The complete cDNA sequences of heat shock protein 90 (hsp90) and of heat shock cognate protein 70 (hsc70) were cloned by reverse transcription polymerase chain reaction from the rice stem borer, Chilo suppressalis Walker. They potentially encode a 717-amino-acids (hsp90) and a 652-amino-acids (hsc70) protein, with calculated molecular weight of 82.5 and 71.3 kDa, respectively. The deduced amino acid sequence of hsp90 showed the highest homology of 97.2% to Spodoptera frugiperda hsp90. The closest match of C. suppressalis hsc70 was with Manduca sexta hsc70 at 98.0% identity. Expression of hsp90 in diapausing larvae was higher than that in non-diapausing larvae. No such up-regulation in diapausing larvae was observed for hsc70. In non-diapausing larvae, but not in diapausing ones, hsp90 expression was up-regulated by cold acclimation. Hsc70 expression slightly decreased during cold acclimation irrespective of the state of diapause. Involvement of hsp90 and hsc70 in larval diapause and cold tolerance acquisition in C. suppressalis is discussed.

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.

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Overwintering larvae of the rice stem borer, Chilo suppressalis accumulate glycerol and are freezing tolerant to about -25°C. However, non-diapausing larvae cannot accumulate glycerol and are killed by freezing. We compared the extent of tissue damage, the effects of glycerol concentration, and the transport of glycerol and water in fat body tissues from these larvae at selected freezing temperatures. Tissues from overwintering larvae, but not non-diapausing larvae, survive when frozen at -20°C with 0.25M glycerol, but the protection afforded by glycerol is offset by the water-channel inhibitor mercuric chloride. Glycerol in higher concentration (0.75M) affords some protection even to the fat body of non-diapausing larvae. Radiotracer assays of overwintering larvae show that water leaves the tissues during freezing while glycerol enters, and that mercuric chloride disrupts this process. Transport is also disrupted after lethal freezing at -35°C. Therefore, membrane transport of water and glycerol is involved in the avoidance of freezing injury to fat body cells of the rice stem borer, apparently by mediating the replacement of water with glycerol in freezing-tolerant tissues.

SummaryUsing the grafting procedure, we examined the transmission of post-transcriptional gene silencing (PTGS) in Nicotiana benthamiana which had been transformed with the coat protein gene, including the 3¢ non-translated region of the sweet potato feathery mottle potyvirus. Transmission of PTGS from silenced lines to non-silenced ones was bidirectional, but occurred ef®ciently from root stocks to scions. The level of transgene methylation in non-silenced scions grafted onto silenced root stocks was not increased. When grafted scions which had become silenced were removed from silenced root stocks and regrafted onto non-silenced or vector-transformed root stocks, PTGS was maintained. However, their progeny did not show PTGS. Previously we reported that our transgenic lines had different target speci®cities of PTGS for RNA degradation: one line recognized only the 3¢ part of the transgene mRNA while others involved the whole transgene mRNA (Sonoda et al., 1999, Phytopathology, 89, 385±391). Using these lines, we showed that target speci®city of PTGS induced in non-silenced scions after grafting was determined by that in silenced root stocks. However, unexpectedly, target speci®city of PTGS induced in silenced scions after grafting was not changed by grafting onto silenced root stocks showing different target speci®city, indicating that the second PTGS from silenced root stocks was not superimposed to silenced scions.

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We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3' non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5' 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5' or 3' silencing inducer rootstocks. When non-silenced scions were grafted onto 5' silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3' region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3' silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3' region of the transgene and did not spread to the 5' region. In addition, results from crossing experiments, involving non-silenced and 3' silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5'-3' direction, not in the 3'-5' direction, along the transgene mRNA.