Cellular events which occur prior to cell collapse were examined in the root cortex of rice (Oryza sativa L.) during aerenchyma formation. Cell collapse started at a speci®c position in the mid cortex. These cells were distinct in shape from those located towards the periphery. Furthermore, cell collapse was preceded by acidi®cation and the loss of plasma-membrane integrity in cells of the mid cortex. Subsequent death of neighboring cells followed a radial path. Microinjection of molecules of dierent sizes conjugated with uorescein isothiocyanate (FITC) showed a molecular exclusion limit of between 9.3 and 19.6 kDa in the root cortex. Furthermore, large molecules, i.e. those around 9.3 kDa, were predominantly transferred in a radial direction, which coincided with the path of sequential cell death.Abbreviations: F-dextran = FITC conjugated dextran; FITC = uorescein isothiocyanate; LY = Lucifer Yellow Correspondence to: M. Kawai;
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.
SummaryHeme activator protein (HAP), also known as nuclear factor Y or CCAAT binding factor (HAP/NF-Y/CBF), has important functions in regulating plant growth, development and stress responses. The expression of rice HAP gene (OsHAP2E) was induced by probenazole (PBZ), a chemical inducer of disease resistance. To characterize the gene, the chimeric gene (OsHAP2E::GUS) engineered to carry the structural gene encoding b-glucuronidase (GUS) driven by the promoter from OsHAP2E was introduced into rice. The transgenic lines of OsHAP2Ein::GUS with the intron showed high GUS activity in the wounds and surrounding tissues. When treated by salicylic acid (SA), isonicotinic acid (INA), abscisic acid (ABA) and hydrogen peroxide (H 2 O 2 ), the lines showed GUS activity exclusively in vascular tissues and mesophyll cells. This activity was enhanced after inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. The OsHAP2E expression level was also induced after inoculation of rice with M. oryzae and X. oryzae pv. oryzae and after treatment with SA, INA, ABA and H 2 O 2, respectively. We further produced transgenic rice overexpressing OsHAP2E. These lines conferred resistance to M. oryzae or X. oryzae pv. oryzae and to salinity and drought. Furthermore, they showed a higher photosynthetic rate and an increased number of tillers. Microarray analysis showed up-regulation of defence-related genes. These results suggest that this gene could contribute to conferring biotic and abiotic resistances and increasing photosynthesis and tiller numbers.
The complete nucleotide sequence of the genomic RNA of cucumber green mottle mosaic virus watermelon strain SH (CGMMV-SH) was determined using cloned cDNA. This sequence is 6421 nucleotides long containing at least four open reading frames, which correspond to 186K, 129K, 29K and 17.3K proteins. The 17.3K protein is the coat protein. Sequence analysis shows that CGMMV-SH is very closely related to another watermelon strain, CGMMV-W, although three amino acid substitutions in the 29K protein were found between these strains. The sequence was also compared to those of other tobamoviruses, tobacco mosaic virus (TMV) vulgare, TMV-L (a tomato strain) and tobacco mild green mosaic virus reported by other groups. It shows 55 to 56% identity with these viruses. The size and location of the open reading frames are very similar to those of TMV but the 129K and 186K proteins are composed of 1142 and 1646 amino acids, being larger than those of TMV by 27 and 31 amino acids, respectively. The deduced amino acid sequences of these proteins are highly homologous to those of TMV, especially in the readthrough downstream region of the 186K protein.
Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus-resistant plants.
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