgression lines (ILs) possessing a combination of seven to ten genes/QTLs for different biotic and abiotic stresses have been developed using marker-assisted selection (MAS) breeding method in the background of Swarna with drought QTLs. These ILs were superior to the respective recurrent parent in agronomic performance and also possess preferred grain quality with intermediate to high amylose content (AC) (23-26%). Out of these, three ILs viz., IL1 (Pi9+ Xa4+ xa5+ Xa21+ Bph17+ Gm8+ qDTY1.1+ qDTY3.1), IL6 (Pi9+ Xa4+ xa5+ Xa21+ Bph3+ Bph17+ Gm4+ Gm8+ qDTY1.1+ qDTY3.1) and IL7 (Pi9+ Xa4+ xa5+ Bph3+ Gm4+ qDTY1.1+ qDTY3.1) had shown resistance\tolerance for multiple biotic and abiotic stresses both in the field and glasshouse conditions. Overall, the ILs were high yielding under various stresses and importantly they also performed well in non-stress conditions without any yield penalty. CONCLUSION: The current study clearly illustrated the success of MAS in combining tolerance to multiple biotic and abiotic stresses while maintaining higher yield potential and preferred grain quality. Developed ILs with seven to ten genes in the current study showed superiority to recurrent parent Swarna+drought for multiple-biotic stresses (blast, BLB, BPH and GM) together with yield advantages of 1.0 t ha- 1 under drought condition, without adverse effect on grain quality traits under non-stress.

Expression Profile of Defense Genes in Rice Lines Pyramided with Resistance Genes Against Bacterial Blight, Fungal Blast and Insect Gall Midge.

BACKGROUND: Rice, a major food crop of the world, endures many major biotic stresses like bacterial blight (BB), fungal blast (BL) and the insect Asian rice gall midge (GM) that cause significant yield losses. Progress in tagging, mapping and cloning of several resistance (R) genes against aforesaid stresses has led to marker assisted multigene introgression into elite cultivars for multiple and durable resistance. However, no detailed study has been made on possible interactions among these genes when expressed simultaneously under combined stresses. RESULTS: Our studies monitored expression profiles of 14 defense related genes in 11 rice breeding lines derived from an elite cultivar with different combination of R genes against BB, BL and GM under single and multiple challenge. Four of the genes found implicated earlier under combined GM and BB stress were confirmed to be induced (≥ 2 fold) in stem tissue following GM infestation; while one of these, cytochrome P450 family protein, was also induced in leaf in plants challenged by either BB or BL but not together. Three of the genes highlighted earlier in plants challenged by both BB and BL were also found induced in stem under GM challenge. Pi54 the target R gene against BL was also found induced when challenged by GM. Though expression of some genes was noted to be inhibited under combined pest challenge, such effects did not result in compromise in resistance against any of the target pests. CONCLUSION: While R genes generally tended to respond to specific pest challenge, several of the downstream defense genes responded to multiple pest challenge either single, sequential or simultaneous, without any distinct antagonism in expression of resistance to the target pests in two of the pyramided lines RPNF05 and RPNF08.

RNA-seq of Rice Yellow Stem Borer Scirpophaga incertulas Reveals Molecular Insights During Four Larval Developmental Stages.

The yellow stem borer (YSB), Scirpophaga incertulas, is a prominent pest in rice cultivation causing serious yield losses. The larval stage is an important stage in YSB, responsible for maximum infestation. However, limited knowledge exists on the biology and mechanisms underlying the growth and differentiation of YSB. To understand and identify the genes involved in YSB development and infestation, so as to design pest control strategies, we performed de novo transcriptome analysis at the first, third, fifth, and seventh larval developmental stages employing Illumina Hi-seq. High-quality reads (HQR) of ∼229 Mb were assembled into 24,775 transcripts with an average size of 1485 bp. Genes associated with various metabolic processes, i.e., detoxification mechanism [CYP450, GSTs, and carboxylesterases (CarEs)], RNA interference (RNAi) machinery (Dcr-1, Dcr-2, Ago-1, Ago-2, Sid-1, Sid-2, Sid-3, and Sid-1-related gene), chemoreception (CSPs, GRs, OBPs, and ORs), and regulators [transcription factors (TFs) and hormones] were differentially regulated during the developmental stages. Identification of stage-specific transcripts made it possible to determine the essential processes of larval development. Comparative transcriptome analysis revealed that YSB has not evolved much with respect to the detoxification mechanism, but showed the presence of distinct RNAi machinery. The presence of strong specific visual recognition coupled with chemosensory mechanisms supports the monophagous nature of YSB. Designed expressed sequenced tags-simple-sequence repeats (EST-SSRs) will facilitate accurate estimation of the genetic diversity of YSB. This is the first report on characterization of the YSB transcriptome and the identification of genes involved in key processes, which will help researchers and industry to devise novel pest control strategies. This study also opens up a new avenue to develop next-generation resistant rice using RNAi or genome editing approaches.

Silencing of CYP6 and APN Genes Affects the Growth and Development of Rice Yellow Stem Borer, Scirpophaga incertulas.

RNAi is a powerful tool to target the insect genes involved in host-pest interactions. Key insect genes are the choice for silencing to achieve pest derived resistance where resistance genes are not available in gene pool of host plant. In this study, an attempt was made to determine the effect of dsRNA designed from two genes Cytochrome P450 derivative (CYP6) and Aminopeptidase N (APN) of rice yellow stem borer (YSB) on growth and development of insect. The bioassays involved injection of chemically synthesized 5' FAM labeled 21-nt dsRNA into rice cut stems and allowing the larvae to feed on these stems which resulted in increased mortality and observed growth and development changes in larval length and weight compared with its untreated control at 12-15 days after treatment. These results were further supported by observing the reduction in transcripts expression of these genes in treated larvae. Fluorescence detection in treated larvae also proved that dsRNA was readily taken by larvae when fed on dsRNA treated stems. These results from the present study clearly show that YSB larvae fed on dsRNA designed from Cytochrome P450 and Aminopeptidase N has detrimental effect on larval growth and development. These genes can be deployed to develop YSB resistance in rice using RNAi approach.

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.