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

Sinha DK, Atray I, Bentur JS, Nair S - BMC Plant Biol. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110 067, India. deepak22sinha@yahoo.co.in.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Heterologous expression of OoDAD1 and Western analysis. a Coomassie blue stained sodium dodecyl sulphate polyacrylamide gel showing over-expression of recombinant OoDAD1 in BL21 (DE3) pLysE Escherichia coli-based expression system using pET 28a expression vector. Lanes: 1, lysate of induced, transformed cells with OoDAD1; 2, supernatent of induced, transformed cells with OoDAD1; 3, pellet of induced, transformed cells with OoDAD1. M, protein ladder. b Western analysis of polyacrylamide gel shown in (a) and electrotransferred to a nitrocellulose membrane and probed with anti-His tag antibodies (see Experimental Procedures). Lane designation is same as in (a). Arrows indicate the location of the His-tagged OoDAD1. Numbers on the left represent molecular weights in kilodaltons (kDa)
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Fig5: Heterologous expression of OoDAD1 and Western analysis. a Coomassie blue stained sodium dodecyl sulphate polyacrylamide gel showing over-expression of recombinant OoDAD1 in BL21 (DE3) pLysE Escherichia coli-based expression system using pET 28a expression vector. Lanes: 1, lysate of induced, transformed cells with OoDAD1; 2, supernatent of induced, transformed cells with OoDAD1; 3, pellet of induced, transformed cells with OoDAD1. M, protein ladder. b Western analysis of polyacrylamide gel shown in (a) and electrotransferred to a nitrocellulose membrane and probed with anti-His tag antibodies (see Experimental Procedures). Lane designation is same as in (a). Arrows indicate the location of the His-tagged OoDAD1. Numbers on the left represent molecular weights in kilodaltons (kDa)

Mentions: The use of pET 28a vector for protein expression resulted in the production of His-tagged OoDAD1 protein with a molecular weight of 13.2 kDa, (including the seven His-residues in the N-Terminal region) as expected. Anti-His antibodies were used to confirm the expression and the size of the protein (Fig. 5).Fig. 5


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

Sinha DK, Atray I, Bentur JS, Nair S - BMC Plant Biol. (2015)

Heterologous expression of OoDAD1 and Western analysis. a Coomassie blue stained sodium dodecyl sulphate polyacrylamide gel showing over-expression of recombinant OoDAD1 in BL21 (DE3) pLysE Escherichia coli-based expression system using pET 28a expression vector. Lanes: 1, lysate of induced, transformed cells with OoDAD1; 2, supernatent of induced, transformed cells with OoDAD1; 3, pellet of induced, transformed cells with OoDAD1. M, protein ladder. b Western analysis of polyacrylamide gel shown in (a) and electrotransferred to a nitrocellulose membrane and probed with anti-His tag antibodies (see Experimental Procedures). Lane designation is same as in (a). Arrows indicate the location of the His-tagged OoDAD1. Numbers on the left represent molecular weights in kilodaltons (kDa)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4591563&req=5

Fig5: Heterologous expression of OoDAD1 and Western analysis. a Coomassie blue stained sodium dodecyl sulphate polyacrylamide gel showing over-expression of recombinant OoDAD1 in BL21 (DE3) pLysE Escherichia coli-based expression system using pET 28a expression vector. Lanes: 1, lysate of induced, transformed cells with OoDAD1; 2, supernatent of induced, transformed cells with OoDAD1; 3, pellet of induced, transformed cells with OoDAD1. M, protein ladder. b Western analysis of polyacrylamide gel shown in (a) and electrotransferred to a nitrocellulose membrane and probed with anti-His tag antibodies (see Experimental Procedures). Lane designation is same as in (a). Arrows indicate the location of the His-tagged OoDAD1. Numbers on the left represent molecular weights in kilodaltons (kDa)
Mentions: The use of pET 28a vector for protein expression resulted in the production of His-tagged OoDAD1 protein with a molecular weight of 13.2 kDa, (including the seven His-residues in the N-Terminal region) as expected. Anti-His antibodies were used to confirm the expression and the size of the protein (Fig. 5).Fig. 5

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110 067, India. deepak22sinha@yahoo.co.in.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus