The titers of rice tungro bacilliform virus dictate the expression levels of genes related to cell wall dynamics in rice plants affected by tungro disease.

Rice tungro disease (RTD) is a devastating disease of rice caused by combined infection with rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV), with one of the main symptoms being stunting. To dissect the molecular events responsible for RTD-induced stunting, the expression patterns of 23 cell-wall-related genes were examined in different rice lines with the same titers of RTSV but different titers of RTBV and in lines where only RTBV was present. Genes encoding cellulose synthases, expansins, glycosyl hydrolases, exostosins, and xyloglucan galactosyl transferase showed downregulation, whereas those encoding defensin or defensin-like proteins showed upregulation with increasing titers of RTBV. RTSV titers did not affect the expression levels of these genes. A similar relationship was seen for the reduction in the cellulose and pectin content and the accumulation of lignin. In silico analysis of promoters of the genes indicated a possible link to transcription factors reported earlier to respond to viral titers in rice. These results suggest a common network in which the genes related to the cell wall components are affected during infection with diverse viruses in rice.

Assessment of resistance to rice tungro disease in popular rice varieties in India by introgression of a transgene against Rice tungro bacilliform virus.

Rice crops in South and Southeast Asian countries suffer critical yield losses due to rice tungro disease caused by joint infection with rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV). Previously, for generating RNA interference-based transgenic resistance against tungro viruses, RTBV ORF IV was used as a transgene to develop RTBV resistance in a popular high-yielding scented rice variety. The transgene from this line was then introgressed into five popular high-yielding but tungro-susceptible rice varieties by marker-assisted backcross breeding with a view to combine the resistant trait with the agronomic traits. The present work includes a resistance assay of the BC3F5 lines of these varieties under glasshouse conditions. Out of a total of 28 lines tested, each consisting of 12 individual plants, eight lines showed significant amelioration in height reduction and 100- to 1000-fold reduction in RTBV titers. The RNAi-mediated resistance was clearly manifested by the presence of virus-derived small RNA (vsRNA) specific for RTBV ORF IV in the transgenic backcrossed lines.

Physical interaction of RTBV ORFI with D1 protein of Oryza sativa and Fe/Zn homeostasis play a key role in symptoms development during rice tungro disease to facilitate the insect mediated virus transmission.

Rice tungro disease is caused by the combined action of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). The RTBV is involved in the development of symptoms while RTSV is essential for virus transmission. We attempted to study the mode of action of RTBV in the development of symptoms. The tungro disease symptoms were attributed to viral interference in chlorophyll and carotenoids biosynthesis, photosynthesis machinery, iron/zinc homeostasis, and the genes encoding the enzymes associated with these biological processes of rice. The adverse effects of virus infection in photosystem II (PSII) activity was demonstrated by analyzing the Fv/Fm ratio, expression of psbA and cab1R genes, and direct interaction of RTBV ORF I protein with the D1 protein of rice. Since ORF I function is not yet known in the RTBV life cycle, this is the first report showing its involvement in regulating host photosynthesis process and symptoms development.

Interactions of Rice tungro bacilliform pararetrovirus and its protein P4 with plant RNA-silencing machinery.

Small interfering RNA (siRNA)-directed gene silencing plays a major role in antiviral defense. Virus-derived siRNAs inhibit viral replication in infected cells and potentially move to neighboring cells, immunizing them from incoming virus. Viruses have evolved various ways to evade and suppress siRNA production or action. Here, we show that 21-, 22-, and 24-nucleotide (nt) viral siRNAs together constitute up to 19% of total small RNA population of Oryza sativa plants infected with Rice tungro bacilliform virus (RTBV) and cover both strands of the RTBV DNA genome. However, viral siRNA hotspots are restricted to a short noncoding region between transcription and reverse-transcription start sites. This region generates double-stranded RNA (dsRNA) precursors of siRNAs and, in pregenomic RNA, forms a stable secondary structure likely inaccessible to siRNA-directed cleavage. In transient assays, RTBV protein P4 suppressed cell-to-cell spread of silencing but enhanced cell-autonomous silencing, which correlated with reduced 21-nt siRNA levels and increased 22-nt siRNA levels. Our findings imply that RTBV generates decoy dsRNA that restricts siRNA production to the structured noncoding region and thereby protects other regions of the viral genome from repressive action of siRNAs, while the viral protein P4 interferes with cell-to-cell spread of antiviral silencing.

A negative element in the downstream region of the Rice tungro bacilliform virus promoter is orientation- and position-independent and is active with heterologous promoters.

The promoter of an Indian isolate of the pararetrovirus Rice tungro bacilliform virus (RTBV-WB) contains a negative element downstream of the transcription start site (TSS), between nucleotide residues +58 and +195 (Mathur and Dasgupta, 2007). To further characterize the element, we show, by using transient gus reporter gene assays in the cells of onion peel, rice calli and Arabidopsis leaves, that it down-regulates heterologous promoters CaMV35S and Maize ubiquitin. Quantitative measurements of transient GUS activity indicated more than 90% inhibition of reporter gene expression by the negative element. We also show, by reversing the orientation of the element downstream and by placing it in a position upstream to a constitutively expressing RTBV promoter, that the negative element is orientation- and position-independent, pointing towards its activity at the transcriptional and not post-transcriptional level.

Suppression of two tungro viruses in rice by separable traits originating from cultivar Utri Merah.

Rice tungro disease (RTD) is caused by Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus (RTBV) transmitted by green leafhoppers. Rice cv. Utri Merah is highly resistant to RTD. To define the RTD resistance of Utri Merah, near-isogenic lines (NIL, BC(5) or BC(6)) developed from Utri Merah and susceptible cv. Taichung Native 1 (TN1) were evaluated for reactions to RTSV and RTBV. TW16 is an NIL (BC(5)) resistant to RTD. RTBV was able to infect both TN1 and TW16 but the levels of RTBV were usually significantly lower in TW16 than in TN1. Infection of RTSV was confirmed in TN1 by a serological test but not in TW16. However, the global gene-expression pattern in an RTSV-resistant NIL (BC(6)), TW16-69, inoculated with RTSV indicated that RTSV can also infect the resistant NIL. Infection of RTSV in TW16 was later confirmed by reverse-transcription polymerase chain reaction but the level of RTSV was considerably lower in TW16 than in TN1. Examination for virus accumulation in another NIL (BC(6)), TW16-1029, indicated that all plants of TW16-1029 were resistant to RTSV, whereas the resistance to RTBV and symptom severity were segregating among the individual plants of TW16-1029. Collectively, these results suggest that RTD resistance of Utri Merah involves suppression of interacting RTSV and RTBV but the suppression trait for RTSV and for RTBV is inherited separately.

Downstream promoter sequence of an Indian isolate of Rice tungro bacilliform virus alters tissue-specific expression in host rice and acts differentially in heterologous system.

An Indian isolate of Rice tungro bacilliform virus from West Bengal (RTBV-WB) showed significant nucleotide differences in its putative promoter region when compared with a previously characterized isolate from Philippines. The transcription start site of RTBV-WB was mapped followed by assessing the activity and tissue-specificity of the full-length (FL) promoter (-231 to +645) and several of its upstream and downstream deletions by studying the expression of beta-Glucuronidase (GUS) reporter gene in transgenic rice (Oryza sativa L. subsp. indica) plants at various stages of development. In addition to the expected vascular-specific expression pattern, studied by histochemical staining, GUS enzymatic assay and northern and RT-PCR analysis, two novel patterns were revealed in some of the downstream deleted versions; a non-expressing type, representing no expression at any stage in any tissue and constitutive type, representing constitutive expression at all stages in most tissues. This indicated the presence of previously unreported positive and negative cis-regulatory elements in the downstream region. The negative element and a putative enhancer region in the upstream region specifically bound to rice nuclear proteins in vitro. The FL and its deletion derivatives were also active in heterologous systems like tobacco (Nicotiana tabacum) and wheat (Triticum durum). Expression patterns in tobacco were different from those observed in rice suggesting the importance of upstream elements in those systems and host-specific regulation of the promoter in diverse organisms. Thus, the RTBV-WB FL promoter and its derivatives contain an array of cis-elements, which control constitutive or tissue- and development-specific gene expression in a combinatorial fashion.

Role of the C-terminal domains of rice (Oryza sativa L.) bZIP proteins RF2a and RF2b in regulating transcription.

Rice (Oryza sativa L.) transcription factors RF2a and RF2b are bZIP (basic leucine zipper) proteins that interact with, and activate transcription from the RTBV (rice tungro bacilliform virus) promoter. Here we characterize the C-terminal domains of RF2a and RF2b: these domains are rich in glutamine and proline/glutamine, respectively. Affinity pull-down assays demonstrated that the C-terminal domains of RF2a and RF2b can associate to form either homodimers or heterodimers; however, they do not interact with other domains of RF2a or RF2b. Results of in vitro transcription assays using a rice whole-cell extract demonstrate that the C-terminal domains of both RF2a and RF2b activate transcription from the RTBV promoter. In addition, dimerization of the RF2a C-terminal domain is involved in regulating the transcription activation function of RF2a. The predicted helical region within the RF2a C-terminal glutamine-rich domain was determined to be involved in inter-molecular dimerization, and contributed to the regulatory functions of RF2a in these assays.