The complete mitochondrial genome of the Asian rice gall midge, Orseolia oryzae (Diptera; Cecidomyiidae) was sequenced, annotated and analysed in the present study. The circular genome is 15,286 bp with 13 protein-coding genes, 22 tRNAs and 2 ribosomal RNA genes, and a 578 bp non-coding control region. All protein coding genes used conventional start codons and terminated with a complete stop codon. The genome presented many unusual features: (1) rearrangement in the order of tRNAs as well as protein coding genes; (2) truncation and unusual secondary structures of tRNAs; (3) presence of two different repeat elements in separate non-coding regions; (4) presence of one pseudo-tRNA gene; (5) inversion of the rRNA genes; (6) higher percentage of non-coding regions when compared with other insect mitogenomes. Rearrangements of the tRNAs and protein coding genes are explained on the basis of tandem duplication and random loss model and why intramitochondrial recombination is a better model for explaining rearrangements in the O. oryzae mitochondrial genome is discussed. Furthermore, we evaluated the number of iterations of the tandem repeat elements found in the mitogenome. This led to the identification of genetic markers capable of differentiating rice gall midge biotypes and the two Orseolia species investigated.
The Asian rice gall midge, Orseolia oryzae, is a major dipteran pest of rice, with many known biotypes. The present investigation was initiated to understand the molecular mechanisms of infestation for developing novel integrated pest management strategies. We isolated and characterized a gene, nucleoside diphosphate kinase (OoNDPK), from the rice gall midge, encoding a protein with 169 amino acid residues and with a secretory signal sequence - an observation that assumes significance as salivary gland secretions have been implicated to play a major role in insect-plant interactions. Furthermore, up-regulation (> 18 folds) of OoNDPK was observed in the salivary glands of maggots feeding on susceptible host in contrast to those feeding on resistant host. Phylogenetic analysis revealed similarity of OoNDPK with its dipteran orthologues. 3DLigandSite analysis, of the predicted OoNDPK and its orthologues, revealed phenylalanine and tyrosine residues to be specifically present in NDPK proteins from the plant feeders. Results suggest secretion of OoNDPK into the host plant and its probable involvement in gall midge-rice interaction. Using the coleoptile cell elongation assay, we demonstrated that the recombinant OoNDPK is capable of causing elongation of rice coleoptile cells. Additionally, heterologous expression of OoNDPK in Escherichia coli increased the tolerance of these cells to salt (NaCl; up to 1 mM), hinting at the involvement of this gene in abiotic stress response as well.
BackgroundThe Asian rice gall midge (Orseolia oryzae) is a destructive insect pest of rice. Gall midge infestation in rice triggers either compatible or incompatible interactions leading to survival or mortality of the feeding maggots, respectively. In incompatible interactions, generation of plant allelochemicals/defense molecules and/or inability of the maggots to continue feeding on the host initiate(s) apoptosis within the maggots. Unraveling these molecular events, triggered within the maggots as a response to feeding on resistant hosts, will enable us to obtain a better understanding of host resistance. The present study points towards the likely involvement of a defender against apoptotic cell death gene (DAD1) in the insect in response to the host defense.ResultsThe cDNA coding for the DAD1 orthologue in the rice gall midge (OoDAD1) consisted of 339 nucleotides with one intron of 85 bp and two exons of 208 and 131 nucleotides. The deduced amino acid sequence of OoDAD1 showed a high degree of homology (94.6 %) with DAD1 orthologue from the Hessian fly (Mayetiola destructor) —a major dipteran pest of wheat. Southern hybridization analysis indicated that OoDAD1 was present as a single copy in the genomes of the Asian rice gall midge biotypes (GMB) 1, 4 and 4 M. In the interactions involving GMB4 with Jaya (susceptible rice host) the expression level of OoDAD1 in feeding maggots gradually increased to 3-fold at 96hai (hours after infestation) and peaked to 3.5-fold at 96hai when compared to that at 24 hai. In contrast, expression in maggots feeding on RP2068 (resistant host) showed a steep increase of more than 8-fold at 24hai and this level was sustained at 48, 72 and 96hai when compared with the level in maggots feeding on Jaya at 24hai. Recombinant OoDAD1, expressed in E. coli cells, when injected into rice seedlings induced a hypersensitive response (HR) in the resistant rice host, RP2068, but not in the susceptible rice variety, Jaya.ConclusionsThe results indicate that the expression of OoDAD1 is triggered in the feeding maggots probably due to the host resistance response and therefore, is likely an important molecule in the initial stages of the interaction between the midge and its rice host.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0618-y) contains supplementary material, which is available to authorized users.
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