Exploring Host Resistance against Chilli Leaf Curl Disease in a Tolerant Chilli Genotype.

In tropical countries, combating leaf curl disease in hot peppers has become important in improvement programs. Leaf curl disease is caused by whitefly (Bemisia tabaci) transmitted begomoviruses, which mainly include chilli leaf curl virus (ChiLCV). However, multiple begomoviruses have also been found to be associated with this disease. The Capsicum annuum line, DLS-Sel-10, was found to be a tolerant source against this disease during field screening. In this study, we characterized the resistance of DLS-sel-10 against chilli leaf curl virus (ChiLCV) in comparison to the susceptible cultivar Phule Mukta (PM), focusing on the level, stage, and nature of resistance. Comprehensive investigations involved screening of DLS-Sel-10 against the whitefly vector ChiLCV. The putative tolerant line displayed reduced virus infection at the seedling stage, with increasing resistance during vegetative, flowering, and fruiting stages. Both DLS-Sel-10 and PM could be infected with ChiLCV, although DLS-Sel-10 remained symptomless. Insect feeding assays revealed DLS-Sel-10 as a less preferred host for whiteflies compared to PM. In conclusion, DLS-Sel-10 demonstrated tolerance not only to ChiLCV but also served as an unfavorable host for the whitefly vector. The study highlighted an age-dependent increase in tolerance within DLS-Sel-10, showcasing its potential for effective leaf curl disease management in chilli.

Expanding Possibilities for Foreign Gene Expression by Cucumber Green Mottle Mosaic Virus Genome-Based Bipartite Vector System.

Expanding possibilities for foreign gene expression in cucurbits, we present a novel approach utilising a bipartite vector system based on the cucumber green mottle mosaic virus (CGMMV) genome. Traditional full-length CGMMV vectors face limitations such as a restricted cargo capacity and unstable foreign gene expression. To address these challenges, we developed two 'deconstructed' CGMMV genomes, DG-1 and DG-2. DG-1 features a major internal deletion, resulting in the loss of crucial replicase enzyme domains, rendering it incapable of self-replication. However, a staggered infiltration of DG-1 in CGMMV-infected plants enabled successful replication and movement, facilitating gene-silencing experiments. Conversely, DG-2 was engineered to enhance replication rates and provide multiple cloning sites. Although it exhibited higher replication rates, DG-2 remained localised within infiltrated tissue, displaying trans-replication and restricted movement. Notably, DG-2 demonstrated utility in expressing GFP, with a peak expression observed between 6 and 10 days post-infiltration. Overall, our bipartite system represents a significant advancement in functional genomics, offering a robust tool for foreign gene expression in Nicotiana benthamiana.

Emerging evidence of seed transmission of begomoviruses: implications in global circulation and disease outbreak.

Begomoviruses (family Geminiviridae) are known for causing devastating diseases in fruit, fibre, pulse, and vegetable crops throughout the world. Begomoviruses are transmitted in the field exclusively through insect vector whitefly (Bemisia tabaci), and the frequent outbreaks of begomoviruses are attributed largely due to the abundance of whitefly in the agri-ecosystem. Begomoviruses being phloem-borne were known not be transmitted through seeds of the infected plants. The recent findings of seed transmission of begomoviruses brought out a new dimension of begomovirus perpetuation and dissemination. The first convincing evidence of seed transmission of begomoviruses was known in 2015 for sweet potato leaf curl virus followed by several begomoviruses, like bhendi yellow vein mosaic virus, bitter gourd yellow mosaic virus, dolichos yellow mosaic virus, mungbean yellow mosaic virus, mungbean yellow mosaic India virus, pepper yellow leaf curl Indonesia virus, tomato leaf curl New Delhi virus, tomato yellow leaf curl virus, tomato yellow leaf curl Sardinia virus, and okra yellow mosaic Mexico virus. These studies brought out two perspectives of seed-borne nature of begomoviruses: (i) the presence of begomovirus in the seed tissues derived from the infected plants but no expression of disease symptoms in the progeny seedlings and (ii) the seed infection successfully transmitted the virus to cause disease to the progeny seedlings. It seems that the seed transmission of begomovirus is a feature of a specific combination of host-genotype and virus strain, rather than a universal phenomenon. This review comprehensively describes the seed transmitted begomoviruses reported in the last 9 years and the possible mechanism of seed transmission. An emphasis is placed on the experimental results that proved the seed transmission of various begomoviruses, factors affecting seed transmission and impact of begomovirus seed transmission on virus circulation, outbreak of the disease, and management strategies.

Bottle gourd IC-0262269, a super-susceptible genotype to tomato leaf curl Palampur virus.

While conducting field trial of 82 genotypes of bottle gourd at Delhi during 2020-2021, a particular genotype, IC-0262269 was found to be affected by chlorotic curly stunt disease (CCSD). The affected plants were severely stunted and bearing very small chlorotic and crinkle leaves. The disease incidence in the said genotype was as high as 80% among different replicated trial blocks. The application of PCR using a generic primers specific to begomoviruses, as well as species-specific PCR diagnostics to six tomato-infecting begomoviruses: tomato leaf curl New Delhi virus (ToLCNDV), tomato leaf curl Palampur virus (ToLCPalV), tomato leaf curl Joydebpur virus (ToLCJoV), tomato leaf curl Gujrat virus (ToLCGuV), tomato leaf curl Bangalore virus (ToLCBV), and chilli leaf curl virus (ChiLCV) showed that, only ToLCPalV could be detected in the genotype IC-0262269. Following, rolling circle amplification, cloning and sequencing of full-length DNA-A and DNA-B genome of an isolate BoG1-ND from the genotype IC-0262269 revealed association of ToLCPalV with the disease. The successful agro-infection of the cloned genome of BoG1-ND (DNA-A and DNA-B) in the plants of Nicotiana benthamiana and bottle gourd demonstrated that ToLCPalV is the causal begomovirus of CCSD. The study provides the first evidence of the natural occurrence of ToLCPalV in bottle gourd crop and also showed that the bottle gourd genotype IC-0262269 is super-susceptible to ToLCPalV. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03838-y.

Genetic diversity of Potato virus M (PVM) in the major potato growing region in the Indo-Gangetic plain and characterization of a distinct strain of PVM occurring in India.

Potato virus M (PVM) is one of the most prevalent viruses infecting potatoes worldwide, showing a wide range of diversity in their populations; however, the diversity and genome information of PVM occurring in India is hardly known. The present study serologically detected the PVM in 22.8% of leaf samples collected from the potato fields, generated 13 coat protein (CP) genes and one complete genome sequence for the isolates from India, and identified four differential hosts confirming PVM-Del-144 as a distinct strain of PVM occurring in India. The phylogenetic analyses conducted based on the CP gene sequences (14 from India and 176 from other countries) suggested the existence of three evolutionary divergent lineages (PVM-o, PVM-d, and a new divergent group) in the PVM population, where isolates from India belong to only two clusters (PVM-o and PVM-d) within four sub-clusters. High levels of nucleotide diversity (0.124) and genetic distance (0.142) recorded among the isolates from India may be due to the deviation from the neutral evolution and experiencing population expansion in the past. The complete genome of the isolate Del-144 (KJ194171; 8,526 nucleotides) shared 92.2-93.9% nt sequence identity with the population of PVM-o, whereas it shared only 70.2-72.1% identity with PVM-d. In the phylogenetic analyses, Del-144 clustered with the isolates of PVM-o; however, it formed a separate branch away from all other isolates, indicating the diversity of the strain. Overall, this study revealed the diversity of the isolates of PVM from India and reported the first complete genome sequence of a distinct strain of PVM occurring in India.

Accelerated Long-Fragment Circular PCR for Genetic Manipulation of Plant Viruses in Unveiling Functional Genomics.

Molecular cloning, a crucial prerequisite for engineering plasmid constructs intended for functional genomic studies, relies on successful restriction and ligation processes. However, the lack of unique restriction sites often hinders construct preparation, necessitating multiple modifications. Moreover, achieving the successful ligation of large plasmid constructs is frequently challenging. To address these limitations, we present a novel PCR strategy in this study, termed 'long-fragment circular-efficient PCR' (LC-PCR). This technique involves one or two rounds of PCR with an additional third-long primer that complements both ends of the newly synthesized strand of a plasmid construct. This results in self-circularization with a nick-gap in each newly formed strand. The LC-PCR technique was successfully employed to insert a partial sequence (210 nucleotides) of the phytoene desaturase gene from Nicotiana benthamiana and a full capsid protein gene (770 nucleotides) of a begomovirus (tomato leaf curl New Delhi virus) into a 16.4 kb infectious construct of a tobamovirus, cucumber green mottle mosaic virus (CGMMV), cloned in pCambia. This was done to develop the virus-induced gene silencing vector (VIGS) and an expression vector for a foreign protein in plants, respectively. Furthermore, the LC-PCR could be applied for the deletion of a large region (replicase enzyme) and the substitution of a single amino acid in the CGMMV genome. Various in planta assays of these constructs validate their biological functionality, highlighting the utility of the LC-PCR technique in deciphering plant-virus functional genomics. The LC-PCR is not only suitable for modifying plant viral genomes but also applicable to a wide range of plant, animal, and human gene engineering under in-vitro conditions. Additionally, the LC-PCR technique provides an alternative to expensive kits, enabling quick introduction of modifications in any part of the nucleotide within a couple of days. Thus, the LC-PCR proves to be a suitable 'all in one' technique for modifying large plasmid constructs through site-directed gene insertion, deletion, and mutation, eliminating the need for restriction and ligation.

Geographical distribution of tomato-infecting begomoviruses in major cucurbits in India: a diagnostic analysis using begomovirus species specific PCR.

Cucurbits are an essential summer-season vegetable crops, but they are highly vulnerable from a range of abiotic and biotic factors. One of the significant biotic factors posing a growing menace to the production of major cucurbits in India is the emergence of tomato-infecting begomoviruses. In this study, we utilized PCR-based species-specific primers, developed earlier in our laboratory for the detection of begomoviruses infecting tomato and chilli plants, to identify begomoviruses in cucurbits across various regions of India. Leaf samples from major cucurbits were collected from different regions of Haryana, Delhi, Uttar Pradesh, Chhattisgarh, Maharashtra, Telangana and Karnataka, during the year 2020-2021. Total nucleic acid (TNA) was extracted from the samples and subjected to PCR using a generic primer specific to begomoviruses. The samples that exhibited positive amplification were further tested using six different species-specific primers targeting specific begomovirus species, namely Tomato leaf curl New Delhi virus (ToLCNDV), Tomato leaf curl Palampur virus (ToLCPalV), Tomato leaf curl Bangalore virus (ToLCBV), Tomato leaf curl Joydebpur virus (ToLCJoV), Tomato leaf curl Gujarat virus (ToLCGuV), and Chilli leaf curl virus (ChiLCV). The PCR analysis revealed that among the 551 plant samples tested, a total of 124 samples exhibited positive amplification using the universal begomovirus PCR. Specifically, 47 samples tested positive for ToLCNDV, 73 samples were positive for ToLCPalV and only one sample showed positive amplification for ChiLCV. However, none of the samples tested positive for ToLCJoV, ToLCGuV and ToLCBV. These findings from our study indicate the prevalence of ToLCNDV and ToLCPalV in major cucurbits across India. Furthermore, the study highlights the varied distribution of begomoviruses in major cucurbits between northern and southern regions of India.

Exigency of Plant-Based Vaccine against COVID-19 Emergence as Pandemic Preparedness.

After two years since the declaration of COVID-19 as a pandemic by the World Health Organization (WHO), more than six million deaths have occurred due to SARS-CoV-2, leading to an unprecedented disruption of the global economy. Fortunately, within a year, a wide range of vaccines, including pathogen-based inactivated and live-attenuated vaccines, replicating and non-replicating vector-based vaccines, nucleic acid (DNA and mRNA)-based vaccines, and protein-based subunit and virus-like particle (VLP)-based vaccines, have been developed to mitigate the severe impacts of the COVID-19 pandemic. These vaccines have proven highly effective in reducing the severity of illness and preventing deaths. However, the availability and supply of COVID-19 vaccines have become an issue due to the prioritization of vaccine distribution in most countries. Additionally, as the virus continues to mutate and spread, questions have arisen regarding the effectiveness of vaccines against new strains of SARS-CoV-2 that can evade host immunity. The urgent need for booster doses to enhance immunity has been recognized. The scarcity of "safe and effective" vaccines has exacerbated global inequalities in terms of vaccine coverage. The development of COVID-19 vaccines has fallen short of the expectations set forth in 2020 and 2021. Furthermore, the equitable distribution of vaccines at the global and national levels remains a challenge, particularly in developing countries. In such circumstances, the exigency of plant virus-based vaccines has become apparent as a means to overcome supply shortages through fast manufacturing processes and to enable quick and convenient distribution to millions of people without the reliance on a cold chain system. Moreover, plant virus-based vaccines have demonstrated both safety and efficacy in eliciting robust cellular immunogenicity against COVID-19 pathogens. This review aims to shed light on the advantages and disadvantages of different types of vaccines developed against SARS-CoV-2 and provide an update on the current status of plant-based vaccines in the fight against the COVID-19 pandemic.

Size variations of mesoporous silica nanoparticle control uptake efficiency and delivery of AC2-derived dsRNA for protection against tomato leaf curl New Delhi virus.

KEY MESSAGE: We report the size dependent uptake of dsRNA loaded MSNPs into the leaves and roots of Nicotiana benthamiana plants and accessed for their relative reduction in Tomato leaf curl New Delhi viral load. A non-GMO method of RNA interference (RNAi) has been recently in practice through direct delivery of double stranded RNA into the plant cells. Tomato leaf curl New Delhi virus (ToLCNDV), a bipartitie begomovirus, is a significant viral pathogen of many crops in the Indian subcontinent. Conventional RNAi cargo delivery strategies for instance uses viral vectors and Agrobacterium-facilitated delivery, exhibiting specific host responses from the plant system. In the present study, we synthesized three different sizes of amine-functionalized mesoporous silica nanoparticles (amino-MSNPs) to mediate the delivery of dsRNA derived from the AC2 (dsAC2) gene of ToLCNDV and showed that these dsRNA loaded nanoparticles enabled effective reduction in viral load. Furthermore, we demonstrate that amino-MSNPs protected the dsRNA molecules from nuclease degradation, while the complex was efficiently taken up by the leaves and roots of Nicotiana benthamiana. The real time gene expression evaluation showed that plants treated with nanoparticles of different sizes ~ 10 nm (MSNPDEA), ~ 32 nm (MSNPTEA) and ~ 66 nm (MSNPNH3) showed five-, eleven- and threefold reduction of ToLCNDV in N. benthamiana, respectively compared to the plants treated with naked dsRNA. This work clearly demonstrates the size dependent internalization of amino-MSNPs and relative efficacy in transporting dsRNA into the plant system, which will be useful in convenient topical treatment to protect plants against their pathogens including viruses. Mesoporous silica nanoparticles loaded with FITC, checked for its uptake into Nicotiana benthamiana.

Delivery of progeny virus from the infectious clone of cucumber green mottle mosaic virus and quantification of the viral load in different host plants.

Cucumber green mottle mosaic virus (CGMMV, genus Tobamovirus) is a widely occurring tobamovirus in cucurbits. The genome of CGMMV has been used previously for the expression of foreign genes in the plant. High throughput delivery and high viral titer are important requirements of foreign protein expression in plant through virus genome-based vector, in this study, Agrobacterium containing infectious construct of CGMMV was infiltrated through syringe, vacuum and high-speed spray to N. benthamiana, cucumber and bottle gourd leaves. The success rate of systemic infection of CGMMV agro-construct through all three methods was higher (80-100%) in N. benthamiana compared to the cucurbits (40-73.3%). To determine the high-throughput delivery of CGMMV in the plant system, four delivery methods viz. rubbing, syringe infiltration, vacuum infiltration and high-speed spray using the progeny virus derived through CGMMV agro-construct were compared in the three different plant species. Based on the rate of systemic infection and time required to perform delivery by different methods, vacuum infiltration was found most efficient for the high-throughput delivery of CGMMV. The quantification of CGMMV through qPCR revealed that CGMMV load varied considerably in leaf and fruit tissues depending with the time of infection. Immediately after expression of symptoms, a high load of CGMMV (~ 1 µg/100 mg of tissues) was noticed in young leaves of N. benthamiana and cucumber. In bottle gourd leaves, the CGMMV load was far low compared to N. benthamiana and cucumber plants. In the fruit tissues of cucumber and bottle gourd higher virus load was observed in mature fruit but not in immature fruit. The findings of the present study will serve as an important base line information to produce foreign protein through CGMMV genome-vector. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03630-y.

Evaluation of sowing dates for managing yellow mosaic disease caused by mungbean yellow mosaic India virus in mungbean.

Yellow mosaic disease, a most important destructive disease of mungbean production caused by Mungbean yellow mosaic India virus (MYMIV) under North Indian conditions. However, management of this deadly disease is still becoming the biggest challenge due to breaking of resistance under changing climatic conditions. Hence, a field experiment was conducted at IARI, New Delhi, India during Kharif 2021 and Spring-Summer 2022 to understand the sowing date influence on incidence of MYMIV in mungbean resistant (Pusa 1371) and susceptible (Pusa 9531) cultivars. The results revealed the higher disease incidence percentage (PDI) in the first sowing (15-20th July) of Kharif and third sowing (5-10th April) of Spring-Summer season. The mean PDI ranged from 25-41% to 11.80-13.54% for resistant followed by 23.13-49.84% and 14.40-21.45% in susceptible cultivar during Kharif and Spring-Summer season respectively. The detection of MYMIV through DAC-ELISA at 405 nm showed the absorbance values of 0.40-0.60 in susceptible and < 0.45 in resistant cultivar during the Kharif and 0.40-0.45 in Spring-Summer season. The PCR analysis with MYMIV and MYMV specific primers indicated the presence of only MYMIV and absence of MYMV in the present studied mungbean cultivars. The PCR analysis with DNA-B specific primers resulted in the amplification of 850 bp from both susceptible and resistant cultivars during the first sowing of Kharif whereas amplification was observed only in susceptible cultivar with second and third sowings of Kharif and all the three sowings of Spring-Summer season. The experiment results revealed that the most suitable date of sowing for mungbean will be before 30th March during Spring-Summer and after third week of July (30th July to 10th August) during the Kharif season under Delhi conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03621-z.

Leaf Curl Epidemic Risk in Chilli as a Consequence of Vector Migration Rate and Contact Rate Dynamics: A Critical Guide to Management.

Chilli is an important commercial crop grown in tropical and subtropical climates. The whitefly-transmitted chilli leaf curl virus (ChiLCV) is a serious threat to chilli cultivation. Vector migration rate and host-vector contact rate, the major drivers involved in the epidemic process, have been pinpointed to link management. The complete interception of migrant vectors immediately after transplantation has been noted to increase the survival time (to remain infection free) of the plants (80%) and thereby delay the epidemic process. The survival time under interception (30 days) has been noted to be nine weeks (p < 0.05), as compared to five weeks, which received a shorter period of interception (14-21 days). Non-significant differences in hazard ratios between 21- and 30-day interceptions helped optimize the cover period to 26 days. Vector feeding rate, estimated as a component of contact rate, is noted to increase until the sixth week with host density and decline subsequently due to plant succulence factor. Correspondence between the peak time of virus transmission or inoculation rate (at 8 weeks) and contact rate (at 6 weeks) suggests that host succulence is of critical importance in host-vector interactions. Infection proportion estimates in inoculated plants at different leaf stages have supported the view that virus transmission potential with plant age decreases, presumably due to modification in contact rate. The hypothesis that migrant vectors and contact rate dynamics are the primary drivers of the epidemic has been proved and translated into rules to guide management strategies.

Engineering giant excitonic coupling in bioinspired, covalently bridged BODIPY dyads.

Strong excitonic coupling in photosynthetic systems is believed to enable efficient light absorption and quantitative charge separation, motivating the development of artificial multi-chromophore arrays with equally strong or even stronger excitonic coupling. However, large excitonic coupling strengths have typically been accompanied by fast non-radiative recombination, limiting the potential of the arrays for solar energy conversion as well as other applications such as fluorescent labeling. Here, we report giant excitonic coupling leading to broad optical absorption in bioinspired BODIPY dyads that have high photostability, excited-state lifetimes at the nanosecond scale, and fluorescence quantum yields of nearly 50%. Through the synthesis, spectroscopic characterization, and computational modeling of a series of dyads with different linking moieties, we show that the strongest coupling is obtained with diethynylmaleimide linkers, for which the coupling occurs through space between BODIPY units with small separations and slipped co-facial orientations. Other linkers allow for broad tuning of both the relative through-bond and through-space coupling contributions and the overall strength of interpigment coupling, with a tradeoff observed in general between the strength of the two coupling mechanisms. These findings open the door to the synthesis of molecular systems that function effectively as light-harvesting antennas and as electron donors or acceptors for solar energy conversion.

Effects of Solvent Dielectric on Thermally Activated Delayed Fluorescence: A Predictive Computational Polarization Consistent Approach.

We study computationally thermally activated delayed fluorescence (TADF) in donor-acceptor compounds. The relevant electronic excited states that are strongly affected by the dielectric environment are treated by a polarization consistent framework. The high fidelity potential energy surfaces are used following a quantum-mechanical Fermi's golden rule (FGR) picture to calculate rates of intersystem crossing (ISC) and reverse intersystem crossing (RISC). To demonstrate the potency of the approach, we consider isomers of benzonitrile functionalized tert-butyl-substituted dimethylacridine (DMAC-BN), which were recently found to perform well as TADF emitters. The calculated excited state energies that appear to reproduce well measured spectral trends with respect to the dielectric constant are used to parametrize ISC/RISC FGR rates. The calculated rates reproduce well measured rates, whereas semiclassical based rates are grossly underestimated. In particular, we find in agreement with the recent experimental study [Phys. Rev. Appl.2019, 12, 044021] that the ortho and meta isomers are significantly more effective as TADF emitters. The computational framework provides valuable insight at the molecular level into RISC rates and therefore can contribute to the design of materials of increased TADF efficiency.

Host biology and genomic properties of Plumeria mosaic virus, a tobamovirus discovered in a temple tree in India co-infecting with frangipani mosaic virus.

Temple tree (Plumeria rubra f. acutifolia), an important fragrant-flower tree extensively used in the urban landscaping is known to be infected with a tobamovirus, frangipani mosaic virus (FrMV). In this study, we describe another tobamovirus, Plumeria mosaic virus (PluMV) infecting temple tree in India. PluMV was isolated from an old temple tree co-infected with FrMV. The presence of another tobamovirus was initially realized based on the distinct symptoms on Gomphrena globosa (globe amaranth), a non-host of FrMV. PluMV was highly transmissible through simple rub-inoculation. In host-range study, brinjal (Solanum melongena), chilli (Capsicum annuum), datura (Datura stramonium), globe amaranth and tobacco (Nicotiana benthamiana, N. glutinosa, N. tabacum cv. Xanthi) could differentiate PluMV from FrMV. The complete genome sequence of PluMV was determined (6,688 nucleotides [nt], GenBank KJ395757), which showed the genome structure typical of tobamovirus encoding four proteins: small replicase (3,549 nt/130 kDa), large replicase (5,061 nt/188 kDa), movement protein (770 nt/29 kDa) and coat protein (527 nt/19 kDa). The 5' and 3' UTR of PluMV contained 91 and 284 nt, respectively. The PluMV genome was 45 nts longer than that of FrMV and shared only 71.4-71.6% sequence identity with FrMV and < 50% sequence identity with the rest of the other members of the genus Tobamovirus. PluMV shared a close but a divergent evolutionary relationship with FrMV. Based on the species demarcation guidelines of ICTV (<90% genome sequence identity), PluMV was considered as a new tobamovirus species. As PluMV was serologically related with FrMV, differential diagnostic assays such as simplex and duplex RT-PCR were developed, which revealed that PluMV naturally existed in both the species of temple tree, P. rubra f. acutifolia and P. rubra f. obtusa in India either alone or in mixed infection with FrMV.

Random distribution of nucleotide polymorphism throughout the genome of tomato-infecting begomovirus species occurring in India: implication in PCR based diagnosis.

Multiple begomovirus species are known to cause leaf curl disease in tomato in India. In order to develop specific and generic PCR based diagnostics for the tomato-infecting begomoviruses, in this study, we attempted to design primers initially based on the multiple alignment of the complete genome sequence of DNA-A component. However, the specific nucleotide stretches adequate for preparing specific primers could not be obtained. Alternatively, the online Primer-BLAST tool that offers designing of target-specific PCR primers was attempted to prepare specific primers targeting three clones (DNA-A) of tomato-infecting begomovirus species (Tomato leaf curl New Delhi virus, Tomato leaf curl Palampur virus and Tomato leaf curl Joydebpur virus) selected based on their sequence identity and phylogenetic relatedness. The primers derived from Primer-BLAST tool showed high level of cross-reaction among these begomovirus species and therefore were not able to differentiate these target begomovirus species. In order to understand the reason of cross-reactivity further sequence analysis revealed the high occurrence of single nucleotide variations (SNVs) compared to the multi-nucleotide stretches. There was no SNV hot-spot in the genome, rather the SNVs were randomly distributed throughout the genome of these begomovirus species. This pattern of nucleotide diversities among these tomato-infecting begomoviruses seriously implicated on developing specific PCR diagnostics. On the contrary, sequence analysis showed high sequence conservancy, which enabled to develop a generic PCR diagnostic for these begomoviruses. Our study, thus showed that the genome sequence diversity pattern among the tomato-infecting begomoviruses in India poses challenges in developing PCR based specific diagnostics. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00785-9.

Direct Foliar Application of dsRNA Derived From the Full-Length Gene of NSs of Groundnut Bud Necrosis Virus Limits Virus Accumulation and Symptom Expression.

Groundnut bud necrosis virus (GBNV) is the most significant member of the genus Orthotospovirus occurring in the Indian subcontinent. There is hardly any effective measure to prevent GBNV in crop plants. In order to develop GBNV infection prevention procedure, we examined the effect of the direct foliar application of double-stranded RNA (dsRNA) derived from the full-length NSs gene (1,320 nucleotides) of GBNV. The bacterially expressed dsRNA to the non-structural (dsNSs) gene of GBNV was purified and delivered to plants as an aqueous suspension containing 0.01% Celite for evaluating its efficacy in preventing GBNV infection in systemic host, Nicotiana benthamiana as well as in local lesion and systemic host, cowpea cv. Pusa Komal (Vigna unguiculata). The dsNSs application and challenge-inoculation were conducted in three different combinations, where plants were challenge-inoculated with GBNV a day after, immediately, and a day before the application of dsNSs. N. benthamiana plants, which were not treated with dsRNA showed severe systemic wilting and death by 9-16 days post-inoculation (dpi). The non-treated cowpea plants exhibited many chlorotic and necrotic lesions on the cotyledonary leaves followed by systemic necrosis and death of the plants by 14-16 dpi. The dsNSs treated plants in all the combinations showed significant reduction of disease severity index in both N. benthamiana and cowpea. The treatment combination where the GBNV inoculation was conducted immediately after the dsNSs treatment was found to be the most effective treatment in preventing symptom expression. The viral RNA analysis by real time PCR also showed 20 and 12.5 fold reduction of GBNV in cowpea and N. benthamiana, respectively. Our results suggest that the foliar application of dsRNA derived from the full-length NSs gene of GBNV through Celite is successful in delivering long dsRNA leading to effective prevention of GBNV infection.

Molecular and biological characterization of soybean yellow mottle mosaic virus severe strain infecting soybean in India.

The complete nucleotide sequence and genome organization of soybean yellow mottle mosaic virus severe strain causing bright yellow mosaic, mottling and puckering symptoms in soybean (Glycine max) from India was determined. The monopartite single stranded genomic RNA is 3974 nuclotides long and has the potential to encode six viral proteins viz., p25, p83, p8, p10, p39 and p25. The SYMMV-Sb isolate differed from mungbean strain with 69 nucleotides and nine aminoacids dispersed over the various ORFs. Comparative sequence analysis revealed that SYMMV-Sb shared 98% nt sequence identity at complete genome level and 96-100% at all ORFs level with SYMMV mungbean strain from India and 71-92% identity with SYMMV Korean soybean isolate, whereas it showed very low sequence identity with other tombusviridae members (2-53%). The phylogenetic analysis showed the clustering of SYMMV-Sb along with other members of genus Gammacarmovirus. The SYMMV-Sb isolate produced chlorotic blotches, mild and veinal mottling, necrosis and puckering symptoms in various leguminous host plants. The symptomatalogy of the soybean isolate was differed from mungbean strain as earlier induced severe symptoms on soybean and mild symptoms on mungbean. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02925-2.

Sub-cellular localization of suppressor proteins of tomato leaf curl New Delhi virus.

Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, is the most important among the 14 species of begomoviruses infecting tomato in Indian subcontinent. Begomovirus is known to  evade RNA silencing of host plants through suppressor proteins. However, in case of ToLCNDV, the suppressor proteins have not been studied well. The objective of the study is to know the sub-cellular localization of three suppressor proteins encoded by AV2, AC2 and AC4 ORFs of ToLCNDV in Nicotiana benthamiana. AV2, AC2 and AC4 ORFs of ToLCNDV were  cloned and sequenced (accession numbers MW423574, MW423576, MW423575, respectively) from a ToLCNDV isolate characterized earlier (accession number MW429271) and GFP tagged constructs were prepared in a plant expressing binary vector pEarleygate103. Bioinformatics analysis using Peptide 2.0 server predicted that all these proteins have more basic amino acid residues then acidic amino acid and AV2 protein has more hydrophobic amino acid residues. ScanProsite server predicted presence of different fuctional motifs in these proteins amongst which presence of kinase motif was observed in all of them. Virus mPLoc server predicted their subcellular localization. The suppressor gene constructs were agroinfiltrated on to leaves of one month old N. benthamiana plants and their subcellular localization has been studied through confocal microscopy. Results have shown that AV2 localizes in the host cell membrane and nucleus, AC2 in the nucleus and AC4 in the host cell membrane. Earlier reports with other begomoviruses also showed similar localization behaviour of these suppressor protein except AV2, where it was shown to be present in cytoplasm. Such localization study will help understand the mechanism of their suppression activity.

Overexpression of an insect virus encoded silencing suppressor does not enhance plants' susceptibility to its natural virus.

RNA silencing plays a key role in shielding plant and animal hosts against viral invasion and infection. Viruses encode RNA silencing suppressors (RSS) to block small RNA guided silencing of viral transcripts. The B2 protein encoded by Flock House virus (FHV) is a well-characterized RSS that facilitates infection in insects. It has been shown to act as a functional RSS in plants. FHVB2 over-expressing tobacco plants were used to study the effect of RSS on plant susceptibility to Tobacco mosaic virus (TMV), its natural pathogen. The major symptoms observed in TMV-infected transgenic plants were greenish mosaic, puckering and distortion of leaves, but the infected transgenic leaves were able to resist chlorophyll loss. The infected leaves of transgenic plants showed no significant difference in accumulation of virus when compared with that of the wild type plants. FHVB2 plants showed higher levels of H2O2 and the ROS scavenging enzymes, APX and SOD. This suggests that interference of FHVB2 with RNA silencing machinery may activate alternative defense pathways in the plants so that they are not overly sensitive to TMV infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-020-00644-5.