Is the cucumber mosaic virus 2b silencing suppressor protein regulated by direct interactions with the viral 1a protein?
Lead Research Organisation:
University of Cambridge
Department Name: Plant Sciences
Abstract
PhD project strategic theme: Bioscience for sustainable agriculture and food
Proposed title for PhD: Using the cucumber mosaic virus 2b silencing suppressor protein and the viral 1a protein interaction to identify the relationship between silencing pathways and plant insect resistance mechanisms.
The cucumber mosaic virus (CMV) 2b silencing suppressor protein inhibits RNA silencing mediated by short-interfering (si)RNAs (a potent antiviral mechanism) and micro(mi)RNA-regulated plant development. These two activities of the 2b protein are distinct: suppression of antiviral silencing depends on 2b's ability to bind double-stranded siRNA, while its effect on miRNA-regulated development depend largely on the interaction of 2b with Argonaut (AGO)1 protein. The effect of the 2b protein on an anti-insect defence system controlled by AGO1 are antagonized by another CMV protein the 1a protein, a replication-associated protein with no previously known connection to silencing. Recent evidence suggests that this effect is achieved through a direct interaction between the 2b and 1a viral proteins. The aim of this PhD is to characterise the interaction between 1a and 2b using 1a and 2b-derived wild-type and mutant constructs for protein binding studies including bimolecular fluorescence complementation and FLAG pull-downs. These techniques, along with fluorescent microscopy techniques, will also be used to identify the localisation and cell biology of the 1a and 2b viral proteins. This PhD will attempt to determine if 1a also affects the silencing suppressor activity of 2b or just its effects on miRNA pathways. Finally, this PhD will use 1a and 2b-derived wild-type and mutant constructs in aphid assays to assess the effects of disrupted silencing pathways on insect resistance in plants.
Proposed title for PhD: Using the cucumber mosaic virus 2b silencing suppressor protein and the viral 1a protein interaction to identify the relationship between silencing pathways and plant insect resistance mechanisms.
The cucumber mosaic virus (CMV) 2b silencing suppressor protein inhibits RNA silencing mediated by short-interfering (si)RNAs (a potent antiviral mechanism) and micro(mi)RNA-regulated plant development. These two activities of the 2b protein are distinct: suppression of antiviral silencing depends on 2b's ability to bind double-stranded siRNA, while its effect on miRNA-regulated development depend largely on the interaction of 2b with Argonaut (AGO)1 protein. The effect of the 2b protein on an anti-insect defence system controlled by AGO1 are antagonized by another CMV protein the 1a protein, a replication-associated protein with no previously known connection to silencing. Recent evidence suggests that this effect is achieved through a direct interaction between the 2b and 1a viral proteins. The aim of this PhD is to characterise the interaction between 1a and 2b using 1a and 2b-derived wild-type and mutant constructs for protein binding studies including bimolecular fluorescence complementation and FLAG pull-downs. These techniques, along with fluorescent microscopy techniques, will also be used to identify the localisation and cell biology of the 1a and 2b viral proteins. This PhD will attempt to determine if 1a also affects the silencing suppressor activity of 2b or just its effects on miRNA pathways. Finally, this PhD will use 1a and 2b-derived wild-type and mutant constructs in aphid assays to assess the effects of disrupted silencing pathways on insect resistance in plants.
