Some biological and genomic properties of rice tungro bacilliform badnavirus and rice tungro spherical waikavirus from Nepal

A survey of rice fields during the main growing seasons in 81 locations from 21 districts of the Southern Terai region of Nepal indicated that rice tungro was primarily restricted to the Hardinath (Janakpur) and Parwanipur (Bara) regions. The tungro incidence in Hardinath ranged from 17% to 51% and in Parwanipur from 6% to 61% causing about 89% grain yield loss in Hardinath. Both rice tungro bacilliform badnavirus (RTBV) and rice tungro spherical picornavirus (RTSV) were found in tungro isolates collected from Hardinath and Parwanipur. These isolates were transmitted by Nephotettix virescens and leaf extracts reacted to antisera against RTBV and RTSV. In a dot blot hybridisation assay, leaf extracts of 12 weed species collected from the tungro-affected area in Hardinath and Parwanipur also reacted with RTBV DNA probes. On mass inoculation of 15 popular rice cultivars most became more than 50% infected and only cv. Radha 9 had low (22.2%) infection. RTBV DNA and the coat protein region of RTSV from the Hardinath isolate were cloned and partially characterised. A comparative analyses by restriction endonuclease digestion, cross hybridisation, the polymerase chain reaction and partial sequencing indicated that the Nepalese RTBV DNA clone and the cDNA clones of the RTSV RNA were more similar to the various tungro isolates from the Indian subcontinent than to those from the Philippines.     

Inheritance of tolerance to rice tungro bacilliform virus (RTBV) in rice (Oryza sativa L.)

Summary From a large number of rice varieties tested, no variety was identified as resistant to tungro bacilliform virus (RTBV). Only in Utri Merah was the RTBV multiplication restrictive, whereas other varieties such as Kataribhog and Pankhari 203 were identified as tolerant. These varieties were crossed with a susceptible variety. TN1, to study the inheritance of restrictive multiplication and tolerance to RTBV. After 3 weeks of inoculation with RTBV, F₁; F₂, and F₃ progenies were assessed by enzyme-linked immunosorbent assay (ELISA). The RTBV concentration in all F₁ populations was intermediate between parents. The frequency distribution of F₂ seedlings with various levels of RTBV concentration indicated that the RTBV tolerance is controlled by multiple genes. The RTBV concentrations in F₁ and F₂ progenies from the Utri Merah x TN1 cross revealed that restrictive multiplication of RTBV in Utri Merah is a polygenic character. The continuous variation observed in F₂ populations from crosses between tolerant varieties and Utri merah indicated no allelic relationships between tolerant and restrictive multiplication traits.Part of PhD thesis submitted by senior author to University of Kebangsaan Malaysia, Bangi     

Reconstruction of putative DNA virus from endogenous rice tungro bacilliform virus-like sequences in the rice genome: implications for integration and evolution

Background  

Plant genomes contain various kinds of repetitive sequences such as transposable elements, microsatellites, tandem repeats and virus-like sequences. Most of them, with the exception of virus-like sequences, do not allow us to trace their origins nor to follow the process of their integration into the host genome. Recent discoveries of virus-like sequences in plant genomes led us to set the objective of elucidating the origin of the repetitive sequences. Endogenous rice tungro bacilliform virus (RTBV)-like sequences (ERTBVs) have been found throughout the rice genome. Here, we reconstructed putative virus structures from RTBV-like sequences in the rice genome and characterized to understand evolutionary implication, integration manner and involvements of endogenous virus segments in the corresponding disease response.     

The rice tungro bacilliform virus gene II product interacts with the coat protein domain of the viral gene III polyprotein

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly.     

Physiology of rice tungro virus disease: Proline accumulation due to infection

Tungro virus infection stimulates proline accumulation in leaves of rice ( Oryza sativa L.), especially in a sensitive cultivar, Taichung Native 1. Disease-induced proline accumulation increases with the severity of the disease. Proline also accumulates in senescing, detached healthy rice leaves. The magnitude of proline accumulation in these leaves was further accentuated by ABA and retarded by kinetin. In the absence of drought stress, virus infection induces severe symptoms (stunting) in a drought tolerant cultivar (Lalnakanda 41) when compared to cultivars with intermediate (MW 10) and high sensitivity (Cauvery) to drought. Thus tungro virus mimics water stress in inducing proline accumulation in rice leaves. In both cases a common factor, ABA, may mediate proline accumulation. In drought stress, proline accumulation is associated with tolerance, while in virus stress, proline accumulation is connected with sensitivity. It is, therefore, clear that proline cannot always act to relieve physiological stress.     

Comparative transmission of, and varietal reaction to, three isolates of rice tungro virus disease

Comparative transmission by leafhoppers of three tungro isolates obtained from the Philippines, India and Malaysia, and of an infectious clone of the Philippine isolate of rice tungro bacilliform virus (RTBV) by agroinoculation, was conducted on 12 rice cultivars. The symptoms, including height of inoculated plants were recorded and the efficiency of RTBV and rice tungro spherical virus (RTSV) transmission was determined by enzyme-linked immunosorbent assay. In most cases, the reduction of height and leaf symptoms of plants infected with RTBV and/or RTSV by the three isolates were similar in any given cultivar. On cultivar ASD 7 , the Malaysian isolate showed more severe yellow orange leaf discolouration symptoms than the Indian isolate which in turn had more severe leaf discolouration than the Philippine isolate. On the other hand, cultivars ASD 7 and Ptb 18 produced the most severe yellow orange leaf discolouration when agroinoculated with an infectious RTBV clone of the Philippine isolate. There was some variation in the transmission profile of the two tungro viruses among the three isolates. However, there was no one clear set of characteristics by which one could use cultivars to distinguish isolates. The amount of viral DNA in agroinfected plants of cultivars Utri merah, Balimau putih, Utri Rajapan and ARC 11554 was low, while the amount was high in cultivars TN1, ASD7, Ptb 18 and TKM 6. There was high correlation between the amount of viral coat protein by ELISA and viral nucleic acid by DNA hybridisation on 10 agroinoculated rice cultivars; this might indicate that similar proportions of the total RTBV DNA are encapsidated in each cultivar.     

Inheritance studies of SSR and ISSR molecular markers and phylogenetic relationship of rice genotypes resistant to tungro virus

Multivariate analyses were performed using 13 morphological traits and 13 molecular markers (10 SSRs and three ISSRs) to assess the phylogenetic relationship among tungro resistant genotypes. For morphological traits, the genotypes were grouped into six clusters, according to D² statistic and Canonical vector analysis. Plant height, days to flowering, days to maturity, panicle length, number of spikelet per panicle, number of unfilled grain per panicle and yield were important contributors to genetic divergence in 14 rice genotypes. Based on Nei's genetic distance for molecular studies, seven clusters were formed among the tungro resistant and susceptible genotypes. Mantel's test revealed a significant correlation (r = 0.834*) between the morphological and molecular data. To develop high yielding tungro resistant varieties based on both morphological and molecular analyses, crosses could be made with susceptible (BR10 and BR11) genotypes with low yielding but highly resistant genotypes, Sonahidemota, Kumragoir, Nakuchimota, Khaiyamota, Khairymota and Kachamota. The chi-square analysis for seven alleles (RM11, RM17, RM20, RM23, RM80, RM108 and RM531) of SSR and five loci (RY1, MR1, MR2, MR4 and GF5) of three ISSR markers in F₂ population of cross, BR11 × Sonahidemota, showed a good fit to the expected segregation ratio (1:2:1) for a single gene model.     

Sequence-specific binding of simian virus 40 A protein to nonorigin and cellular DNA.

The simian virus 40 A protein (T antigen) recognized and bound to the consensus sequence 5'-GAGGC-3' in DNA from many sources. Sequence-specific binding to single pentanucleotides in randomly chosen DNA predominated over binding to nonspecific sequences. The asymmetric orientation of protein bound to nonorigin recognition sequences also resembled that of protein bound to the origin region of simian virus 40 DNA. Sequence variations in the DNA adjacent to single pentanucleotides influenced binding affinities even though methylation interference and protection studies did not reveal specific interactions outside of pentanucleotides. Thus, potential locations of A protein bound to any DNA can be predicted although the determinants of binding affinity are not yet understood. Sequence-specific binding of A protein to cellular DNA would provide a mechanism for specific alterations of host gene expression that facilitate viral function.     

Prevention of rice tungro virus disease and control of the vector with granular insecticides

Thirteen granular insecticides were screened under glasshouse conditions for their ability to kill the vector, Nephotettix virescens, and to prevent tungro virus infection. Carbofuran, bendiocarb, isoprocarb, BPMC, disulfoton, mephosfolan and phorate caused 100% vector mortality, but only the first four reduced virus infection to an acceptable level; the remainder killing the vector too slowly to prevent virus transmission. Seven of these compounds were evaluated in the field on two test cultivars, Traichung Native 1 and Pankaj. Carbofuran, isoprocarb and BPMC gave acceptable control of the disease on both cultivars and diazinon on cv. Pankaj only.     

Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region

Latently infected B lymphocytes continuously express an Epstein-Barr Virus nuclear antigen (EBNA-1) required in trans for maintenance of the plasmid state of the EBV genome. Filter binding assays and DNAase I footprinting analyses revealed that the carboxy-terminal domain of EBNA-1 protects binding sites at three different loci in the 172,000 bp EBV genome. Two of these loci correspond to essential elements within an 1800 bp segment defined as the minimal region required for plasmid maintenance (ori-P). Binding to each of 20 X 30 bp tandem repeats in the "sink" locus protects 25 bp centered over a 12 bp palindromic consensus sequence TAGCATATGCTA. The nearby dyad symmetry "origin" locus contains two 46 bp protected regions each encompassing two paired core binding sites. The demonstration of sequence-specific binding at multiple loci suggests that EBNA-1 has pleiotropic functions, which may include control of copy number and segregation of the EBV plasmids as well as initiation of replication.     

Phosphorylation-mediated regulation of a rice ABA responsive element binding factor

OREB1 is a rice ABRE binding factor characterized by the presence of multiple highly-conserved phosphorylation domains (C1, C2, C3, and C4) and two kinase recognition motifs, RXXS/T and S/TXXE/D, within different functional domains. An in vitro kinase assay showed that OREB1 is phosphorylated not only by the SnRK2 kinase, but also by other Ser/Thr protein kinases, such as CaMKII, CKII, and SnRK3. Furthermore, the N-terminal phosphorylation domain C1 was found to be differentially phosphorylated by the SnRK2/SnRK3 kinase and by hyperosmotic/cold stress, suggesting that the C1 domain may function in decoding different signals. The phosphorylation-mediated regulation of OREB1 activity was investigated through mutation of the SnRK2 recognition motif RXXS/T within each phosphorylation module. OREB1 contains a crucial nine-amino acid transactivation domain located near the phosphorylation module C1. Deletion of the C1 domain increased OREB1 activity, whereas mutation of Ser 44, Ser 45, and Ser 48 of the C1 domain to aspartates decreased OREB1 activity. In the C2 domain, a double mutation of Ser 118 and Ser 120 to alanines suppressed OREB1 activity. These findings strongly suggest that selective phosphorylation of the C1 or C2 modules may positively or negatively regulate OREB1 transactivation. In addition, mutation of Ser 385 of the C4 domain to alanines completely abolished the interaction between OREB1 and a rice 14-3-3 protein, GF14d, suggesting that SnRK2-mediated phosphorylation may regulate this interaction. These results indicate that phosphorylation domains of OREB1 are not functionally redundant and regulate at least three different functions, including transactivation activity, DNA binding, and protein interactions. The multisite phosphorylation of OREB1 is likely a key for the fine control of its activity and signal integration in the complex stress signaling network of plant cells.     

Presequence binding factor-dependent and -independent import of proteins into mitochondria.

A cytosolic protein factor(s) is involved in the import of precursor proteins into mitochondria. PBF (presequence binding factor) is a protein factor which binds to the precursor form (pOTC) of rat ornithine carbamoyltransferase (OTC) but not to the mature OTC, and is required for the mitochondrial import of pOTC. The precursors for aspartate aminotransferase and malate dehydrogenase as well as pOTC synthesized in a reticulocyte lysate were efficiently imported into the mitochondria. However, the precursors synthesized in the lysate depleted for PBF by treatment with pOTC-Sepharose were not imported. Readdition of the purified PBF to the depleted lysate fully restored the import. pOTC synthesized in the untreated lysate sedimented as a complex with a broad peak of around 9 S, whereas pOTC synthesized in the PBF-depleted lysate sedimented at an expected position of monomer (2.5 S). When the purified PBF was readded to the depleted lysate, pOTC sedimented as a complex of about 7 S. In contrast to most mitochondrial proteins, rat 3-oxoacyl-CoA thiolase is synthesized with no cleavable presequence and an NH2-terminal portion of the mature protein functions as a mitochondrial import signal. The thiolase synthesized in the PBF-depleted lysate could be efficiently imported into the mitochondria, and readdition of PBF had little effect on the import. The thiolase synthesized in the untreated, the PBF-depleted, or the PBF-readded lysate sedimented at an expected position of monomer (2.5 S). These observations provide support for the existence of PBF-dependent and -independent pathways of mitochondrial protein import.