Journey of begomovirus betasatellite molecules: from satellites to indispensable partners.

Betasatellites are a group of circular, single-stranded DNA molecules that are frequently found to be associated with monopartite begomoviruses of the family Geminiviridae. Betasatellites require their helper viruses for replication, movement, and encapsidation and they are often essential for induction of typical disease symptoms. The ßC1 protein encoded by betasatellites is multifunctional that participates in diverse cellular events. It interferes with several cellular processes like normal development, chloroplasts, and innate immune system of plants. Recent research has indicated ßC1 protein interaction with cellular proteins and its involvement in modulation of the host's cell cycle and symptom determination. This article focuses on the functional mechanisms of ßC1 and its interactions with other viral and host proteins.

Structure activity relationship (SAR) and quantitative structure activity relationship (QSAR) studies showed plant flavonoids as potential inhibitors of dengue NS2B-NS3 protease.

BACKGROUND: Due to dengue virus disease, half of the world population is at severe health risk. Viral encoded NS2B-NS3 protease complex causes cleavage in the nonstructural region of the viral polyprotein. The cleavage is essentially required for fully functional viral protein. It has already been reported that if function of NS2B-NS3 complex is disrupted, viral replication is inhibited. Therefore, the NS2B-NS3 is a well-characterized target for designing antiviral drug. RESULTS: In this study docking analysis was performed with active site of dengue NS2B-NS3 protein with selected plant flavonoids. More than 100 flavonoids were used for docking analysis. On the basis of docking results 10 flavonoids might be considered as the best inhibitors of NS2B-NS3 protein. The interaction studies showed resilient interactions between ligand and receptor atoms. Furthermore, QSAR and SAR studies were conducted on the basis of NS2B-NS3 protease complex docking results. The value of correlation coefficient (r) 0.95 shows that there was a good correlation between flavonoid structures and selected properties. CONCLUSION: We hereby suggest that plant flavonoids could be used as potent inhibitors of dengue NS2B-NS3 protein and can be used as antiviral agents against dengue virus. Out of more than hundred plant flavonoids, ten flavonoid structures are presented in this study. On the basis of best docking results, QSAR and SAR studies were performed. These flavonoids can directly work as anti-dengue drug or with little modifications in their structures.

Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly.

RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions.

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

BACKGROUND: Geminiviruses cause major losses to several economically important crops. Pedilanthus leaf curl virus (PeLCV) is a pathogenic geminivirus that appeared in the last decade and is continuously increasing its host range in Pakistan and India. This study reports the identification and characterization of PeLCV-Petunia from ornamental plants in Pakistan, as well as geographical, phylogenetic, and recombination analysis. METHODS: Viral genomes and associated satellites were amplified, cloned, and sequenced from Petunia atkinsiana plants showing typical geminivirus infection symptoms. Virus-satellite complex was analyzed for phylogenetic and recombination pattern. Infectious clones of isolated virus and satellite molecules were constructed using a partial dimer strategy. Infectivity analysis of PeLCV alone and in combination with Digera yellow vein betasatellite (DiYVB) was performed by Agrobacterium infiltration of Nicotiana benthamiana and Petunia atkinsiana plants with infectious clones. RESULTS: PeLCV, in association with DiYVB, was identified as the cause of leaf curl disease on P. atkinsiana plants. Sequence analysis showed that the isolated PeLCV is 96-98% identical to PeLCV from soybean, and DiYVB has 91% identity to a betasatellite identified from rose. Infectivity analysis of PeLCV alone and in combination with DiYVB, performed by Agrobacterium infiltration of infectious clones in N. benthamiana and P. atkinsiana plants, resulted in mild and severe disease symptoms 14 days after infiltration, respectively, demonstrating that these viruses are natural disease-causing agents. Southern blot hybridization indicated successful replication of the virus-betasatellite complex in the infected plants. Phylogenetic analysis suggests that PeLCV originated from Pakistan and later spread to India. Recombination analysis predicted that PeLCV is a donor parent for recombination and evolution of two important begomoviruses, Papaya leaf curl virus (PaLCuV) and Radish leaf curl virus (RaLCuV). The molecular phylogeny of genes encoding coat protein (CP) and replication associated protein (Rep) depict a complex evolutionary pattern of the viruses, with wide diversity in both of the genes. CONCLUSIONS: This study presents PeLCV and DiYVB as a new natural combination resulting in leaf curl disease on P. atkinsiana plants. Phylogenetic analysis, in addition to recent agricultural reports, identify PeLCV as an emerging broad host range Begomovirus that is resident in Pakistan and, more recently, has also spread to India. Recombination analysis showed that PeLCV was involved in a natural recombinational event leading to the evolution of two recombinant begomoviruses, RaLCuV and PaLCuV.

Molecular analysis of partial VP-2 gene amplified from rectal swab samples of diarrheic dogs in Pakistan confirms the circulation of canine parvovirus genetic variant CPV-2a and detects sequences of feline panleukopenia virus (FPV).

BACKGROUND: The infection in dogs due to canine parvovirus (CPV), is a highly contagious one with high mortality rate. The present study was undertaken for a detailed genetic analysis of partial VP2 gene i.e., 630 bp isolated from rectal swab samples of infected domestic and stray dogs from all areas of district Faisalabad. Monitoring of viruses is important, as continuous prevalence of viral infection might be associated with emergence of new virulent strains. METHODS: In the present study, 40 rectal swab samples were collected from diarrheic dogs from different areas of district Faisalabad, Pakistan, in 2014-15 and screened for the presence of CPV by immunochromatography. Most of these dogs were stray dogs showing symptoms of diarrhea. Viral DNA was isolated and partial VP2 gene was amplified using gene specific primer pair Hfor/Hrev through PCR. Amplified fragments were cloned in pTZ57R/T (Fermentas) and completely sequenced. Sequences were analyzed and assembled by the Lasergene DNA analysis package (v8; DNAStar Inc., Madison, WI, USA). RESULTS: The results with immunochromatography showed that 33/40 (82%) of dogs were positive for CPV. We were able to amplify a fragment of 630 bp from 25 samples. In 25 samples the sequences of CPV-2a were detected showing the amino acid substitution Ser297Ala and presence of amino acid (426-Asn) in partial VP2 protein. Interestingly the BLAST analysis showed the of feline panleukopenia virus (FPV) sequences in 3 samples which were already positive for new CPV-2a, with 99% sequence homology to other FPV sequences present in GenBank. CONCLUSIONS: Phylogenetic analysis showed clustering of partial CPV-VP-2 gene with viruses from China, India, Japan and Uruguay identifying a new variant, whereas the 3 FPV sequences showed immediate ancestral relationship with viruses from Portugal, South Africa and USA. Interesting observation was that CPV are clustering away from the commercial vaccine strains. In this work we provide a better understanding of CPV prevailing in Pakistan at molecular level. The detection of FPV could be a case of real co-infection or a case of dual presence, due to ingestion of contaminated food.

Effect of high temperature on grain filling period, yield, amylose content and activity of starch biosynthesis enzymes in endosperm of basmati rice.

BACKGROUND: High temperature during grain filling affects yield, starch amylose content and activity of starch biosynthesis enzymes in basmati rice. To investigate the physiological mechanisms underpinning the effects of high temperature on rice grain, basmati rice was grown under two temperature conditions - 32 and 22 °C - during grain filling. RESULTS: High temperature decreased the grain filling period from 32 to 26 days, reducing yield by 6%, and caused a reduction in total starch (3.1%) and amylose content (22%). Measurable activities of key enzymes involved in sucrose to starch conversion, sucrose synthase, ADP-glucose pyrophosphorylase, starch phosphorylase and soluble starch synthase in endosperms developed at 32 °C were lower than those at 22 °C compared with similar ripening stage on an endosperm basis. In particular, granule-bound starch synthase (GBSS) activity was significantly lower than corresponding activity in endosperms developing at 22 °C during all developmental stages analyzed. CONCLUSION: Results suggest changes in amylose/amylopectin ratio observed in plants grown at 32 °C was attributable to a reduction in activity of GBSS, the sole enzyme responsible for amylose biosynthesis.

A betasatellite-dependent begomovirus infects ornamental rose: characterization of begomovirus infecting rose in Pakistan.

The Begomovirus genus of the family Geminiviridae comprises the largest group of geminiviruses. The list of begomoviruses is continuously increasing as a result of improvement in the methods for identification. Ornamental rose plants (Rosa chinensis) with highly stunted growth and leaf curling were found in Faisalabad, Pakistan. Plants were analyzed for begomovirus infection, through rolling circle amplification and PCR methods. Based on complete genome sequence homologies with other begomoviruses, a new begomovirus species infecting the rose plants was discovered. In this paper, we propose a new species name, Rose leaf curl virus (RoLCuV), for the virus. RoLCuV showed close identity (83 %) with Tomato leaf curl Pakistan virus, while associated betasatellite showed 96 % identity with Digera arvensis yellow vein betasatellite (DiAYVB), justifying a new isolate for the betasatellite. Recombination analysis of newly identified begomovirus revealed it as a recombinant of tomato leaf curl Pakistan virus from its coat protein region. The infectious molecules for virus/satellite were prepared and inoculated through Agrobacterium tumefaciens to N. benthamiana plants. RoLCuV alone was unable to induce any level of symptoms on N. benthamiana plants, but co-inoculation with cognate betasatellite produced infection symptoms. Further investigation to understand the trans-replication ability of betasatellites revealed their flexibility to interact with Rose leaf curl virus.

Yeast screens for host factors in positive-strand RNA virus replication based on a library of temperature-sensitive mutants.

RNA viruses exploit host cells by altering cellular pathways, recruiting host factors, remodeling intracellular membranes and escaping host antiviral responses. Model hosts, such as Saccharomyces cerevisiae (yeast), are valuable to identify host factors involved in viral RNA replication. The many advantages of using yeast include the availability of various yeast mutant libraries, such as (i) single gene-deletion library; (ii) the essential gene library (yTHC); and (iii) the yeast ORF over-expression library. Here, we have used a novel temperature-sensitive (ts) mutant library of essential yeast genes to identify 118 host proteins affecting replication of Tomato bushy stunt virus, in yeast model host. Testing 787 ts mutants led to the identification of host factors, of which 72 proteins facilitated TBSV replication in yeast and 46 proteins were inhibitory. Altogether, ~85% of the identified proteins are novel host factors affecting tombusvirus replication. The ts mutant library screen also led to the identification of 17 essential genes, which have been documented before, thus confirming the importance of these genomic screens. Overall, we show the power of ts mutant library in identification of host factors for RNA virus replication.

REPercussions: how geminiviruses recruit host factors for replication.

Circular single-stranded DNA viruses of the family Geminiviridae encode replication-associated protein (Rep), which is a multifunctional protein involved in virus DNA replication, transcription of virus genes, and suppression of host defense responses. Geminivirus genomes are replicated through the interaction between virus Rep and several host proteins. The Rep also interacts with itself and the virus replication enhancer protein (REn), which is another essential component of the geminivirus replicase complex that interacts with host DNA polymerases α and δ. Recent studies revealed the structural and functional complexities of geminivirus Rep, which is believed to have evolved from plasmids containing a signature domain (HUH) for single-stranded DNA binding with nuclease activity. The Rep coding sequence encompasses the entire coding sequence for AC4, which is intricately embedded within it, and performs several overlapping functions like Rep, supporting virus infection. This review investigated the structural and functional diversity of the geminivirus Rep.

Patterns of Genetic Diversity among Alphasatellites Infecting Gossypium Species.

Alphasatellites are small single-stranded circular DNA molecules associated with geminiviruses and nanoviruses. In this study, a meta-analysis of known alphasatellites isolated from the genus Gossypium (cotton) over the last two decades was performed. The phylogenetic and pairwise sequence identity analysis suggested that cotton-infecting begomoviruses were associated with at least 12 different alphasatellites globally. Three out of twelve alphasatellite were associated with cotton leaf curl geminiviruses but were not isolated from cotton plants. The cotton leaf curl Multan alphasatellite, which was initially isolated from cotton, has now been reported in several plant species, including monocot plants such as sugarcane. Our recombination analysis suggested that four alphasatellites, namely cotton leaf curl Lucknow alphasatellites, cotton leaf curl Multan alphasatellites, Ageratum yellow vein Indian alphasatellites and Ageratum enation alphasatellites, evolved through recombination. Additionally, high genetic variability was detected among the cotton-infecting alphasatellites at the genome level. The nucleotide substitution rate for the replication protein of alphasatellites (alpha-Rep) was estimated to be relatively high (~1.56 × 10-3). However, unlike other begomoviruses and satellites, the first codon position of alpha-Rep rapidly changed compared to the second and third codon positions. This study highlights the biodiversity and recombination of alphasatellites associated with the leaf curl diseases of cotton crops.

Drought Stress Mitigating Morphological, Physiological, Biochemical, and Molecular Responses of Guava (Psidium guajava L.) Cultivars.

Guava (Psidium guajava L.), a major fruit crop of the sub-tropical region, is facing a production decline due to drought stress. Morphophysiological responses to drought stress and underlying transcriptional regulations in guava are, largely, unknown. This study evaluated the drought stress tolerance of two guava cultivars, viz. "Gola" and "Surahi," at morphological and physiological levels regulated differentially by ESTs (Expressed Sequence Tags). The treatments comprises three moisture regimes, viz. To = 100% (control), T1 = 75%, and T2 = 50% of field capacity. There was an overall decrease in both morphological and physiological attributes of studied guava cultivars in response to drought stress. Nonetheless, the water use efficiency of the "Surahi" cultivar increased (41.86%) speculating its higher drought tolerance based on enhanced peroxidase (402%) and catalase (170.21%) activities under 50% field capacity (T2). Moreover, higher proline and flavonoid contents reinforced drought stress retaliation of the "Surahi" cultivar. The differential expression of a significant number of ESTs in "Surahi" (234) as compared to "Gola" (117) cultivar, somehow, regulated its cellular, biological, and molecular functions to strengthen morphophysiological attributes against drought stress as indicated by the upregulation of ESTs related to peroxidase, sucrose synthase (SUS), alcohol dehydrogenase (ADH), and ubiquitin at morphological, biochemical, and physiological levels. In conclusion, the drought stress acclimation of pear-shaped guava cultivar "Surahi" is due to the increased activities of peroxidase (POD) and catalase (CAT) complimented by the upregulation of related ESTs.

Magnetofection approach for the transformation of okra using green iron nanoparticles.

Climate change, pesticide resistance, and the need for developing new plant varieties have galvanized biotechnologists to find new solutions in order to produce transgenic plants. Over the last decade scientists are working on green metallic nanoparticles to develop DNA delivery systems for plants. In the current study, green Iron nanoparticles were synthesized using leaf extract of Camellia sinensis (green tea) and Iron Chloride (FeCl3), the characterization and Confirmation was done using UV-VIS Spectroscopy, FTIR, SEM, and TEM. Using these nanoparticles, a novel method of gene transformation in okra plants was developed, with a combination of different Magnetofection factors. Maximum gene transformation efficiency was observed at the DNA to Iron-nanoparticles ratio of 1:20, by rotation of mixture (Plasmid DNA, Iron-nanoparticles, and seed embryo) at 800 rpm for 5 h. Using this approach, the transformation of the GFP (green fluorescent protein) gene was successfully carried out in Abelmoschus esculentus (Okra plant). The DNA transformation was confirmed by observing the expression of transgene GFP via Laser Scanning Confocal Microscope (LSCM) and PCR. This method is highly economical, adaptable, genotype independent, eco-friendly, and time-saving as well. We infer that this approach can be a potential solution to combat the yield and immunity challenges of plants against pathogens.

Interaction between bacterial endophytes and host plants.

Endophytic bacteria are mainly present in the plant's root systems. Endophytic bacteria improve plant health and are sometimes necessary to fight against adverse conditions. There is an increasing trend for the use of bacterial endophytes as bio-fertilizers. However, new challenges are also arising regarding the management of these newly discovered bacterial endophytes. Plant growth-promoting bacterial endophytes exist in a wide host range as part of their microbiome, and are proven to exhibit positive effects on plant growth. Endophytic bacterial communities within plant hosts are dynamic and affected by abiotic/biotic factors such as soil conditions, geographical distribution, climate, plant species, and plant-microbe interaction at a large scale. Therefore, there is a need to evaluate the mechanism of bacterial endophytes' interaction with plants under field conditions before their application. Bacterial endophytes have both beneficial and harmful impacts on plants but the exact mechanism of interaction is poorly understood. A basic approach to exploit the potential genetic elements involved in an endophytic lifestyle is to compare the genomes of rhizospheric plant growth-promoting bacteria with endophytic bacteria. In this mini-review, we will be focused to characterize the genetic diversity and dynamics of endophyte interaction in different host plants.

Emergence of Asian endemic begomoviruses as a pandemic threat.

Plant viruses are responsible for the most devastating and commercially significant plant diseases, especially in tropical and subtropical regions. The genus begomovirus is the largest one in the family Geminiviridae, with a single-stranded DNA genome, either monopartite or bipartite. Begomoviruses are transmitted by insect vectors, such as Bemisia tabaci. Begomoviruses are the major causative agents of diseases in agriculture globally. Because of their diversity and mode of evolution, they are thought to be geographic specific. The emerging begomoviruses are of serious concern due to their increasing host range and geographical expansion. Several begomoviruses of Asiatic origin have been reported in Europe, causing massive economic losses; insect-borne transmission of viruses is a critical factor in virus outbreaks in new geographical regions. This review highlights crucial information regarding Asia's four emerging and highly destructive begomoviruses. We also provided information regarding several less common but still potentially important pathogens of different crops. This information will aid possible direction of future studies in adopting preventive measures to combat these emerging viruses.

Interaction of eukaryotic proliferating cell nuclear antigen (PCNA) with the replication-associated protein (Rep) of cotton leaf curl Multan virus and pedilanthus leaf curl virus.

The replication-associated (Rep) proteins of pathogenic begomoviruses, including cotton leaf curl Multan virus (CLCuMuV) and pedilanthus leaf curl virus (PeLCV), interact with the DNA replication machinery of their eukaryotic hosts. The analysis of Rep protein sequences showed that there is 13-28% sequence variation among CLCuMuV and PeLCV isolates, with phylogenetic clusters that can separated at least in part based on the country of origin of the respective viruses. To identify specific host factors involved in the virus replication cycle, we conducted yeast two-hybrid assays to detect possible interactions between the CLCuMuV and PeLCV Rep proteins and 30 protein components of the Saccharomyces cerevisiae DNA replication machinery. This showed that the proliferating cell nuclear antigen (PCNA) protein of S. cerevisiae interacts with Rep proteins from both CLCuMuV and PeLCV. We used the yeast PCNA sequence in BLAST comparisons to identify two PCNA orthologs each in Gossypium hirsutum (cotton), Arabidopsis thaliana (Arabidopsis), and Nicotiana benthamiana (tobacco). Sequence comparisons showed 38-40% identity between the yeast and plant PCNA proteins, and > 91% identity among the plant PCNA proteins, which clustered together in one phylogenetic group. The expression of the six plant PCNA proteins in the yeast two-hybrid system confirmed interactions with the CLCuMuV and PeLCV Rep proteins. Our results demonstrate that the interaction of begomovirus Rep proteins with eukaryotic PCNA proteins is strongly conserved, despite significant evolutionary variation in the protein sequences of both of the interacting partners.

Distinct evolutionary histories of the DNA-A and DNA-B components of bipartite begomoviruses.

BACKGROUND: Viruses of the genus Begomovirus (family Geminiviridae) have genomes consisting of either one or two genomic components. The component of bipartite begomoviruses known as DNA-A is homologous to the genomes of all geminiviruses and encodes proteins required for replication, control of gene expression, overcoming host defenses, encapsidation and insect transmission. The second component, referred to as DNA-B, encodes two proteins with functions in intra- and intercellular movement in host plants. The origin of the DNA-B component remains unclear. The study described here was initiated to investigate the relationship between the DNA-A and DNA-B components of bipartite begomoviruses with a view to unraveling their evolutionary histories and providing information on the possible origin of the DNA-B component. RESULTS: Comparative phylogenetic and exhaustive pairwise sequence comparison of all DNA-A and DNA-B components of begomoviruses demonstrates that the two molecules have very distinct molecular evolutionary histories and likely are under very different evolutionary pressures. The analysis highlights that component exchange has played a far greater role in diversification of begomoviruses than previously suspected, although there are distinct differences in the apparent ability of different groups of viruses to utilize this "sexual" mechanism of genetic exchange. Additionally we explore the hypothesis that DNA-B originated as a satellite that was captured by the monopartite progenitor of all extant bipartite begomoviruses and subsequently evolved to become the integral (essential) genome component that we recognize today. The situation with present-day satellites associated with begomoviruses provides some clues to the processes and selection pressures that may have led to the "domestication" of a wild progenitor of the DNA-B component. CONCLUSIONS: The analysis has highlighted the greater genetic variation of DNA-B components, in comparison to the DNA-A components, and that component exchange is more widespread than previously demonstrated and confined to viruses from the Old World. Although the vast majority of New World and some Old World begomoviruses show near perfect co-evolution of the DNA-A and DNA-B components, this is not the case for the majority of Old World viruses. Genetic differences between Old and New World begomoviruses and the cultivation of exotic crops in the Old World are likely factors that have led to this dichotomy.

Maintenance of an old world betasatellite by a new world helper begomovirus and possible rapid adaptation of the betasatellite.

Begomoviruses (family Geminiviridae) cause major losses to crops throughout the tropical regions of the world. Begomoviruses originating from the New World (NW) and the Old World (OW) are genetically distinct. Whereas the majority of OW begomoviruses have monopartite genomes and whereas most of these associate with a class of symptom-modulating satellites (known as betasatellites), the genomes of NW begomoviruses are exclusively bipartite and do not associate with satellites. Here, we show for the first time that a betasatellite (cotton leaf curl Multan betasatellite [CLCuMuB]) associated with a serious disease of cotton across southern Asia is capable of interacting with a NW begomovirus. In the presence of CLCuMuB, the symptoms of the NW cabbage leaf curl virus (CbLCuV) are enhanced in Nicotiana benthamiana. However, CbLCuV was unable to interact with a second betasatellite, chili leaf curl betasatellite. Although CbLCuV can transreplicate CLCuMuB, satellite accumulation levels in plants were low. However, progeny CLCuMuB isolated after just one round of infection with CbLCuV contained numerous mutations. Reinoculation of one such progeny CLCuMuB with CbLCuV to N. benthamiana yielded infections with significantly higher satellite DNA levels. This suggests that betasatellites can rapidly adapt for efficient transreplication by a new helper begomovirus, including begomoviruses originating from the NW. Although the precise mechanism of transreplication of betasatellites by begomoviruses remains unknown, an analysis of betasatellite mutants suggests that the sequence(s) required for maintenance of CLCuMuB by one of its cognate begomoviruses (cotton leaf curl Rajasthan virus) differs from the sequences required for maintenance by CbLCuV. The significance of these findings and, particularly, the threat that betasatellites pose to agriculture in the NW, are discussed.

Molecular characterization and phylogenetic analysis of tomato leaf curl Palampur virus, a bipartite begomovirus, associated with Cucumis sativus L. in Pakistan.

Leaf samples of Cucumis Sativus L. (C. sativus) (Family; Cucurbitaceae) showing vein thickening, mild leaf curling and leaf enations were collected from the farmer's field. Amplification of the full-length viral molecules was performed through rolling circle amplification (RCA). Cloning of the full-length viral molecules was done through standard cloning procedure followed by sequencing. Sequence similarity analysis and phylogenetic studies showed that the virus associated with leaf curling and enations in C. sativus was a bipartite begomovirus, where DNA-A and DNA-B showed highest nucleotide sequence homology of 98% and 97% to tomato leaf curl Palampur virus (ToLCPMV) from India. Attempts to isolate betasatellites and alphasatellites through PCR using RCA product as template, did not result in any amplification. A maximum likelihood phylogenetic tree grouped DNA-A and B components with other isolates from India. SDT was used to find the pairwise identity scores of different sequences of ToLCPMV present in the database. Phylogenetic analysis showed that sequences of ToLCPMV DNA-A and B components in this study share high degree of homology with existing viruses and are isolates of ToLCPMV-India. Infectious molecules of both components (Accessions, MG252783 and MG252784, respectively) were constructed for infectivity analysis to fulfill the Koch's postulate. Infectivity analysis revealed that ToLCPMV DNA-A is infectious to model host plant Nicotiana benthamiana and viral accumulation was confirmed through Southern blot analysis. Accumulation of DNA-B was confirmed through PCR. Infectivity analysis was also conducted using the original host, C. sativus, but plants were unable to survive the agroinoculation. To our knowledge this is the first report of ToLCPMV associated with C. sativus L. in Pakistan.

RNAi-mediated male sterility of tobacco by silencing TA29.

The superior performance of F1 hybrids has a significant impact on agricultural productivity. For commercial application, the availability of an efficient system for obtaining male-sterile lines of crops is an essential prerequisite. Here we have investigated the use of RNA interference (RNAi) technology to silence a male-specific gene in the model host tobacco. TA29 is expressed exclusively in anthers at the time of microspore development. About 10 out of 13 tobacco lines transformed with a hairpin RNAi construct containing TA29 sequences were male sterile. Transgenic plants were phenotypically indistinguishable from non-transgenic plants. At the anthesis stage, pollen grains from transgenic, male-sterile plants were aborted and lysed in comparison to the round and fully developed pollen in non-transgenic plants. Microscopic analysis of anthers showed selective degradation of tapetum in transgenic plants with no microspore development. One week after self-pollination, the ovules of non-transgenic plants were double the size of those in transgenic plants, due to successful self-fertilization. Male sterile transgenic plants set seed normally, when cross-pollinated with pollen from non-transgenic plants, confirming no adverse effect on the female parts of the flower. These results show that silencing of male-specific genes by RNAi is potentially a useful tool for generating male-sterile lines for producing hybrid seed.

Diversity, Mutation and Recombination Analysis of Cotton Leaf Curl Geminiviruses.

The spread of cotton leaf curl disease in China, India and Pakistan is a recent phenomenon. Analysis of available sequence data determined that there is a substantial diversity of cotton-infecting geminiviruses in Pakistan. Phylogenetic analyses indicated that recombination between two major groups of viruses, cotton leaf curl Multan virus (CLCuMuV) and cotton leaf curl Kokhran virus (CLCuKoV), led to the emergence of several new viruses. Recombination detection programs and phylogenetic analyses showed that CLCuMuV and CLCuKoV are highly recombinant viruses. Indeed, CLCuKoV appeared to be a major donor virus for the coat protein (CP) gene, while CLCuMuV donated the Rep gene in the majority of recombination events. Using recombination free nucleotide datasets the substitution rates for CP and Rep genes were determined. We inferred similar nucleotide substitution rates for the CLCuMuV-Rep gene (4.96X10-4) and CLCuKoV-CP gene (2.706X10-4), whereas relatively higher substitution rates were observed for CLCuMuV-CP and CLCuKoV-Rep genes. The combination of sequences with equal and relatively low substitution rates, seemed to result in the emergence of viral isolates that caused epidemics in Pakistan and India. Our findings also suggest that CLCuMuV is spreading at an alarming rate, which can potentially be a threat to cotton production in the Indian subcontinent.