P4 protein of an Indian isolate of rice tungro bacilliform virus modulates gene silencing.

Plant hosts and their viral pathogens are engaged in a constant cycle of defense and counter-defense as part of a molecular arms race, principal among them being the plant RNAi defense and the viral RNAi suppressor counter-defense. Rice tungro bacilliform virus (RTBV), member of the family Caulimoviridae, genus Tungrovirus, species Tungrovirus oryzae, infects rice in South- and Southeast Asia and causes severe symptoms of stunting, yellow-orange discoloration and twisting of leaf tips. To better understand the possible counter-defensive roles of RTBV against the host RNAi defense system, we explored the ability of the P4 protein of an Indian isolate of RTBV to act as a possible modulator of RNAi. Using a transient silencing and silencing suppression assay in Nicotiana benthamiana, we show that P4 not only displays an RNAi suppressor function, but also potentially enhances RNAi. The results also suggests that the N-terminal 168 amino acid residues of P4 are sufficient to maintain RNAi suppressor activity. Taken together with the earlier reports this work strengthens the view that the P4 protein carries out RNAi suppressor and a potential RNAi enhancer function.

Silencing suppressor protein PRT of rice tungro bacilliform virus interacts with the plant RNA silencing-related protein SGS3.

BACKGROUND: Rice tungro bacilliform virus (RTBV) is a double stranded DNA containing virus which causes the devastating tungro disease of rice in association with an RNA virus, rice tungro spherical virus. RNA silencing is an evolutionarily conserved antiviral defence pathway in plants as well as in several classes of higher organisms. Many viruses, in turn, encode proteins which are termed Viral Suppressor of RNA Silencing (VSR) because they downregulate or suppress RNA silencing. RESULTS: Using an RNA silencing suppressor assay we show that RTBV protease (PRT) acts as a mild VSR. A truncated version of PRT gene abolished the silencing suppression activity. We also show in planta interaction of PRT with the SGS3 protein of Solanum tuberosum and Arabidopsis thaliana using bimolecular fluorescence complementation assay (BIFC). Transient expression of PRT in Nicotiana benthamiana caused an increased accumulation of the begomovirus Sri Lankan cassava mosaic virus (SLCMV) DNA-A, which indicated a virulence function imparted on an unrelated virus. CONCLUSION: The finding supports the idea that PRT acts as suppressor of RNA silencing and this action may be mediated by its interaction with SGS3.

RTBV-Based VIGS Vector for Functional Genomics in Rice: Methodology, Advances, Challenges, and Future Implications.

The availability of protocols for virus-induced gene silencing (VIGS) in rice has opened up an important channel for the elucidation of gene functions in this important crop plant. Here, we present an updated protocol of a VIGS system based on Rice tungro bacilliform virus (RTBV) for gene silencing in rice. We present complete updated protocols for VIGS in rice, compare the system with other existing ones for monocots, identify some of the challenges faced by this system and discuss ways in which the vector could be improved for better silencing efficiency.

Analysis of codon usage patterns and influencing factors in rice tungro bacilliform virus.

Rice tungro bacilliform virus (RTBV) belongs to genus Tungrovirus within the family Caulimoviridae harbors circular double-stranded DNA (dsDNA). Rice tungro disease (RTD) caused by RTBV, responsible for severe rice yield losses in South and Southeast Asia. Here, we performed a systematic evolutionary and codon usage bias (CUB) analysis of RTBV genome sequences. We analysed different bioinformatics techniques to calculate the nucleotide compositions, the relative synonymous codon usage (RSCU), and other indices. The results indicated slightly or low codon usage bias in RTBV isolates. Mutation and natural selection pressures have equally contributed to this low codon usage bias. Additionally, multiple factors such as host, geographical distribution also affect codon usage patterns in RTBV genomes. RSCU analysis revealed that RTBV shows mutation bias and prefers A and U ended codons to code amino acids. Codon usage patterns of RTBV were also found to be influenced by its host. This indicates that RTBV have evolved codon usage patterns that are specific to its host. The findings from this study are expected to increase our understanding of factors leading to viral evolution and fitness with respect to hosts and the environment.

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.

Ancient Endogenous Pararetroviruses in Oryza Genomes Provide Insights into the Heterogeneity of Viral Gene Macroevolution.

Endogenous viral sequences in eukaryotic genomes, such as those derived from plant pararetroviruses (PRVs), can serve as genomic fossils to study viral macroevolution. Many aspects of viral evolutionary rates are heterogeneous, including substitution rate differences between genes. However, the evolutionary dynamics of this viral gene rate heterogeneity (GRH) have been rarely examined. Characterizing such GRH may help to elucidate viral adaptive evolution. In this study, based on robust phylogenetic analysis, we determined an ancient endogenous PRV group in Oryza genomes in the range of being 2.41-15.00 Myr old. We subsequently used this ancient endogenous PRV group and three younger groups to estimate the GRH of PRVs. Long-term substitution rates for the most conserved gene and a divergent gene were 2.69 × 10-8 to 8.07 × 10-8 and 4.72 × 10-8 to 1.42 × 10-7 substitutions/site/year, respectively. On the basis of a direct comparison, a long-term GRH of 1.83-fold was identified between these two genes, which is unexpectedly low and lower than the short-term GRH (>3.40-fold) of PRVs calculated using published data. The lower long-term GRH of PRVs was due to the slightly faster rate decay of divergent genes than of conserved genes during evolution. To the best of our knowledge, we quantified for the first time the long-term GRH of viral genes using paleovirological analyses, and proposed that the GRH of PRVs might be heterogeneous on time scales (time-dependent GRH). Our findings provide special insights into viral gene macroevolution and should encourage a more detailed examination of the viral GRH.

Small RNA-based interactions between rice and the viruses which cause the tungro disease.

Rice tungro disease is caused by a complex of two viruses, Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). To examine the RNAi-based defence response in rice during tungro disease, we characterized the virus-derived small RNAs and miRNAs by Deep Sequencing. We found that, while 21 nt/22 nt (nucleotide) siRNAs are predominantly produced in a continuous, overlapping and asymmetrical manner from RTBV, siRNA accumulation from RTSV were negligible. Additionally, 54 previously known miRNAs from rice, predicted to be regulating genes involved in plant defence, hormone signaling and developmental pathways were differentially expressed in the infected samples, compared to the healthy ones. This is the first study of sRNA profile of tungro virus complex from infected rice plants. The biased response of the host antiviral machinery against the two viruses and the differentially-expressed miRNAs are novel observations, which entail further studies.

Simultaneous resistance against the two viruses causing rice tungro disease using RNA interference.

Rice tungro is the most important viral disease affecting rice in South and Southeast Asia, caused by two viruses rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV). Transgenic resistance using RNA-interference (RNAi) has been reported individually against RTBV and RTSV earlier. Here we report the development of transgenic rice plants expressing RNAi against both RTBV and RTSV simultaneously. A DNA construct carrying 300 bp of RTBV DNA and 300 bp of RTSV cDNA were cloned as the two arms in hairpin orientation in a binary plasmid background to generate RNAi against both viruses simultaneously. Transgenic rice plants were raised using the above construct and their resistance against RTBV and RTSV was quantified at the T1 plants. Levels of both the viral nucleic acids showed a fall of 100- to 500-fold in the above plants, compared with the non-transgenic controls, coupled with the amelioration of stunting. The transgenic plants also retained higher chlorophyll levels than the control non-transgenic plants after infection with RTBV and RTSV. Small RNA analysis of virus inoculated transgenic plants indicated the presence of 21 nt and 22 nt siRNAs specific to RTBV and RTSV. The evidence points towards an active RNAi mechanism leading to resistance against the tungro viruses in the plants analysed.

Phenotyping of VIGS-mediated gene silencing in rice using a vector derived from a DNA virus.

KEY MESSAGE: Target genes in rice can be optimally silenced if inserted in antisense or hairpin orientation in the RTBV-derived VIGS vector and plants grown at 28 °C and 80% humidity after inoculation. Virus induced gene silencing (VIGS) is a method used to transiently silence genes in dicot as well as monocot plants. For the important monocot species rice, the Rice tungro bacilliform virus (RTBV)-derived VIGS system (RTBV-VIGS), which uses agroinoculation to initiate silencing, has not been standardized for optimal use. Here, using RTBV-VIGS, three sets of conditions were tested to achieve optimal silencing of the rice marker gene phytoene desaturase (pds). The effect of orientation of the insert in the RTBV-VIGS plasmid (sense, antisense and hairpin) on the silencing of the target gene was then evaluated using rice magnesium chelatase subunit H (chlH). Finally, the rice Xa21 gene, conferring resistance against bacterial leaf blight disease (BLB) was silenced using RTBV-VIGS system. In each case, real-time PCR-based assessment indicated approximately 40-80% fall in the accumulation levels of the transcripts of pds, chlH and Xa21. In the case of pds, the appearance of white streaks in the emerging leaves, and for chlH, chlorophyll levels and F v/F m ratio were assessed as phenotypes for silencing. For Xa21, the resistance levels to BLB were assessed by measuring the lesion length and the percent diseased areas of leaves, following challenge inoculation with Xanthomonas oryzae. In each case, the RTBV-MVIGS system gave rise to a discernible phenotype indicating the silencing of the respective target gene using condition III (temperature 28 °C, humidity 80% and 1 mM MES and 20 µM acetosyringone in secondary agrobacterium culture), which revealed the robustness of this gene silencing system for rice.

Virus-induced gene silencing (VIGS) for functional genomics in rice using Rice tungro bacilliform virus (RTBV) as a vector.

The large-scale functional analysis of genes in plants depends heavily on robust techniques for gene silencing. Virus-induced gene silencing (VIGS) is a transient gene silencing method for plants, triggered by the inoculation of a modified viral vector carrying a fragment of the gene targeted for silencing. Here we describe a VIGS protocol for rice, based on the Rice tungro bacilliform virus (RTBV, a DNA virus). We present an updated and detailed protocol for silencing of the gene encoding Phytoene desaturase in rice, using the RTBV-VIGS system.

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.

Virus-induced gene silencing for rice using agroinoculation.

Virus-induced gene silencing (VIGS) is a reverse genetics technique that is based on the RNA-mediated defense against viruses in plants. VIGS is a method of gene knockdown triggered by a replicating viral nucleic acid engineered to carry a host gene to be silenced. While there are a number of excellent VIGS vectors available for dicots, only a few are available for monocots. Here, we describe the detailed method of the use of a newly developed VIGS vector for rice, based on the rice-infecting Rice tungro bacilliform virus, a pararetrovirus with dsDNA genome. Using a method based on Agrobacterium-mediated injection of the VIGS construct at the meristematic region of young rice plants, silencing of target genes can be achieved and the silenced phenotype can be visualized in 3 weeks.

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.

Evolutionary force of AT-rich repeats to trap genomic and episomal DNAs into the rice genome: lessons from endogenous pararetrovirus.

In plant genomes, the incorporation of DNA segments is not a common method of artificial gene transfer. Nevertheless, various segments of pararetroviruses have been found in plant genomes in recent decades. The rice genome contains a number of segments of endogenous rice tungro bacilliform virus-like sequences (ERTBVs), many of which are present between AT dinucleotide repeats (ATrs). Comparison of genomic sequences between two closely related rice subspecies, japonica and indica, allowed us to verify the preferential insertion of ERTBVs into ATrs. In addition to ERTBVs, the comparative analyses showed that ATrs occasionally incorporate repeat sequences including transposable elements, and a wide range of other sequences. Besides the known genomic sequences, the insertion sequences also represented DNAs of unclear origins together with ERTBVs, suggesting that ATrs have integrated episomal DNAs that would have been suspended in the nucleus. Such insertion DNAs might be trapped by ATrs in the genome in a host-dependent manner. Conversely, other simple mono- and dinucleotide sequence repeats (SSR) were less frequently involved in insertion events relative to ATrs. Therefore, ATrs could be regarded as hot spots of double-strand breaks that induce non-homologous end joining. The insertions within ATrs occasionally generated new gene-related sequences or involved structural modifications of existing genes. Likewise, in a comparison between Arabidopsis thaliana and Arabidopsis lyrata, the insertions preferred ATrs to other SSRs. Therefore ATrs in plant genomes could be considered as genomic dumping sites that have trapped various DNA molecules and may have exerted a powerful evolutionary force.

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.

Short ORF-dependent ribosome shunting operates in an RNA picorna-like virus and a DNA pararetrovirus that cause rice tungro disease.

Rice tungro disease is caused by synergistic interaction of an RNA picorna-like virus Rice tungro spherical virus (RTSV) and a DNA pararetrovirus Rice tungro bacilliform virus (RTBV). It is spread by insects owing to an RTSV-encoded transmission factor. RTBV has evolved a ribosome shunt mechanism to initiate translation of its pregenomic RNA having a long and highly structured leader. We found that a long leader of RTSV genomic RNA remarkably resembles the RTBV leader: both contain several short ORFs (sORFs) and potentially fold into a large stem-loop structure with the first sORF terminating in front of the stem basal helix. Using translation assays in rice protoplasts and wheat germ extracts, we show that, like in RTBV, both initiation and proper termination of the first sORF translation in front of the stem are required for shunt-mediated translation of a reporter ORF placed downstream of the RTSV leader. The base pairing that forms the basal helix is required for shunting, but its sequence can be varied. Shunt efficiency in RTSV is lower than in RTBV. But in addition to shunting the RTSV leader sequence allows relatively efficient linear ribosome migration, which also contributes to translation initiation downstream of the leader. We conclude that RTSV and RTBV have developed a similar, sORF-dependent shunt mechanism possibly to adapt to the host translation system and/or coordinate their life cycles. Given that sORF-dependent shunting also operates in a pararetrovirus Cauliflower mosaic virus and likely in other pararetroviruses that possess a conserved shunt configuration in their leaders it is tempting to propose that RTSV may have acquired shunt cis-elements from RTBV during their co-existence.

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.

The large intergenic region of Rice tungro bacilliform virus evolved differentially among geographically distinguished isolates.

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus. The large intergenic region (LIGR) of RTBV having a single transcriptional promoter produces more than genome length pregenomic RNA (pgRNA) which directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA. By computer-aided analysis of LIGR, the 11 RTBV isolates sequenced so far were compared with respect to structural organization of promoter and pgRNA 5'-leader. The results revealed only 74.90% identity at LIGR between 'Southeast Asian' (SEA) and 'South Asian' (SA) isolates of RTBV indicating considerable variation between two groups which was also reflected during analysis of promoter and leader sequence. The predicted promoter region of SA isolates exhibited major variations in terms of transcription start site and consensus sequences of cis motifs expecting further exploitation of promoter region of SA isolates. The reduced length of leader sequence along with less numbers and different arrangements of small open reading frames (sORFs) in case of SA isolates might have some alterations in the control of expression of ORF II and III between the two groups. In spite of these variations, the leader sequence of both SEA and SA type isolates showed formation of stable secondary or stem-loop structure having identical features for efficient translation. The conservation of sORF1 at seven nucleotides upstream of stable stem-loop, CU-rich sequence following the sORF1 stop codon and AU-rich shunt landing sequence immediately downstream of the secondary structure suggested conservation of ribosomal shunt mechanism in all RTBV isolates irrespective of their geographical distribution.

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.