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).

Feeding on resistant rice leads to enhanced expression of defender against apoptotic cell death (OoDAD1) in the Asian rice gall midge.

BACKGROUND: The 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. RESULTS: The 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 96 hai (hours after infestation) and peaked to 3.5-fold at 96 hai 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 24 hai and this level was sustained at 48, 72 and 96 hai when compared with the level in maggots feeding on Jaya at 24 hai. 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. CONCLUSIONS: The 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.

Virulent Diuraphis noxia Aphids Over-Express Calcium Signaling Proteins to Overcome Defenses of Aphid-Resistant Wheat Plants.

The Russian wheat aphid, Diuraphis noxia, an invasive phytotoxic pest of wheat, Triticum aestivum, and barley, Hordeum vulgare, causes huge economic losses in Africa, South America, and North America. Most acceptable and ecologically beneficial aphid management strategies include selection and breeding of D. noxia-resistant varieties, and numerous D. noxia resistance genes have been identified in T. aestivum and H. vulgare. North American D. noxia biotype 1 is avirulent to T. aestivum varieties possessing Dn4 or Dn7 genes, while biotype 2 is virulent to Dn4 and avirulent to Dn7. The current investigation utilized next-generation RNAseq technology to reveal that biotype 2 over expresses proteins involved in calcium signaling, which activates phosphoinositide (PI) metabolism. Calcium signaling proteins comprised 36% of all transcripts identified in the two D. noxia biotypes. Depending on plant resistance gene-aphid biotype interaction, additional transcript groups included those involved in tissue growth; defense and stress response; zinc ion and related cofactor binding; and apoptosis. Activation of enzymes involved in PI metabolism by D. noxia biotype 2 aphids allows depletion of plant calcium that normally blocks aphid feeding sites in phloem sieve elements and enables successful, continuous feeding on plants resistant to avirulent biotype 1. Inhibition of the key enzyme phospholipase C significantly reduced biotype 2 salivation into phloem and phloem sap ingestion.

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.

Selection of reference genes for expression analysis in Diuraphis noxia (Hemiptera: Aphididae) fed on resistant and susceptible wheat plants.

The Russian wheat aphid (RWA), Diuraphis noxia Kurdjumov, is a major global pest of wheat and barley production that causes enormous economic damage. Few studies have been conducted to explore and decipher the molecular basis of RWA strategies to evade plant defense mechanisms. Gene expression studies of RWA in response to wheat genotypes carrying different RWA resistance genes have been initiated in our group; however, a secure and accurate understanding of RWA gene expression is dependent on identification of suitable reference genes. This study analyzed expression profiles of five potential reference genes selected and sequenced during RNA sequencing experiments. The expression of genes coding for actin and ribosomal protein L27 was comparatively less variable in RWA fed on different wheat hosts. Results of geNorm, NormFinder, and BestKeeper expression analyses support the use of actin and ribosomal protein L27 in RT-qPCR studies of RWA gene expression in studies involving RWA-wheat interactions.