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.

Further support of genetic conservation in Indian isolates of Rice tungro bacilliform virus by sequence analysis of an isolate from North-Western India.

The genomic sequence of an isolate of Rice tungro bacilliform virus (RTBV), collected from the state of Punjab (Pb), a non-endemic tungro region from North-Western India was determined. In silico comparison of the 7931-bp sequence with isolates from Southeast Asia and the three previously characterized Indian isolates, revealed not only similar genome size to other Indian isolates but also high degree of homology both at nucleotide (>93 %) and amino acid (>96 %) levels among them. On the other hand, like the other Indian isolates, RTBV-Pb showed much lower nucleotide (<87 %) and amino acid (<90 % in most of the open reading frames) identities with the Southeast Asian isolates owing to several nucleotide substitutions and indels. In-depth annotation comparisons reinforce the hypothesis that Indian isolates of RTBV have diverged sufficiently from the Southeast Asian ones to form a separate group.

The molecular diversity and evolution of Rice tungro bacilliform virus from Indian perspective.

Rice tungro disease is caused by a combination of two viruses: Rice tungro spherical virus and Rice tungro bacilliform virus (RTBV). This study was performed with the objective to decipher the molecular variability and evolution of RTBV isolates present in the tungro-affected states of Indian subcontinent. Phylogenetic analysis based on ORF-I, ORF-II, and ORF-IV sequences showed distinct divergence of Indian RTBV isolates into two groups; one consisted isolates from Hyderabad (Andhra Pradesh), Cuttack (Orissa), and Puducherry and another from West Bengal, Chinsura West Bengal, and Kanyakumari (Tamil Nadu). The results obtained from phylogenetic analysis were further supported with the single nucleotide polymorphisms (SNPs), insertion and deletion (INDELs) and evolutionary distance analysis. In addition, sequence difference count matrix revealed a maximum of 56 (ORF-I), 13 (ORF-II) and 73 (ORF-IV) nucleotides differences among all the Indian RTBV isolates taken in this study. However, at the protein level these differences were not significant as revealed by K (a)/K (s) ratio calculation. Sequence identity at nucleotide and amino acid level was 92-100 % (ORF-I), 96-100 % (ORF-II), 94-100 % (ORF-IV) and 86-100 % (ORF-I), 98-100 % (ORF-II) and 95-100 % (ORF-IV), respectively, among Indian isolates of RTBV. The divergence of RTBV isolates into two independent clusters of Indian and non-Indian was shown with the help of the data obtained from phylogeny, SNPs, and INDELs, evolutionary distance analysis, and conserved motifs analysis. The important role of ORF-I and ORF-IV in RTBV diversification and adaptation to different rice growing regions is also discussed.

Development of SYBR Green I based real-time PCR assays for quantitative detection of Rice tungro bacilliform virus and Rice tungro spherical virus.

Rice tungro disease, caused by simultaneous infection of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV), is an important cause of reduced rice harvests in South and Southeast Asia. Although various biological, serological and molecular techniques have been reported previously for the detection of RTBV and RTSV, a method that determines accurately the exact viral load in a tungro affected plant is still not available. The present study describes a method for the absolute quantitation of RTBV and RTSV using SYBR Green I based real-time PCR. The number of copies of RTBV DNA and RTSV RNA present in a tungro affected rice plant at two different time points after inoculation was determined. The sensitivity of real-time PCR based detection was found 10(3)- and 10(5)-folds higher than dot-blot hybridization and standard PCR assays respectively. In addition, the method was used for the simultaneous detection of RTBV and RTSV in a single reaction on the basis of melt curve analysis.

Analysis of the complete DNA sequence of rice tungro bacilliform virus from southern India indicates it to be a product of recombination.

The complete nucleotide sequence of an isolate of rice tungro bacilliform virus (RTBV), collected from Kanyakumari, India, where RTBV was reported recently for the first time, has been analyzed. Sequence comparison revealed that the RTBV isolate from Kanyakumari (RTBV-KK) has a high degree of identity to the two previously reported RTBV sequences from India, RTBV-AP and RTBV-WB, which had been collected from field locations about 10 years ago and 1000-2000 km away from the collection site of RTBV-KK. Most of the sequence domains reported previously in other RTBV isolates were found to be conserved in RTBV-KK. Closer inspection revealed RTBV-KK to be a possible recombinant between RTBV-AP and RTBV-WB in the genomic region encompassing the coat protein gene.

Phylogenetic analysis of Rice tungro bacilliform virus ORFs revealed strong correlation between evolution and geographical distribution.

A new isolate of Rice tungro bacilliform virus (RTBV) was collected from Chinsura, West Bengal, India. The full genome was sequenced and deposited to GenBank designating the new one as Chinsura isolate. The four open reading frames (ORFs) of the new isolate were compared with those of previously reported 'South-east Asian' (SEA) and 'South Asian' (SA) isolates emphasizing the ORF3, which is the largest and functionally most important gene of RTBV. In the ORFs, Chinsura isolate shared 90.0-100.0% identity at amino acid level with SA isolates, but only 58.76-88.63% identity with SEA isolates for the same. Similarly, the amino acid identity of ORFs between SEA and SA isolates ranged from 58.77 to 88.64, whereas within each group the corresponding value was >96.0%. The phylogenetic analysis based on nucleotide and amino acid sequences of each ORF made two broad clusters of SEA- and SA-types including Chinsura isolate within SA cluster. Moreover, the relative positions and length of functional domains corresponding to movement protein (MP), coat protein (CP), aspartate protease (PR) and reverse transcriptase/ribonuclease H (RT/RNase H) of ORF3 of Chinsura isolate were completely identical with SA isolates. The clustering pattern indicated strong influence of geographical habitat on genomic evolution. Comparison of ORF3 among all the isolates revealed major variations at non-functional regions in between the functional domains and at the hypervariable 3'-terminal end of ORF3, while PR appeared to have evolved differentially in SA isolates expecting further characterization.

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.

RNA-interference in rice against Rice tungro bacilliform virus results in its decreased accumulation in inoculated rice plants.

Rice tungro is a viral disease seriously affecting rice production in South and Southeast Asia. Tungro is caused by the simultaneous infection in rice of Rice tungro bacilliform virus (RTBV), a double-stranded DNA virus and Rice tungro spherical virus (RTSV), a single-stranded RNA virus. To apply the concept of RNA-interference (RNAi) for the control of RTBV infection, transgenic rice plants expressing DNA encoding ORF IV of RTBV, both in sense as well as in anti-sense orientation, resulting in the formation of double-stranded (ds) RNA, were raised. RNA blot analysis of two representative lines indicated specific degradation of the transgene transcripts and the accumulation of small molecular weight RNA, a hallmark for RNA-interference. In the two transgenic lines expressing ds-RNA, different resistance responses were observed against RTBV. In one of the above lines (RTBV-O-Ds1), there was an initial rapid buildup of RTBV levels following inoculation, comparable to that of untransformed controls, followed by a sharp reduction, resulting in approximately 50-fold lower viral titers, whereas the untransformed controls maintained high levels of the virus till 40 days post-inoculation (dpi). In RTBV-O-Ds2, RTBV DNA levels gradually rose from an initial low to almost 60% levels of the control by 40 dpi. Line RTBV-O-Ds1 showed symptoms of tungro similar to the untransformed control lines, whereas line RTBV-O-Ds2 showed extremely mild symptoms.