9. Iftikhar S., Shahid A.A., Javed S., Nasir I.A., Tabassum B., Haider M.S. Essential Oils and Latices as Novel Antiviral Agent Against Potato Leaf Roll Virus and Analysis of Their Phytochemical Constituents Responsible for Antiviral Activity.Journal of Agricultural Science., 2013; 5(7): 167-188. doi:10.5539/jas.v5n7p167
Sequence alignment and phylogenies
To confirm the presence of PVY and PVV in the NGS analyzed samples, isolates of PYV and PVV were ampli- fied with the degenerated primers POT1 and POT2 (Tab. 1). Amplicons were cloned and sent to Macrogen (Seoul, Korea) for Sanger sequencing. Molecular Evolutionary Ge- netics Analysis (MEGA) software (version 4.0) (Tamura et al., 2007) was used for bioinformatics analysis and Clustal W for aligning sequences (Thompson et al., 1994). The evolutionary distances were computed using the maximum composite likelihood method (Tamura et al., 2004), units being the number of base substitutions per site. Evolutio- nary history was inferred using the Neighbour-Joining method (Saitou and Nei, 1987). Statistical confidence was evaluated using a bootstrap test with 1,000 replicates (Fel- senstein, 1985). Pea seed-borne mosaicvirus (PSbMV) was used as the outgroup.
The frequent linkage of resistance gene analogues (RGAs) to known resistance loci has been exploited by many groups to identify genetic markers linked to known R-genes. With the completion of the sequencing of the Arabidopsis and Rice genomes, numerous in silico studies and laboratory analysis have been conducted in order to identify and assess RGAs. The broad strategy in these studies is to search for conserved motifs with the aim of identifying new markers for isolating candidate resistance loci (Chelkowski and Koczyk, 2003; Koczyk and Chelkowski, 2003; Meyers et al., 2003). In species such as barley (Madsen et al., 2003), soybean (Kanazin et al., 1996), potato (Leister et al., 1996) and rice (Leister et al., 1998), genomic locations of isolated RGAs have been determined using linkage maps and this location of an RGA correlated with the location known resistance loci. In species where no detailed genetic maps are available, RGAs can be a useful tool in the preliminary localisation of candidate R genes and thus facilitate the use of the respective RGA for the development of assays for marker assisted selection. In crops, such as the chestnut rose, Xu and colleagues (2005) have successfully used RGAs to characterize and isolate markers linked to resistance to powdery mildew. Where an RGA co-segregates perfectly, it is possible that the isolated fragment represents a portion of a functional gene. In Arabidopsis thaliana, an RGA was found to be a part of the RPP5 gene which confers resistance to powdery mildew (Aarts et al., 1998a).
to the cap can hinder the binding of ribosomes to the mRNA (13). In mutants e and g, the calculated stability of hp1, which is located 17 nucleotides downstream of the cap, is 2 13.5 kcal/mol. Since even a hairpin with a stability of 2 30 kcal/mol does not hinder the ribosome once it has started scanning the messenger (12), it is more likely that in our case the stabilized hairpin interferes with the binding of the ribosome to the cap structure. A single point mutation in this closed hairpin 1 strongly reduces the stability, thereby restoring translation. In mutant f, for example, which is identical to revertant e1 (found in five cases), the hairpin has a stability of 2 7.1 kcal/mol and the efficiency of translation is restored. The same holds for revertant e3 (data not shown). The stability of the hairpin per se, however, is not the only prerequisite for a proper function of hp1. For both an A z U and a G z C pair, there are five point mutations possible that lead to a mismatch (G z U pairs not taken into account). If we do not take into account the base pairs at the extremities of the stem of hp1, for the remaining 7 bp there are 35 different point mutations that would strongly reduce the stability of the hairpin, 11 of which introduce a C-C or C-A mismatch. Interestingly, the eight revertants that we isolated, starting from mutant e, all contain a C-C or C-A mismatch at the position where the wild-type hairpin has a C-C mismatch. This strongly emphasizes the necessity of these pro- tonatable mismatches in this hairpin. This is, to the best of our knowledge, the first example of a functionally important role of such a mismatch in viral RNA. The mismatch at the top of the internal loop appears to be the most important element, as shown by the sequence of the revertants and the virulence of mutant f. Revertant e2 has a phenotype indistinguishable from that of the wild-type virus, although in this case the C-C and C-A mismatches are adjacent. In this respect, revertant e2 is more similar to the hairpins found in the RNA of the tymovi- ruses eggplant mosaicvirus and onionis yellowmosaicvirus (9), where symmetric internal loops of four cytosines are present. It is remarkable that mutant h, which contains only a single C-C mismatch in the entire 5 9 UTR, does not lead to revertants, even after five cycles of propagation, although this mutant causes less severe symptoms than the wild-type virus does. A reversion or another spontaneous mutation in a viral RNA will take place only if the mutant replicates at a certain level, allowing the replicase to make mistakes, and will only be observed if the new mutants are more viable than the original one. The fact that mutant g does not produce revertants may be ascribed to the first reason, and the absence of revertants of mutant h can be ascribed to the second reason. The fact that a protonatable mismatch at the bottom of the internal loop of
The NTPase activity of NTPB-helicase proteins is typically stimulated by nucleic acids (12, 19, 37, 38). To determine whether this is also true of the TYMV protein, three different RNAs were tested for the capacity to enhance the ATPase activity of GST-NTPB. Addition of poly(A) or poly(U) or of a TYMV RNA fragment at 0.2 mg/ml stimulated the ATPase activity about twofold during the first 10 min of the reaction. This effect disappeared in the presence of 200 mM NaCl (data not shown), suggesting that nucleic acid-protein interaction was responsible for the stimulation observed. Similar results have been reported for other proteins belonging to the NTPB- helicase family (7, 19). The level of RNA stimulation of the ATPase activity varies among the members of the three NTPB- helicase SFs. The highest stimulatory effect (3- to 15-fold) has been described for the enzymes belonging to SF2, whereas ; 2-fold stimulation was observed for the nsP2 protein of Sem- liki Forest virus (27) and the NS polyprotein of rubella virus (11), members of SF1, as is TYMV, and poliovirus 2C protein (a member of SF3 ). Despite this variation, all of these viral proteins show single-strand RNA-stimulated ATPase activity typical of RNA helicases.
In RT-qPCR assay, the Ct value is a parameter reflecting the quantity of template presented in the reaction [18,22,26]. Usually, lower Ct values indicate a high con- centration of template and higher Ct values indicate a low concentration of template . This study showed that standard serial dilution curve was obtained with high RT- qPCR efficiency for WYMV (105.5%). The efficiency proved the balance among enzyme, dNTPs, primers and templates. The rationality of linear equation was deter- mined by the coefficient of determination (R^2). Usually, R^2 value needs to be between 0 and 1, higher value indi- cates its higher rationality. In this study, the R^2 values of WYMV was 0.989. We therefore suggested that this RT- qPCR could be used to detect quantitatively WYMV in routine virus diagnosis.
gave 14.85% plant infection and graded as moderately resistant in the category of 11% - 25% infection. Diksha gave 45.67% plant infection and graded as tolerant in the category of 26% - 50% infection. Lush Green was mod- erately susceptible and showed 59.85% plant infection.  screened six varieties of bhindi for resistance to YVMV in the rainy and summer seasons. The incidence of OYVMV was higher during the rainy season. Arka Anamika was highly resistant, Arka Abhag resistant Parbhani kranti and V-6 were moderately resistant to disease. Others were susceptible or highly susceptible.  conducted field trials in which 7 okra cultivars were evaluated for growth and yield characteristics and virus resistance. Arka anamika and Arka Abhag were the most suitable yellow vein mosaicvirus resistant okra for com- mercial cultivation.  studied the genetics of yellow vein mosaicvirus resistance in okra based on 9 genera- tions derived from crosses involving resistant and sus- ceptible cultivars. Virus resistance was evaluated assume- ing the qualitative and quantitative nature of inheritance. Additive gene effects were more significant than domi- nance gene effects.
(MYMV). For this purpose, highly, susceptible variety of mungbean i.e. Kabuli mung was sown in earthen pots of 35cm filled with sand, peat and loam. Treatments were replicated thrice. After 20 days of sowing when leaves become mature, plants were treated exogenously with Salicylic acid (SA) and Benzohiadiazole (BTH) (Sigma, St. Louis, MO, USA) at different concentrations. After 24 hours of application mungbean plants were inoculated with viruliferous whiteflies (B. tabaci) reared on MYMV infected plants. The plants inoculated with virus and sprayed with distilled water without any application of elicitors were used as infected and healthy control respectively. The enzymatic activates i.e., PAL, POD, SOD, CAT and total Phenolic contents were determined after first, second and third week of post inoculation. The disease severity on each plant was calculated based on symptoms using disease rating scale. For the detection of virus titer all the treated plant was tested through DAS-ELISA (Clark and Adam, 1977) using Tomato Yellow Leaf Curl Bigemini virus polyclonal antibodies (AC, Diagnostics, USA) and has cross reactivity with other Gemini viruses.
In many plant virus groups, cell-to-cell movement is medi- ated by a single MP. The first of these proteins to be identified was the 30-kDa protein (p30) of tobacco mosaicvirus (TMV), which was shown to affect viral host range to be essential for virus movement (15, 18), to be localized to plasmodesmata (16, 17, 34, 42, 50), and to alter size exclusion limits of plasmodes- mata (51, 54). In addition, it was shown that p30 is phosphor- ylated in infected protoplasts (53) and in vitro (8). Also, in vitro assays demonstrated that the TMV MP has a nonspecific nucleic acid-binding activity which may be involved in unfold- ing the single-stranded genomic RNA in a manner compatible with transport through plasmodesmata (7). By using deletion analysis, the putative phosphorylation site, the activity-increas- ing size exclusion limit, and the RNA-binding activity have been tentatively mapped to specific domains of the TMV MP (6, 7, 51). More recently, immunofluorescence assays showed an association between p30 and cytoskeletal proteins (23, 32).
losses in the Indian subcontinent (Nene, 1973). Infection of MYMV may cause up to 85–100% yield loss in urdbean have been reported by Singh et al., 2011. Main pathogens causing YMD in India are Mungbean yellowmosaicvirus (MYMV) and Mungbean yellowmosaic India virus (MYMIV) (Varma and Malathi (2003); Malathi and John (2008). The disease is caused by geminivirus with bipartite genomes transmitted by different species of whitefly belongs to the genus begomovirus and family geminiviridae. Nariani (1960) was the first person ever to report the mungbean yellowmosaicvirus disease from the fields of IARI, New Delhi. The disease is characterized by the presence of bright yellow patches on leaves interspersed with green areas, complete yellowing and stunting of the plants. The knowledge on mode of inheritance of YMV resistance is important to formulate appropriate breeding strategy targeted to incorporation useful gene conferring resistance to MYMIV, otherwise agronomically poor cultivar. There are only few contrasting reports were published on mode of inheritance and gene governing the resistance. In blackgram, monogenic dominant nature of resistance was reported by Dahiya et al. (1977), Kaushal and Singh (1988) and Gupta et al. (2005) while it was reported to be digenic recessive by Singh (1980), Dwivedi and Singh (1985) and Verma and Singh (1986). However, digenic dominant natures of inheritance were also reported by Murugan and Nadarajan (2012). The prime objective of this present study is to decipher gene action involved in the inheritance of YMV resistance in black gram by using six generations.
8 involved in the initiation of RNAi by processing the dsRNA into small interfering RNA (siRNA), 21-27 nucleotides long with 5‟-phosphate and 3‟-hydroxyl termini, and two to three nucleotide 3‟ overhang (Andrew and Baulcombe, 1999). siRNA serves as a specific guide for the RNA-induced silencing complex (RISC) to recognize target messenger RNAs by homologous base pairing and directs cleavage of the target mRNAs (Hammond et al. 2002). The main components of the RISC belong to the family of Argonaute proteins that contain two typical domains; a conserved PAZ domain that has an RNA- binding factor and specifically recognizes the 3‟overhang of double stranded siRNA (Yan et al., 2003) and a PIWI domain that contain „slicer‟ catalytic activity being able to cleave RNA in RNA/siRNA complex (Hall, 2005). The signal of RNA silencing produces a systemic effect where the host RNA-dependent-RNA polymerase plays an important role using siRNA as a primer further generating a dsRNA substrate for Dicer. The signal is transmitted through the plasmodesmata and phloem (Jorgensen, 2002). A mobile silencing signal could move either with or ahead of the virus to silence the viral RNA before, or at the same time (Baulcombe, 2004).
RNA silencing or post-transcriptional gene silencing (PTGS), developed during plant evolution, functions as a defense mechanism against foreign nucleic acid inva- sions (viruses, transponsons, transgenes) . Since the phenomenon of RNA silencing was first observed by Napoli , research has been carried out to elucidate its mechanism. PTGS is a mechanism closely related to RNA interference, which is involved in plant defense against virus infection [5,6]. It was found that when a inverted repeated sequences of partial cDNA from a plant virus are introduced into host plants for expres- sion of dsRNA and induction of RNA silencing, the transgenic plants can silence virus corresponding gene and are resistant to virus infection [7,8]. More than 90% of transgenic Nicotiana benthamiana lines were
Model suggesting that a major role of the tRNA mimicry of TYMV RNA is to permit negative regulation of minus-strand synthesis. If the tRNA mimicry of TYMV RNA is not crucially involved in promoting the mechanics of minus-strand initia- tion, what role does it play? The fact that point mutations in the valine identity elements in the TYMV RNA anticodon loop that abolish valylation result in the almost complete ab- rogation of viral amplification (20) indicates that tRNA mim- icry does play a critical role in the TYMV lifecycle. Two pos- sible roles could be in promoting 3⬘-end integrity and RNA stability. Evidence has been presented that host [CTP, ATP]: tRNA nucleotidyltransferase acts as a telomerase to maintain intact CCA 3⬘ termini of brome mosaicvirus RNAs (14), and that function is also likely for TYMV RNA. However, point mutations in the anticodon would not alter interaction with this host enzyme (16), indicating the existence of another crucial role for tRNA mimicry. RNA stability could be promoted by
of the monopartite begomovirus, okra yellow vein mosaicvirus (family: Geminiviridae) and a small satellite DNA b component. This disease and insect vectors cause heavy losses of crop by affecting the quality and yield of the fruits. Infection of 100% plants in a field is very usual and yield losses ranges from 50 to 94% depending on the stage of crop growth at which infection occurs (Kumar et al. 2015). It is quite difficult to control this disease as it is tuff to successful eliminate the white fly. This disease causes heavy loss of okra by affecting the fruit qualities as well as fruit yield (Solankey et al. 2014)
Fig. 2 and 3). However, the yield of high-quality transcripts varied from unit to unit. Generally, high-quality transcripts were more abundant ( ⬃ 50%) for the rightward units AV2/1 and BV1 and the AC2/3 unit and less abundant (10 to 25%) for the leftward units AC1/4 and BC1 (see Table S2 in the sup- plemental material). This might indicate that the latter tran- scripts are less stable. Interestingly, while mapping the AC1 transcript, we found many nonpolyadenylated sequences start- ing upstream of the TATA box (Fig. 4, small arrows). One such upstream start was also found for the rightward AV2/1 tran- scription unit (Fig. 4). Such RNA sequences may represent breakdown products of readthrough transcription on the cir- cular viral DNA. A pair of such sense and antisense transcripts could lead to formation of the double-stranded RNA that triggers RNA silencing and generation of viral small interfer- ing RNAs (siRNAs). Indeed, we have detected such siRNAs derived from the MYMV promoter region (P. V. Shivaprasad, R. Akbergenov, and M. M. Pooggin, unpublished). Virus-de- rived siRNA accumulation during normal infection has also been reported for other begomoviruses (4, 31).