Naoto Nitta

Inoculation with RNAs 1 and 2 of Cucumber Mosaic Virus Induces Viral RNA Replicase Activity in Tobacco Mesophyll Protoplasts

Summary The activity of membrane-bound RNA-dependent RNA polymerase in tobacco mesophyll protoplasts was measured and in vitro products were characterized after inoculation with cucumber mosaic virus (CMV) or with different combinations of the four virus RNAs and satellite RNA. Activity increased rapidly 4 to 6 h after inoculation with virus particles. When the inoculum contained RNA 1, RNA 2 and RNA 3, most of the in vitro products of the enzyme were full-length positive-sense RNAs (RNAs 1, 2, 3 and 4) that migrated as dsRNA; inoculation of RNA 1 or RNA 2 alone did not increase RNA polymerase activity and no virus RNA was produced. After inoculation with a mixture of RNA 1 and RNA 2, however, the enzyme activity increased and full-length positive-sense RNA 1 and RNA 2 were synthesized, and we speculate that the induced enzyme is CMV RNA replicase as a complex comprising the translation products of RNA 1 and RNA 2, membrane components of the host cell and CMV RNA templates. Protoplasts inoculated with RNA 1, RNA 2 and RNA 4 did not synthesize RNA 4 which suggests that positive-sense RNA 4 does not serve as a template for the replicase complex. Satellite RNA was synthesized in protoplasts inoculated with RNA 1, RNA 2 and satellite RNA which suggests that satellite RNA is replicated by the replicase complex.

Fine genetic mapping of the nuclear gene, Rf-1, that restores the BT-type cytoplasmic male sterility in rice (Oryza sativa L.) by PCR-based markers

The Rf-1 gene restores the pollenfertility disturbed by the BT-type malesterile cytoplasm. Nine restrictionfragment length polymorphism (RFLP) markerson chromosome 10, where the Rf-1 geneis located, were converted to PCR-basedmarkers. Genetic analysis was conductedusing the nine markers and a segregatingpopulation consisting of 1042 plants. As aresult, the Rf-1 gene was mappedbetween S12564 Tsp509I and C1361 MwoI. The genetic distance of the twomarker loci was estimated as 0.3 cM. Basedon the map information of the Rf-1gene, it will be possible to introduce alimited chromosomal segment of the Rf-1 region derived from a donor intojaponica rice varieties efficiently andeffectively by marker-assisted selection(MAS). Also, examination of seed purity ofhybrid rice varieties and their parentallines will be facilitated. Furthermore,judging from the genetic distance from theRf-1 locus to S12564 Tsp509Iand C1361 MwoI loci, it seems thatthe Rf-1 gene could be isolated bychromosomal walking from either or both ofthe two marker loci.

Transgenic rice with reduced glutelin content by transformation with glutelin A antisense gene

Rice plants were genetically transformed with a glutelin A antisensegene construct that was characterized by eight tandem repeats of a ca.0.3 kb fragment at the 5′ end of full-length glutelin AcDNA.Two transgenic rice lines, H39-59 and H75-3, homozygous for the glutelin Aantisense gene were selected from T2 plants, and their T4generation was examined. When grown in an open paddy field, T4plantsof both transgenic lines showed 20% to 40% reduction in theglutelin content of seeds and an increase in the prolamine content. The totalseed-protein content of the two transgenic lines was not significantlydifferentfrom that of the original variety, Tsukinohikari. Northern blot analysisrevealed that the steady-state levels of glutelin A transcript also decreasedand those of prolamine transcript increased in both H39-59 and H75-3 transgeniclines, and the changes in glutelin mRNA corresponded to changes observed inglutelins during seed development. Rice wine, sake, brewed from grains of thetransgenic lines was found to contain 20% to 40% less amino acidsand was evaluated as superior in quality to that made from the non-transgenicrice grains.