Rice ragged stunt virus (RRSV) genome organization and RRSV derived synthetic resistance genes

摘要

Rice ragged stunt virus is an important plant reovirus and is the type species of the genus Oryzavirus under the family Reoviridae. We have cloned (cDNA) and sequenced all the 10 ds RNA genome segments having a total size of 26,066 bp. Each segment contains the genus specific conserved terminal sequences (5' GAUAAA - and -GUGC 3') and a single coding region (ORF) with the exception of S4 which contains two ORFs). By sequence comparison, N-terminal amino acid sequencing, production and analysis of segment specific fusion proteins and antibodies, four structural genes (S3, S5, S8 and S9), one polymerase gene (S4a) and two non-structural genes (S7 and S10) have been identified. Segment S9 sequence is conserved among isolates from different geographical locations. Segment S8 encodes a 67K protein which undergoes self-cleavage to produce the 43K major capsid protein, presumably by the activity of the N-terminal 26K protease. In evolutionary terms, RRSV is closer to members of the genus Fijivirus than those of Phytoreovirus. An approach based on pathogen derived resistance (PDR) was used to produce RRSV resistant rice cultivars. Sequences from the coding region of RRSV S5, S7, S8, S9 and S10 were placed in sense or antisense orientation behind the plant expression promoters CaMV35S, RolC, UbU, Actl or RBTV. Rice cultivars Taipei 309 and Chinsurah Boro II were transformed by biolistic and/or Agrobacterium-medinted delivery of one or more of these PDR gene (instructs. A large number of transgenie lineswas produced from calli derived from mature or immature embryos, co-bombarded with the marker gene Iiph encoding hygromycin resistance and RRSV PDR genes or co-cultivated with strains having the binary vector containing these two genes. Both Mendelian and non-Medelian segregations were observed in transgenie progeny, especially with transgenie lines produced by biolistics. Preliminary tests conducted in China on selected transgenie lines indicate that plants with RRSV segment 5 antisense PDR gene show RRSV resistance.