Comparative Cytology of Rice Tungro Viruses in Selected Rice Cultivars

Ultrastructural studies were made using plants of five selected rice cultivars infected with rice tungro viruses. In all cultivars, rice tungro bacilliform virus (RTBV) was found in both xylem and phloem, while rice tungro spherical virus (RTSV) was observed only in the phloem. In tolerant cultivars Balimau Putih and Utri Merah, plants infected with RTBV and RTSV or RTBV alone, had fewer cells containing tungro viruses than plants of the sensitive cultivar Taichung Native 1. In RTBV-sensitive cultivars FK 135 and ASD 8 infected with both viruses or RTBV alone, electron-dense cytoplasm was observed in most cells of the phloem. Abundant phloem necrosis was also observed in FK 135. These observations were correlated with the reaction of each rice cultivar to tungro infection.     

Transformation of rice plants by plant reovirus genes

Rice dwarf phytoreovirus (RDV), and rice ragged stunt oryzairus (RRSV) genes were introduced into rice protoplasts by using the cauliflower mosaic virus 35S promoter, tissue culture techniques and electroporation. The translation products of cDNA to RDV segment 8 were detected in transformed rice. Plants transgenic for RRSV S9 also expressed an mRNA of appropriate size but the protein was not apparently expressed. These latter plants did not show any resistance when inoculated with RRSV; on the contrary, symptom expression was intensified. Since most plant reoviruses are phloem-limited, an alternative promoter could be that of rice tungro bacilliform virus (RTBV), which is itself phloem-limited. When the β-glucuronidase (GUS) gene was coupled to this promoter and introduced into rice, GUS activity was successfully expressed only in the phloem, so the system could be of interest in the reovirus context.     

Promoter elements required for phloem-specific gene expression from the RTBV promoter in rice

Previous studies indicated that a DNA fragment comprising nucleotides (nt) ?164 to +45 of the RTBV promoter is sufficient to drive phloem-specific expression of a reporter gene in transgenic rice plants. In addition, two potential cis elements, Box I (nt ?3 to +5) and Box II (nt ?53 to ?39) were identified by DNA-protein interaction assays. In this study, the results of further in vivo studies involving mutagenesis of selected DNA sequences and analysis of expression of a reporter gene in transgenic rice plants revealed that, in addition to Box I and Box II, other elements are required for phloem-specific gene expression, among which are a direct repeat (ASL Box, nt ?98 to ?79) and a GATA motif (nt ?143 to ?135). All the these elements bind rice nuclear factors specifically, and mutations of the elements were identified that resulted in loss-of-competition in electrophoretic mobility shift assays. A DNA fragment comprising nt ?164 to ?32, which contains Box II, the ASL Box and the GATA motif, conferred phloem-specific reporter gene expression independent of Box I when fused to a heterologous CaMV 35S minimal promoter and introduced to transgenic rice plants. Studies that introduced point mutations suggested that in the context of the ?103 to +45 promoter fragment, Box II and the ASL Box act synergistically to confer tissue-specific gene expression. Similar studies in the ?164 to +45 promoter fragment indicated that the ?164 to ?103 region, which includes the GATA motif, contains sequences that are functionally redundant with those in the ?103 to ?32 region, including the ASL Box and Box II. It is concluded that these regions act additively to direct phloem-specific gene expression.     

Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz)

A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava’s starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.     

Expression and Immunolocalisation of the Snowdrop Lectin,GNA in Transgenic Rice Plants

Transgenic rice (Oryza sativa L.) plants generated through particle bombardment expressed high levels of an insecticidal protein (the snowdrop lectin, GNA) directed against sap-sucking insects. Engineered plants expressed GNA either constitutively or in a tissue specific manner, depending on the nature of the promoter used to drive expression of the gene. We used specific antibodies raised against GNA to localize its expression in phloem tissue in plants engineered with the rice sucrose synthase promoter driving GNA expression. We report here molecular, biochemical and immunological analyses for fifteen independently-derived transformants out of more than 200 plants we generated.