Cotton leaf curl Burewala virus (CLCuBuV)

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Artificial microRNA mediated resistance against the monopartite begomovirus Cotton leaf curl Burewala virus

Artificial microRNA mediated resistance against the monopartite begomovirus Cotton leaf curl Burewala virus

Background: Cotton leaf curl disease, caused by single-stranded DNA viruses of the genus Begomovirus (family Geminiviridae), is a major constraint to cotton cultivation across Pakistan and north-western India. At this time only cotton varieties with moderate tolerance are available to counter the disease. microRNAs (miRNAs) are a class of endogenous small RNA molecules that play an important role in plant development, signal transduction, and response to biotic and a biotic stress. Studies have shown that miRNAs can be engineered to alter their target specificity. Such artificial miRNAs (amiRNAs) have been shown to provide resistance against plant-infecting viruses. Results: Two amiRNA constructs, based on the sequence of cotton miRNA169a, were produced containing 21 nt of the V2 gene sequence of Cotton leaf curl Burewala virus (CLCuBuV) and transformed into Nicotiana benthamiana. The first amiRNA construct (P1C) maintained the miR169a sequence with the exception of the replaced 21 nt whereas in the second (P1D) the sequence of the miRNA169a backbone was altered to restore some of the hydrogen bonding of the mature miRNA duplex. P1C transgenic plants showed good resistance when challenge with CLCuBV; plants being asymptomatic with low viral DNA levels. The resistance to heterologous viruses was lower and correlated with the numbers of sequence mismatches between the amiRNA and the V2 gene sequence. P1D plants showed overall poorer resistance to challenge with all viruses tested.
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Transcript mapping of Cotton leaf curl Burewala virusand its cognate betasatellite, Cotton leaf curl Multan betasatellite

Transcript mapping of Cotton leaf curl Burewala virusand its cognate betasatellite, Cotton leaf curl Multan betasatellite

Background: Whitefly-transmitted geminiviruses (family Geminiviridae, genus Begomovirus) are major limiting factors for the production of numerous dicotyledonous crops throughout the warmer regions of the world. In the Old World a small number of begomoviruses have genomes consisting of two components whereas the majority have single-component genomes. Most of the monopartite begomoviruses associate with satellite DNA molecules, the most important of which are the betasatellites. Cotton leaf curl disease (CLCuD) is one of the major problems for cotton production on the Indian sub-continent. Across Pakistan, CLCuD is currently associated with a single begomovirus (Cotton leaf curl Burewala virus [CLCuBuV]) and the cotton-specific betasatellite Cotton leaf curl Multan betasatellite (CLCuMuB), both of which have recombinant origins. Surprisingly, CLCuBuV lacks C2, one of the genes present in all previously characterized begomoviruses. Virus-specific transcripts have only been mapped for few begomoviruses, including one monopartite begomovirus that does not associate with betasatellites. Similarly, the transcripts of only two betasatellites have been mapped so far. The study described has investigated whether the recombination/mutation events involved in the evolution of CLCuBuV and its associated CLCuMuB have affected their transcription strategies.
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Association of Satellites with a Mastrevirus in Natural Infection: Complexity of Wheat Dwarf India Virus Disease

Association of Satellites with a Mastrevirus in Natural Infection: Complexity of Wheat Dwarf India Virus Disease

Constructs for infectivity of WDIV, begomoviruses, and satellites. The constructs for the infectivity of WDIV (JQ361910) and begomovi- ruses (Ageratum enation virus [AEV; JF728864], Cotton leaf curl Burewala virus [CLCuBuV; JN678802], and Cyamopsis tetragonoloba leaf curl virus [CyTLCuV; GU385879]) were prepared by cloning head-to-tail tandem repeats of the full-length viral genome in pCAMBIA1301, as described previously (39, 40). The clone for the infectivity of Ageratum yellow leaf curl betasatellite (AYLCB; KC305084) was prepared using primers ␤ 01D/ 04D and ␤ 05D/06D (Table 1) in two PCRs. The two PCR products were digested with BamHI and then ligated to generate head-to-tail tandem repeats of the betasatellite DNA. The ligated product was cloned into a pDRIVE cloning vector. One positive clone was digested with XbaI re- striction endonuclease. The product released after digestion was ligated at the XbaI site in the binary vector pCAMBIA1301 (CAMBIA, Canberra, Australia). A clone for the infectivity of Cotton leaf curl Multan betasat- ellite (CLCuMB; HQ257372) was prepared as described for AYLCB. For preparing the constructs for the infectivity of Cotton leaf curl Multan alphasatellite (CLCuMA; KC305093) and Guar leaf curl alphasatellite (GLCuA; KC305095), the pDRIVE vectors having alphasatellites were double digested with HindIII and PstI to yield a 918-bp fragment of the alphasatellite encompassing the predicted origin of replication. The diges- tion product was introduced into pCAMBIA1301 at the HindIII/PstI site to generate a pCAMBIA-alpha 918-bp intermediate clone. The alphasat- ellite cloned into the pDRIVE vector was digested with PstI and cloned into the PstI site of the pCAMBIA-alpha 918-bp intermediate clone to generate a 1.7-mer head-to-tail tandem repeat of the alphasatellite DNA. All of the PCR-amplified products were sequenced to confirm the absence of mutations in the clones. Derivatives of the binary vector pCAM- BIA1301, having constructs for infectivity of WDIV, begomoviruses, alphasatellites, and betasatellites, were mobilized separately into the Agro- bacterium tumefaciens strain GV3101 via the freeze-thaw method (41).
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Selection of target sequences as well as sequence identity determine the outcome of RNAi approach for resistance against cotton leaf curl geminivirus complex

Selection of target sequences as well as sequence identity determine the outcome of RNAi approach for resistance against cotton leaf curl geminivirus complex

The genomes of monopartite begomoviruses encode six proteins with genes in the virion and complemen- tary-sense separated by a non-coding intergenic (IR) region that contains control sequences as well as the virion-sense origin of replication [24]. The genes in the virion-sense encode the V2 protein, which is involved in virus movement and is a suppressor of RNAi, and the coat protein, the only structural protein of geminiviruses that is required to form the characteristic geminate par- ticles, for movement in plants and interacts with the whitefly vector for transmission plant-to-plant. In the complementary-sense the genes encode the replication associated protein (Rep; the only virus encoded protein required for viral DNA replication, which is a rolling- circle replication-initiator protein), the transcriptional activator protein (TrAP; involved in the up-regulation of late (virion-sense) genes, modulating host gene expres- sion and may be a suppressor of RNAi), the replication- enhancer protein (REn; interacts with and enhances Rep activity) and the C4 protein (a suppressor of RNAi that may be involved in virus movement).
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Conformation-Selective Methylation of Geminivirus DNA

Conformation-Selective Methylation of Geminivirus DNA

Geminiviruses with small circular single-stranded DNA genomes replicate in plant cell nuclei by using various double-stranded DNA (dsDNA) intermediates: distinct open circular and covalently closed circular as well as heterogeneous linear DNA. Their DNA may be methylated partially at cytosine residues, as detected previously by bisulfite sequencing and subsequent PCR. In order to determine the methylation patterns of the circular molecules, the DNAs of tomato yellow leaf curl Sardinia virus (TYLCSV) and Abutilon mosaic virus were investigated utilizing bisulfite treatment followed by rolling circle amplification. Shotgun sequencing of the products yielded a randomly distributed 50% rate of C maintenance after the bisulfite reaction for both viruses. However, controls with unmethylated single-stranded bacteriophage DNA resulted in the same level of C maintenance. Only one short DNA stretch within the C2/C3 promoter of TYLCSV showed hyperprotection of C, with the protection rate exceeding the threshold of the mean value plus 1 standard deviation. Similarly, the use of methylation-sensitive restriction enzymes suggested that geminiviruses escape silencing by methyl- ation very efficiently, by either a rolling circle or recombination-dependent replication mode. In contrast, attempts to detect methylated bases positively by using methylcytosine-specific antibodies detected methylated DNA only in heterogeneous linear dsDNA, and methylation-dependent restriction enzymes revealed that the viral heterogeneous linear dsDNA was methylated preferentially.
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A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein

A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein

To analyze whether the K-to-A substitutions also affect Rep replication activity, we then transiently expressed them as Rep-RFP variants in 2IRGFP plants. As the expression levels vary between infiltrated leaves, we always expressed WT Rep in one half of the leaf as an internal control. At 4 days postinfiltration (dpi), we took UV images of leaves to examine GFP accumulation, and tissue was sampled to quantify the ECM levels. Expression of the variants K69A and K99A resulted in strong GFP signals, as did WT Rep (Fig. 7B). Expression of K65A resulted in less GFP signal but still more than the background signal. Expression of the double and triple K-to-A mutants did not result in an increase of the GFP signal over the background signal, indicating that those Rep versions entirely failed to stimulate viral DNA replication. Importantly, both WT Rep-RFP and the variants (including the inactive K-to-A variants) accumulated at similar levels at the expected mass in this experiment (Fig. 7C). We then quantified the ECM levels in DNA extracted from these leaves using quantitative PCR (qPCR) amplifying a unique fragment present only in circular ECMs. The GFP fluorescence signals seen in the UV images were in full accordance with the ECM levels detected in these tissues (Fig. 7D). Combined, these results confirm that in the case of Rep TYLCV-Alm , the K65 (x) residue is
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Diversity, Distribution, and Evolution of Tomato Viruses in China Uncovered by Small RNA Sequencing

Diversity, Distribution, and Evolution of Tomato Viruses in China Uncovered by Small RNA Sequencing

derived reads were enriched at a length of 24 nt and to a lesser extent at 21 nt, while the remaining reads were preferentially enriched at 21 nt and less significantly at 22 nt (Fig. 1B). The bipartite read size distribution underlined the activity of the host immune defense system against viruses, which released large amounts of vsiRNAs of 21 and 22 nt. We analyzed the sRNA sequences using VirusDetect, a program that can efficiently identify both known and novel viruses from deep siRNA sequences (23). In total, we detected 22 viruses, including 21 known viruses and one newly discovered virus, as well as two viroids (Citrus exocortis viroid, genus Pospiviroid, and Potato spindle tuber viroid, genus Pospiviroid) from the 170 samples, and these viruses spanned 12 genera (Fig. 1C). The sample information, sRNA sequences, and the information on the identified viruses are available at the Chinese Tomato Virome Database (http://ted.bti.cornell.edu/ CtomatoVirome/index.html). Positive-sense single-stranded RNA [( ⫹ )ssRNA] viruses were the dominant group, representing 77% of the identified viruses. Potyvirus was the most abundant subgroup in the ( ⫹ )ssRNA viruses, with six species detected from the collected samples (Fig. 1C). We used the nonparametric viral discovery statistic (5, 25) to predict the bounds of the viral community in tomato and assess the completeness of our virus discovery effort. The curve indicated that the community may contain 30 viral species, and the viruses that we discovered from 170 samples represented ⬃ 73% of all species that may be present in tomato in China (Fig. 1D). All the samples were diagnosed with at least one virus, consistent with their sampling based on virus-like symptoms. Moreover, ⬃ 89% of samples contain two or more viruses, with a peak at three (Fig. 1E), suggesting a mixed infection in the majority of the collected samples. Despite the short length of sRNA sequencing reads, the complete genomes of 13 out of 22 detected viruses could be assembled and the genomes of another 5 were nearly complete (genome coverage ⱖ 90%) (Fig. 2), affirming the efficiency of viral genome recovery from sRNA sequences (11). Viruses such as Tomato mosaic virus (ToMV, genus Tobamovirus), Tomato yellow leaf curl virus (TYLCV, genus Begomovirus), Potato virus Y (PVY, genus Potyvirus), Southern tomato virus (STV, genus Amalgavirus), Cucumber mosaic virus (CMV, genus Cucumovirus), Chilli veinal mottle virus (ChiVMV, genus Potyvirus), Tomato mottle mosaic virus (ToMMV, genus Tobamovirus), Tomato chlorosis virus (ToCV, genus Crinivirus), Tomato zonate spot virus (TZSV, genus Tospovi- rus), and Tomato spotted wilt virus (TSWV, genus Tospovirus) have been well docu-
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Assessment of genetic diversity of cotton genotypes for various economic traits against cotton leaf curl disease (CLCuD).

Assessment of genetic diversity of cotton genotypes for various economic traits against cotton leaf curl disease (CLCuD).

Cotton leaf curl disease (CLCuD) is a menace to cotton production in several African and Asian countries, including Pakistan, northwestern India. It was also recently reported in China. This disease is characterized by several whitefly transmitted begomoviruses (Family: Geminiviridae, Genus: Begomovirus) associated with specific satellite molecules (alpha- and betasatellites), which are responsible for symptom development (Sattar et al., 2013). The infected cotton plants (Gossypium L.) show a range of symptoms including vein thickening/ swelling, leaf enations (which develop into leaf-like structures in extreme cases), and cup- shaped leaf curling. In some cases, CLCuD-affected cotton plants appear as lush and green as healthy plants, due to the proliferation of chloroplast-containing tissues (Sharma et al., 2005; Tahir et al., 2011). Plants infected soon after germination are usually severely stunted with compactly rolled leaves and produce no harvestable lint (Farooq et al., 2011). It has been shown that the leaf enations and vein thickening symptoms are due to the presence of cotton leaf curl Multan betasatellite (CLCuMuB) (Qazi et al., 2007; Tahir and Mansoor, 2011).
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Complete sequence and genomic annotation of carrot torradovirus 1

Complete sequence and genomic annotation of carrot torradovirus 1

necrotic leaf curl virus, a new plant virus infecting lettuce and a proposed member of the genus.. Complete genome sequence of motherwort yellow mottle virus, a novel putative member of [r]

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Alfalfa Leaf Curl Virus: an Aphid-Transmitted Geminivirus

Alfalfa Leaf Curl Virus: an Aphid-Transmitted Geminivirus

An agroinfectious clone was prepared from the viral genome 44-1E as previously described for EcmLV (2) and inoculated into 22 Nicotiana benthamiana plants. Although the plants were symp- tomless, the virus was detected in 10 of them 26 days postinocu- lation using PCR with primers targeting a 785-nt fragment of the CP gene of the cloned virus (Luz-CP-F, 5=-TGGAATATTGTGCT GCTTGG-3=, and Luz-CP-R, 5=-ATTTTGGGACTTGTGCTCC A-3=). The same purification procedure used to obtain EcmLV particles (Fig. 1) was used for the agroinfected N. benthamiana plants. However, despite spherical, possibly degraded, structures being observed from the sucrose gradient fraction within which geminivirus particles were expected (according to EcmLV purifi- cation), twinned icosahedral particles typical of geminiviruses could not be detected by transmission electron microscopy. Nev- ertheless, these fractions were determined to be positive for viral DNA by Southern blotting with P 32 -labeled probes prepared by
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MicroRNA profiling of tomato leaf curl new delhi virus (tolcndv) infected tomato leaves indicates that deregulation of mir159/319 and mir172 might be linked with leaf curl disease

MicroRNA profiling of tomato leaf curl new delhi virus (tolcndv) infected tomato leaves indicates that deregulation of mir159/319 and mir172 might be linked with leaf curl disease

activity and have characteristic 5’ cap and 3’ poly-A tail [8,9]. These pri-miRNA transcripts form hairpin like structure and are sequentially processed by the action of RNase III-like proteins, namely HYL1/SER1 and DCL1 in Arabidopsis, to generate miRNA duplexes [6,10]. The mature miRNA enters into a multi-protein complex termed RNA-induced silencing complex (mi-RISC) and guides it to the target mRNAs with complementary sequences. This consequently leads to the target cleavage [8,11] and/or inhibits translation of the targets [12]. In plants, miRNAs have been demonstrated to participate in leaf morphogenesis, phase transition, flower development and root and shoot development [13-18]. It is thus appar- ent that ToLCNDV induced leaf curling in tomato can be utilized as a model system to study the influence of miRNA-mediated biological actions on leaf deformations. In Arabidopsis, few miRs have been demonstrated to critically regulate leaf development viz., miR165/166, miR164 and miR319/159 [19-21]. For instance, miR165/ 166 targeted HD-ZIP III transcription factors (TFs) are involved in determining adaxial and abaxial pattern for- mation [20] while, miR159 and miR319 play important roles in maintaining leaf phenotype by regulating mem- bers of MYB transcription factors and TCP transcription factors, respectively [19]. Similarly, miR164 that targets CUC2 also takes care of leaf patterning by controlling serration of leaf margins [21]. The involvement of these miRNAs in leaf morphology has been demonstrated by raising Arabidopsis transgenic over-expressing miRNAs or targets with mutated miRNA binding sites and these transgenic plants revealed clear leaf development asso- ciated defects. Moreover, evidences support the involve- ment of miRNAs in biotic and abiotic stresses. For instance, miR393 expression is induced under bacterial infection [22]. The F-box auxin receptor proteins tar- geted by miR393 are consequently down-regulated, thereby suppressing auxin signaling pathways and prob- ably conferring resistance against pathogens. On the other hand, miR395, miR399, miR398, etc., have been associated with specific abiotic stresses [7,23,24].
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Detection and analysis of leaf curl virus from Jatropha

Detection and analysis of leaf curl virus from Jatropha

of the phenomenon of RNA silencing [8] Begomoviruses are also, both inducers and targets of RNAi. The begomovirus siRNAs are of 21, 22 and 24 nucleotide in length. Moreover, many segments of the viral DNAs also are methylated in a siRNA dependent manner in response to infection. However, unlike the case in mammalian systems, the host microRNAs that interfere with replication and spread of plant viruses are not known yet.[19] In response to plant antiviral RNA silencing, viruses are not behind in waging an arms race to neutralize host defenses. They have evolved several RNAi evading mechanisms like evolution of siRNA resistant satellite genomes, defective interfering RNAs, loss of target sequences by high mutation rate, formation of RISC-inaccessible secondary structures, associating with protein complexes posing steric hindrance, encapsidation and partitioning their replicative cycles in vesicles, chloroplasts and nucleus . Suppressors can reverse gene silencing effects and allow high transgene expression – a desired goal of molecular farming. Thus, RNAi suppressors and their hosts with antiviral RNAi, the former seems to be having an edge, at least as seen from the human angle. The enormous loss of our crops to begomoviral diseases necessitates development of intervention strategies to efficiently contain the virus. Spray of insecticides to get rid of the virus transmitting whitefly vector, is neither an effective nor an eco-friendly approach. Unfortunately, stable natural resistance sources for begomoviruses are few and plant breeders have not been successful in introgressing these largely multigenic traits into elite cultivars. Hence, modern biotechnology needs to offer an attractive alternative of engineering begomovirus resistance through transgenic route.[33]Pathogen-derived resistance (PDR)
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A Novel DNA Motif Contributes to Selective Replication of a Geminivirus-Associated Betasatellite by a Helper Virus-Encoded Replication-Related Protein

A Novel DNA Motif Contributes to Selective Replication of a Geminivirus-Associated Betasatellite by a Helper Virus-Encoded Replication-Related Protein

Previously, several sequence motifs have been implicated in betasatellite replication. Eini et al. found that a G-box motif lo- cated 143 nt upstream of the ␤C1 start codon is required for effi- cient replication of CLCuMB (40). Studies of tomato leaf curl virus (TLCV) defective satellite DNA showed that an approxi- mately 330-nt region, including the conserved nonanucleotide se- quence TAATATTAC, is essential for replication (41). Notably, this region aligns well with the SCR of AYVB, which has been shown to be essential for betasatellite replication (25). The in- volvement of the SCR in betasatellite replication was also con- firmed in our transient-replication assay in BY-2 protoplasts (Fig. 6). According to the proposed model of geminivirus rolling-circle replication, binding of viral Rep to the replication origin of the double-stranded (dsDNA) genome is a key step in the initiation of viral DNA replication. Rep specifically binds to iterative se- quences, or “iterons,” located upstream of potential stem-loop structures of its cognate genome (21, 34, 42, 43), catalyzes a cleav- age reaction in the conserved nonanucleotide sequence within the origin (18, 21), and ligates DNA at the conserved hairpin structure (18, 19). However, unlike DNA B, betasatellites generally lack sig- nificant sequence homology with their helper virus, and helper virus-related iteron sequences are frequently not found in betasat- ellites. It is unclear how Rep mediates origin recognition and trans replication of betasatellite DNA. Studies with ToLCV-sat identi- fied two high-affinity Rep-binding sites (GGTGTCT) upstream of the conserved rolling circle cruciform structure which are identi- cal to the ToLCV iteron sequence but occur in an inverted orien- tation (24, 44, 45). Surprisingly, mutation of both motifs does not completely abolish the replication of ToLCV or of ToLCV-sat (24). These data suggest that high-affinity Rep replication origin binding is not required for ToLCV-sat replication, consistent with its promiscuous replication by diverse helper viruses, such as Af- rican cassava mosaic virus and beet curly top virus, that recognize different iteron sequences (44). However, it should be noted that both ToLCV and ToLCV-sat Rep binding site mutants accu- mulated at greatly reduced levels in infected plants compared to wild-type clones (24), suggesting that high-affinity Rep
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Virus- Vector Relationship Of Sunflower Leaf Curl Virus (SuLCV) In Relation To Disease Spread

Virus- Vector Relationship Of Sunflower Leaf Curl Virus (SuLCV) In Relation To Disease Spread

The healthy colonies of whiteflies were allowed to feed on SuLCV infected sunflower leaf sample for a specific period of 24 hrs. After 24 hrs of AAP, such viruliferous whiteflies were allowed to feed on healthy sunflower seedlings for different inoculation access periods viz., 30 min, 1 hr, 2 hr, 3 hr, 6 hr, 12 hr and 24 hr (IAP) to inoculate the virus. After, respective periods of inoculation access periods, whiteflies were killed using systemic insecticide Hostathion 40 EC @ 1.5 ml/L. Later inoculated seedlings were maintained in insect proof cages till the expression of symptoms. Observation was recorded on time period of IAP required to achieve minimum and maximum per cent transmission based on number of seedlings exhibits diseases symptoms upon inoculation of virus.
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Enumerations on seed-borne and post-harvest microflora associated with okra [Abelmoschus esculentus (L.) Moench] and their management

Enumerations on seed-borne and post-harvest microflora associated with okra [Abelmoschus esculentus (L.) Moench] and their management

The crop is attacked by several viruses but yellow vein mosaic virus is important attacked on okra [99]. The virus causing okra yellow vein mosaic (OYVMV) is known as yellow vein mosaic virus the most serious disease of okra. If the plants are affected in the early stage of growth there is a total loss so far as yield and quality of fruit. If the plants are infected within 35 days of germination their growth is retarded, few leaves and fruit are formed and the loss may be about 94%. Plants infected 50 and 65 days after germination suffer a loss of 84 and 49% respectively [100-104]. A virus induced mosaic of okra from Nigeria was reported [105]. The virus was transmitted by grafting or mechanical inoculation of okra, cotton, cowpea and Chenopodium. The effect of okra mosaic virus on growth and yield of okra plants varied with the time of inoculation during the early rains [106]. Inoculation 14 and 21 days after emergence (DAE) reduced the average weight of fruits/plants compared with those inoculated 28 DAE and the uninoculated control. Study of the natural incidence of OYVMV disease in relation to different dates of sowing has revealed that the lowest disease incidence occurred on okra sown at the beginning of October (16.7%) and the highest on crops sowing May and June (100%) with incidence in February and March crops of 36.5 and 54.2%, respectively [107]. The incidence of OYVMV on cv. Pusa Sawani varied from 75 to 91% in the plots sowing between early April and the end of June. A strong positive correlation was obtained by Nath and Saikia (1995) [108] between percent disease incidence and white fly (Bemisia tabaci) population (r =0.085) whereas a strong negative correlation was obtained from disease incidence and fruit yield (r=-0.84). In biochemical studies, OYVMV infection increased the levels of total reducing and non-reducing sugars. Starch, Ammonium, Nitrogen and total free amino acids decreased in diseased plants. Levels of chlorophyll and carbohydrates in infected okra declined with increase in severity of OYVMV symptoms, while lipids, nucleic acids level increased in diseased plants [109-111].
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Characterization, phylogeny and recombination analysis of Pedilanthus leaf curl virus Petunia isolate and its associated betasatellite

Characterization, phylogeny and recombination analysis of Pedilanthus leaf curl virus Petunia isolate and its associated betasatellite

Sesbania constitute a monophyletic group (Fig. 3a). A separate branch of PaLCuV radiating from PeLCV was interesting. The recombination analysis revealed that PaLCuV is a recombinant derived from PeLCV and an unknown virus. The majority of virion and complemen- tary strand genes were derived from PeLCV, which justi- fies its position in the phylogenetic tree (Fig. 3). Similarly, RaLCuV (originally from India) was also found to be a recombinant of PeLCV and an unknown virus [25]. However, the nucleotide span was limited to the virion (AV2 and coat protein genes) strand genes. The PeLCV isolates from tomato, euphorbia, Cestrum, carrot, crape and Chenopodium constituted a separate group. With the available data, there was no detectable level of recombination among these isolates. In the phylogenetic tree, four different clusters are evident, where cluster-I represents the PeLCV isolates originating from Pakistan, whereas cluster-II represents the isolates from India. An isolate from cluster-II appeared to be a donor virus for the RaLCuV from India. Cluster-III represents the PeLCV isolates from Pakistan reported from tomato and euphorbia plants. In the fourth cluster, the PeLCV iso- lates from crape and Cestrum make up a separate group. However, the other three isolates represent a separate group of Indian and Pakistani isolates.
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The infective cycle of Cabbage leaf curl virus (CaLCuV) is affected by CRUMPLED LEAF (CRL) gene in Arabidopsis thaliana

The infective cycle of Cabbage leaf curl virus (CaLCuV) is affected by CRUMPLED LEAF (CRL) gene in Arabidopsis thaliana

Using a screen of MGT lines, we have identified genes whose expression is modified upon CaLCuV inoculation. A further characterization of a selected candidate has resulted in the demonstration that the gene CRUMPLED LEAF (CRL) is involved in the infective cycle of the virus. CRL has been previously reported as involved in the mor- phogenesis of all plant organs and the division of plastids [29]. It was also reported that in a crl mutant, the planes of cell division are distorted in shoot apical meristems, root tips and embryos. In addition, the mutant is dwarf and present pale green and crumpled leaves. CRL protein was observed associated with plastid membranes and, more recently, it has been shown that a crl A. thaliana mutant present cells without detectable plastids [29,31]. Although CRL protein is conserved in various species of dicots, monocots and cyanobacterias, no similarity to pro- teins with predicted or known function has been reported. The usefulness of gene trap technology to identify genes responsive to viral infections is additionally supported by the fact that the CRL gene was not identified in screenings designed to detect genes regulated during different virus infections (including CaLCuV) using A. thaliana microar- rays and sDNA-AFLP analysis (AffyID 24849_at; gene At5 g51020) [25,43-45]. The variety of results observed in the screening also suggests that viral induction of some genes can be a highly localized process (in time or space), thus, those genes could be easily missed in analysis with some methodologies (microarrays, differential libraries) due to a dilution of the mRNAs or an inappropriate timing for sample collection.
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Tomato Yellow Leaf Curl Virus V2 Interacts with Host Histone Deacetylase 6 To Suppress Methylation-Mediated Transcriptional Gene Silencing in Plants

Tomato Yellow Leaf Curl Virus V2 Interacts with Host Histone Deacetylase 6 To Suppress Methylation-Mediated Transcriptional Gene Silencing in Plants

Plants employ transcriptional gene silencing (TGS) as a defense against geminivi- ruses (15–17). As a counterdefensive measure, geminiviruses produce unique proteins that serve as transcriptional gene silencing suppressors to interfere with this process. Virus-encoded TGS suppressors act through divergent mechanisms. The most exten- sively studied geminivirus silencing suppressors are the AC2/AL2 protein encoded by members of the genus Begomovirus and the related C2/L2 protein encoded by mem- bers of the genus Curtovirus. Both AC2/AL2 and C2/L2 suppress TGS by a mechanism that correlates with methyl cycle interference through inhibition of adenosine kinase (ADK) activity (18–20). In addition, Beet severe curly top virus (BSCTV) C2 interferes with the host epigenetic defense by attenuating the activity of 26S proteasome-mediated degradation of S-adenosyl-methionine decarboxylase 1 (SAMDC1), highlighting the importance of the methyl cycle for defense against geminiviruses (21). More recently, AC2/C2 was shown to interact with and inhibit the H3K9 histone methyltransferase SUVH4/KYP to attenuate TGS (22, 23). Besides AC2/C2, the ␤ C1 protein encoded by Tomato yellow leaf curl China betasatellite (TYLCCNB) interacts with and inhibits the activity of S-adenosyl homocysteine hydrolase (SAHH) to block the methyl cycle (24). Furthermore, replication-associated proteins (Reps, also known as C1, AL1, or AC1) of several geminiviruses suppress TGS by reducing the expression of plant DNA methyl- transferases, methyltransferase 1 (MET1) and chromomethylase 3 (CMT3) (25). In addi- tion, the AC5 protein encoded by Mungbean yellow mosaic India virus (MYMIV) was shown to prevent TGS by repressing the expression of domains rearranged methyl- transferase 2 (DRM2) (17). Our previous studies have shown that the V2 protein of Tomato yellow leaf curl virus (TYLCV) is a TGS suppressor (26). However, how TYLCV V2 mediates TGS suppression remains unknown.
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I nsect-t ransm itted viruses threaten agriculture

I nsect-t ransm itted viruses threaten agriculture

In the first case, tomato yellow leaf curl, the virus is not present in California, but the vector insect, silverleaf whitefly, is well established.. In the second case, the opposite is [r]

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An efficient in vitro inoculation method for Tomato yellow leaf curl virus

An efficient in vitro inoculation method for Tomato yellow leaf curl virus

inoculated LA 1777 microshoots were symptomless for TYLCD, but tested positive for TYLCV using PCR and RCA (Figure 5). LA 1777 plants grown under greenhouse conditions and subjected to inoculation with viruliferous whiteflies and PCR analysis revealed the presence of both immune (virus is not detectable in the plant) and tolerant (virus is detectable in the plant, but the TYLCD symp- toms are absent) mechanisms against TYLCV [unpub- lished results], which is consistent with previous reports [24]. Several attempts to transmit TYLCV to LA 1777 through grafting with infected tomato plants or natural infection under greenhouse conditions failed [unpub- lished results]. The current method can overcome such limitations related to incompatibility between scion and stock or natural inoculation difficulties due to whitefly non-preference. According to Vidavsky & Czosnek [24], the mechanisms of resistance in LA 1777 are expressed at the whitefly-plant interface (viral transmission) and internally in the plant (TYLCD symptoms development); therefore, by using natural inoculation methods, the resistance at the whitefly-plant interface will mask the resistance toward the virus inside the plant. Using the described in vitro-inoculation method, it was possible to overcome such limitation and it was feasible to uncover the natural resistance of LA 1777 to TYLCV. This is in general agreement with the results of Kheyr-Pour et al. [27], where in vivo agroinoculation was used to break the TYLCV resistance in LA 1777.
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