Establishment And Application Of An In Vivo Cap Donor Delivery System Investigating The Cap Snatching Of Plant Bunyaviruses

Tomato spotted wilt tospovirus(TSWV)and Rice stripe tenuivirus(RSV)are two important plant bunyaviruses.Like animal bunyaviruses,TSWV and RSV also use the cap-snatching mechanism to aquire a cap structure for their m RNA,i.e.,when synthesizing m RNAs,they first cleave the host m RNA at positions 10-20 nucleotides downstream of the cap structure and use the 5' cleavage product(capped-RNA leader sequence)to prime the transcription of their template RNAs.As a result,each viral m RNA contains a short 5'-terminal capped RNA-leader sequence derived from the a host m RNA.However,our understanding of the cap-snatching of TSWV and RSV remains poor.One of the important reasons is the unavailability of a convenient experimental system allowing researchers to present specific m RNA to TSWV and RSV(for cap-snatching)until present.Agroinfiltration is a method widely used to express forein m RNAs in plant cells.This study found that after RSV/TSWV-infected Nicotiana benthamiana leaves were injected with agrobacterium containing the p CHF3 vector,both TSWV and RSV could snatch capped-RNA leader sequences from the GFP m RNA expressed by p CHF3.Based on this,11 vectors expressing different GFP m RNAs were constructed and with which some details of TSWV and RSV cap-snatching were studied inparallel and comparatively.The main results obtained are as follows:(1)Both TSWV and RSV tend to choose capped-RNA leader sequences complementary to the 3' end of their genome strands,indicating that both viruses follow the "base-pairing" principle during cap-snatching;(2)TSWV tends to cleave to obtain 13-15 nt capped-RNA leader sequences,while RSV tends to cleave to obtain 11-13 nt cap sequences,indicating that the two viruses show differences in their prefered lengths of the capped-RNA leader sequences;(3)When the capped-RNA leader sequences is shorter than a certain length,both TSWV and RSV tend to use the prime-and-realign(PAR)mechanism,but TSWV uses the mechanism less frequently than RSV;(4)RSV has a preference for capped-RNA leader sequences with multiple-base complenmentarity to its genome strands.This study validated some of the models previously proposed by relevant researchers.For the first time,this study clearly shows that different bunyaviruses have different preferences for the length of the capped-RNA leader sequences,and for the first time,this study shows that TSWV also uses the prime-and-realign mechanism frequently in transcription using shorter capped-RNA leader sequences as primers just like that has been demonstrated recently for RSV.More importantly,this thesis established a convenient experimental system to deliver specificm RNAs to TSWV and RSV(for cap-snatching),which is valuable for further studies on the cap-snatching of plant bunyaviruses.