Targeting of multiple sequences for resistance against cotton leaf curl disease through RNA interference

Abstract

The purpose of lhis study was to devel op a broad~s ec lrul11 resistance against bego1l1ovirllses based on the concept of pathogen-de ri ved resistan ce. The first step to achieve this obj ecti ve was to in vestigate the di versity or genomi c components of begol1loviruses associated with culti vated as well as nOIl-clilti vatcu hosts. Diversityor gemini virus components on three weeds hosts found commonl y a round field crops was studi ed . These analyses showed that the weeds arc source of multiple begorn ovirus componen ts fou nd on crop plan ts. These weeds carry Inultipl e monopartilc as we I! as bipa rtite begomov iruses and their associated alphasate llites and belasatellites. An interesting fe ature of bctasntellites on weeds was recombination among M,llvaceolls and non ~ Mal vaceolls bctasatellites and suggests tha l lVeeds are importan t source of recombination both in begoll1oviruses and th eir sa tellites. Thus, weeds seems to be the sourccs of emerging viruses dl1e to recombination, multiple infection and even coinCeclion of lWO genera of geminiviruses. Begoll1ovirus genome sequences charactcrized from weeds and ava il able in the databanks were aligned and analyzed, These [lna lyses identified two conse rved regions in the begomov irus sequences. The sequences overlapping co mplem lllary~se se genes (C I, C2. C3) involved in replication and transcription. and irjon~ ense genes (V I. V2) in volved in cncapsidation and movement/pathogenicity. These two regions were targeted th ro ugh RNA si lencing for deve loping ad~ ee lru m resistallc{;! against begoJ11oviruses. Two vinlses were chosen as model. a monopartite begoillovirus Co/Ion teaf cUI'I Mullan w'rlls and a bipartite bego11lovirus, Tomolo lec!f CllrI Nell' Delhi virus to investigate resistance. Two hairpin constructs werc deve loped targeting co mpl ementa ry~ se genes of Cotton leaf curl Multan virus (CLCuMV). One hairpin construct ta rge ting n~cod jll g regulatory sequences of the same virus was <1 150 developcd. In tl'.:lllsient assays all three hairpin construCts blocked the replicat ion of th e target virus and the systemi c spread of virus was inhibited. However, in the presence of bel.:lSatellite only one of the constrllcllargeling overlapping genes involved in virus repli cntion and palhogenicity was able to block replication as well as system io movement of the virus. Tl'allsgellic plants transfonned with this constru ct blocked gClllini virus replication, showi ng effectiveness of lhe approach to develop resistance. iri n~ sc se pre~coa t protein gene lA Y2) of Tomato leaf elll'l New Delhi virus (ToLCN DV). :1 bipartite begomovi rus prevalent th roughout the Indian subcontinent. was also targeted through anti-sense RNA technology.