Exogenous Application of dsRNA for Protection against Tomato Leaf Curl New Delhi Virus.

Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging plant pathogen, fast spreading in Asian and Mediterranean regions, and is considered the most harmful geminivirus of cucurbits in the Mediterranean. ToLCNDV infects several plant and crop species from a range of families, including Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae and Euphorbiaceae. Up to now, protection from ToLCNDV infection has been achieved mainly by RNAi-mediated transgenic resistance, and non-transgenic fast-developing approaches are an urgent need. Plant protection by the delivery of dsRNAs homologous to a pathogen target sequence is an RNA interference-based biotechnological approach that avoids cultivating transgenic plants and has been already shown effective against RNA viruses and viroids. However, the efficacy of this approach against DNA viruses, particularly Geminiviridae family, is still under study. Here, the protection induced by exogenous application of a chimeric dsRNA targeting all the coding regions of the ToLCNDV DNA-A was evaluated in zucchini, an important crop strongly affected by this virus. A reduction in the number of infected plants and a delay in symptoms appearance, associated with a tendency of reduction in the viral titer, was observed in the plants treated with the chimeric dsRNA, indicating that the treatment is effective against geminiviruses but requires further optimization. Limits of RNAi-based vaccinations against geminiviruses and possible causes are discussed.

Molecular markers associated with resistance to squash leaf curl China virus and tomato leaf curl New Delhi virus in tropical pumpkin (Cucurbita moschata Duchesne ex Poir.) breeding line AVPU1426.

Virus diseases are a major production constraint for pumpkin. Recessive resistance to squash leaf curl China virus and tomato leaf curl New Delhi virus has been mapped in Cucurbita moschata (Duchesne ex Poir.) breeding line AVPU1426 to chromosomes 7 and 8, respectively. Molecular markers tightly associated with the resistance loci have been developed and were able to correctly predict resistance and susceptibility with an accuracy of 99% for squash leaf curl China virus resistance and 94.34% for tomato leaf curl New Delhi virus in F2 and back cross populations derived from the original resistance source AVPU1426. The markers associated with resistance are recommended for use in marker-assisted breeding.

Distinct tomato yellow leaf curl Chuxiong virus isolated from whiteflies and plants in China and its symptom determinant and suppressor of post-transcriptional gene silencing.

A begomovirus isolated from whiteflies (Bemisia tabaci) and tomato, sweet potato in China was found to be representative of a distinct begomovirus species, for which the name tomato yellow leaf curl Chuxiong virus (TYLCCxV) is proposed. The results of genomic identification and sequence comparison showed that TYLCCxV shares the highest complete nucleotide sequence identity (88.3%) with croton yellow vein mosaic virus (CroYVMV), and may have originated from the recombination between synedrella leaf curl virus (SyLCV) and squash leaf curl Yunnan virus (SLCuYV). Agrobacterium-mediated inoculation showed that TYLCCxV is highly infectious for a range of plant species, producing upward leaf curling, leaf crumpling, chlorosis, distortion, and stunt symptoms in Solanum lycopersicum plants. The results of Southern blot indicated that TYLCCxV is capable of efficiently replicating two heterologous betasatellites. The inoculation of PVX::C4 on Nicotiana benthamiana induced upward leaf curling and stem elongation symptoms, suggesting that TYLCCxV C4 functions as a symptom determinant. TYLCCxV V2 is an important virulence factor that induces downward leaf curling symptoms, elicits systemic necrosis, and suppresses local and systemic GFP silencing in co-agroinfiltrated N. benthamiana and transgenic 16c plants. Considering the multifunctional virulence proteins V2 and C4, the possibility of TYLCCxV causing devastating epidemics on tomato in China is discussed.

Molecular characterization and pathogenicity of a novel monopartite geminivirus infecting tobacco in China.

The occurrence of geminiviruses causes significant economic losses in many economically important crops. In this study, a novel geminivirus isolated from tobacco in Sichuan province of China, named tomato leaf curl Chuxiong virus (TLCCxV), was characterized by small RNA-based deep sequencing. The full-length of TLCCxV genome was determined to be 2744 nucleotides (nt) encoding six open reading frames. Phylogenetic and genome-wide pairwise identity analysis revealed that TLCCxV shared less than 91% identities with reported geminiviruses. A TLCCxV infectious clone was constructed and successfully infected Nicotiana benthamiana, N. tabacum, N. glutinosa, Solanum lycopersicum and Petunia hybrida plants. Furthermore, expression of the V2, C1 and C4 proteins through a potato virus X vector caused severe chlorosis or necrosis symptom in N. benthamiana. Taken together, we identified a new geminivirus in tobacco plants, and found that V2, C1 and C4 contribute to symptom development.

The C4 Protein of TbLCYnV Promotes SnRK1 ß2 Degradation Via the Autophagy Pathway to Enhance Viral Infection in N. benthamiana.

Geminiviruses are a group of single-stranded DNA viruses that have developed multiple strategies to overcome host defenses and establish viral infections. Sucrose nonfermenting-1-related kinase 1 (SnRK1) is a key regulator of energy balance in plants and plays an important role in plant development and immune defenses. As a heterotrimeric complex, SnRK1 is composed of a catalytic subunit α (SnRK1 α) and two regulatory subunits, ß and γ. Previous studies on SnRK1 in plant defenses against microbial pathogens have mainly focused on SnRK1 α. In this study, we validated the interaction between the C4 protein encoded by tobacco leaf curl Yunnan virus (TbLCYnV) and the regulatory subunit ß of Nicotiana benthamiana SnRK1, i.e., NbSnRK1 ß2, and identified that the Asp22 of C4 is critical for TbLCYnV C4-NbSnRK1 ß2 interactions. NbSnRK1 ß2 silencing in N. benthamiana enhances susceptibility to TbLCYnV infection. Plants infected with viral mutant TbLCYnV (C4D22A), which contains the mutant version C4 (D22A) that is incapable of interacting with NbSnRK1 ß2, display milder symptoms and lower viral accumulation. Furthermore, we discovered that C4 promotes NbSnRK1 ß2 degradation via the autophagy pathway. We herein propose a model by which the geminivirus C4 protein causes NbSnRK1 ß2 degradation via the TbLCYnV C4-NbSnRK1 ß2 interaction to antagonize host antiviral defenses and facilitates viral infection and symptom development in N. benthamiana.

Transcriptome and small RNAome profiling uncovers how a recombinant begomovirus evades RDRγ-mediated silencing of viral genes and outcompetes its parental virus in mixed infection.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) causes severe disease of cultivated tomatoes. Geminiviruses replicate circular single-stranded genomic DNA via rolling-circle and recombination-dependent mechanisms, frequently generating recombinants in mixed infections. Circular double-stranded intermediates of replication also serve as templates for Pol II bidirectional transcription. IS76, a recombinant derivative of TYLCV with a short sequence in the bidirectional promoter/origin-of-replication region acquired from a related begomovirus, outcompetes TYLCV in mixed infection and breaks disease resistance in tomato Ty-1 cultivars. Ty-1 encodes a γ-clade RNA-dependent RNA polymerase (RDRγ) implicated in Dicer-like (DCL)-mediated biogenesis of small interfering (si)RNAs directing gene silencing. Here, we profiled transcriptome and small RNAome of Ty-1 resistant and control susceptible plants infected with TYLCV, IS76 or their combination at early and late infection stages. We found that RDRγ boosts production rates of 21, 22 and 24 nt siRNAs from entire genomes of both viruses and modulates DCL activities in favour of 22 and 24 nt siRNAs. Compared to parental TYLCV, IS76 undergoes faster transition to the infection stage favouring rightward transcription of silencing suppressor and coat protein genes, thereby evading RDRγ activity and facilitating its DNA accumulation in both single and mixed infections. In coinfected Ty-1 plants, IS76 efficiently competes for host replication and transcription machineries, thereby impairing TYLCV replication and transcription and forcing its elimination associated with further increased siRNA production. RDRγ is constitutively overexpressed in Ty-1 plants, which correlates with begomovirus resistance, while siRNA-generating DCLs (DCL2b/d, DCL3, DCL4) and genes implicated in siRNA amplification (α-clade RDR1) and function (Argonaute2) are upregulated to similar levels in TYLCV- and IS76-infected susceptible plants. Collectively, IS76 recombination facilitates replication and promotes expression of silencing suppressor and coat proteins, which allows the recombinant virus to evade the negative impact of RDRγ-boosted production of viral siRNAs directing transcriptional and posttranscriptional silencing.

First report of Ageratum yellow vein virus infecting papaya in Lampung, Indonesia.

BACKGROUND: Papaya (Carica papaya) is a tropical fruit of great economic and nutritional importance, loved for its sweet and delicious flesh. However, papaya cultivation faces serious challenges in the form of Begomovirus attacks. Begomoviruses are a group of viruses that pose a serious threat to plants worldwide. Including papaya, Begomovirus has become a significant threat to papaya production in various parts of the world and has been identified in several regions in Indonesia. METHODS: DNA was extracted from seven samples representing different papaya growing areas using a Plant Genomic DNA Mini Kit. Genomic DNA from the samples was subjected to PCR using universal primers of AC2, AC1, SPG1 and SPG2. The PCR products then sequenced using the dideoxy (Sanger) approach. The obtained sequence then compared to the gene bank using BLAST software available at NCBI. Multiple sequence alignment and phylogenetic tree construction were analyzed using the MEGA11 program. RESULTS: Detection based on viral nucleic acid in papaya plants in Pesawaran, Lampung Province with seven sampling points using universal primers SPG1/SPG2 showed positive results for Begomovirus infection with visible DNA bands measuring ± 900 bp. Direct nucleotide sequencing using SPG1/SPG2 primers for the AC2 and AC1 genes of the Begomovirus and confirmed by the BLAST program showed that papaya samples were infected with Ageratum yellow vein virus (AYVV). The phylogenetic results show that AYVV from papaya samples has a close relationship with the AYVV group from several other countries, with 98% homology. CONCLUSION: In the papaya cultivation area in Pesawaran, Lampung province, it was identified as Begomovirus, Ageratum yellow vein virus (AYVV) species and is closely related to the AYVV group from several other countries. Overall, our study further suggests that Ageratum acts as an alternative host and reservoir for Begomovirus.

Breakdown of Ty-1-Based Resistance to Tomato Yellow Leaf Curl Virus in Tomato Plants at High Temperatures.

The global dissemination of the Israel (IL) and mild (Mld) strains of tomato yellow leaf curl virus (TYLCV) (family Geminiviridae, genus Begomovirus) is a major threat to tomato production in many regions worldwide. The use of resistant hybrid cultivars bearing the dominant resistance genes Ty-1, Ty-3, and Ty-3a has become a common practice for controlling tomato yellow leaf curl disease (TYLCD) caused by TYLCV. However, TYLCD symptoms have been sporadically observed in resistant cultivars grown in seasons when temperatures are high. In this study, we used TYLCV-resistant cultivars with confirmed presence of Ty-1, which were determined using newly developed allele-specific markers based on polymorphisms within the locus. These Ty-1-bearing resistant tomato plants and susceptible plants were infected with TYLCV and grown at moderate or high temperatures. Under high-temperature conditions, the Ty-1-bearing tomato cultivar Momotaro Hope (MH) infected with TYLCV-IL had severe TYLCD symptoms, which were almost equivalent to those of the susceptible cultivar. However, MH plants infected with TYLCV-Mld were symptomless or had slight symptoms under the same temperature condition. The quantitative analysis of the TYLCV-IL viral DNA content revealed a correlation between symptom development and viral DNA accumulation. Furthermore, under high-temperature conditions, TYLCV-IL caused severe symptoms in multiple commercial tomato cultivars with different genetic backgrounds. Our study provided the scientific evidence for the experientially known phenomenon by tomato growers, and it is anticipated that global warming, associated with climate change, could potentially disrupt the management of TYLCV in tomato plants mediated by the Ty-1 gene.

From Metagenomics to Ecogenomics: NGS-Based Approaches for Discovery of New Circular DNA Single-Stranded Viral Species.

Viruses comprise the most abundant genetic material in the biosphere; however, global viral genomic population (virome) has been largely underestimated. Recently, high-throughput sequencing (HTS) has provided a powerful tool for the detection of known viruses and the discovery of novel viral species from environmental and individual samples using metagenomics and ecogenomics approaches, respectively. Viruses with circular DNA single-stranded (ssDNA) genomes belonging to the begomovirus genera (family Geminiviridae) constitute the largest group of emerging plant viruses worldwide. The knowledge of begomoviruses viromes is mostly restricted to crop plant systems; nevertheless, it has been described that noncultivated plants specifically at the interface between wild and cultivated plants are important reservoirs leading to viral evolution and the emergence of new diseases. Here we present a protocol that allows the identification and isolation of known and novel begomoviruses species infecting cultivated and noncultivated plant species. The method consists of circular viral molecules enrichment by rolling circle amplification (RCA) from begomovirus-positive total plant DNA, followed by NGS-based metagenomic sequencing. Subsequently, metagenomic reads are processed for taxonomic classification using Viromescan software and a customized Geminiviridae family database, and begomovirus-related reads are used for contigs assembly and annotation using Spades software and Blastn algorithm, respectively. Then, the obtained begomovirus-related signatures are used as templates for specific primers design and implemented for PCR-based ecogenomic identification of individual samples harboring the corresponding viral species. Lastly, full-length begomovirus genomes are obtained by RCA-based amplification from total plant DNA of selected individual samples, cloning, and viral molecular identity corroborated by Sanger sequencing. Conclusively, the identification and isolation of a novel monopartite begomovirus species native to the New World (NW) named Gallium leaf deformation virus (GLDV) is shown.

Cotton microbiome profiling and Cotton Leaf Curl Disease (CLCuD) suppression through microbial consortia associated with Gossypium arboreum.

The failure of breeding strategies has caused scientists to shift to other means where the new approach involves exploring the microbiome to modulate plant defense mechanisms against Cotton Leaf Curl Disease (CLCuD). The cotton microbiome of CLCuD-resistant varieties may harbor a multitude of bacterial genera that significantly contribute to disease resistance and provide information on metabolic pathways that differ between the susceptible and resistant varieties. The current study explores the microbiome of CLCuD-susceptible Gossypium hirsutum and CLCuD-resistant Gossypium arboreum using 16 S rRNA gene amplification for the leaf endophyte, leaf epiphyte, rhizosphere, and root endophyte of the two cotton species. This revealed that Pseudomonas inhabited the rhizosphere while Bacillus was predominantly found in the phyllosphere of CLCuV-resistant G. arboreum. Using salicylic acid-producing Serratia spp. and Fictibacillus spp. isolated from CLCuD-resistant G. arboreum, and guided by our analyses, we have successfully suppressed CLCuD in the susceptible G. hirsutum through pot assays. The applied strains exhibited less than 10% CLCuD incidence as compared to control group where it was 40% at 40 days post viral inoculation. Through detailed analytics, we have successfully demonstrated that the applied microbes serve as a biocontrol agent to suppress viral disease in Cotton.

Molecular Signature of a Novel Alternanthera Yellow Vein Virus Variant Infecting the Ageratum conyzoides Weed in Oman.

Alternanthera yellow vein virus (AlYVV), a monopartite begomovirus, has been identified infecting a diverse range of crops and native plants in Pakistan, India, and China. However, distinctive yellow vein symptoms, characteristic of begomovirus infection, were observed on the Ageratum conyzoides weed in Oman, prompting a thorough genomic characterization in this study. The results unveiled a complete genome sequence of 2745 base pairs and an associated betasatellite spanning 1345 base pairs. In addition, Sequence Demarcation Tool analyses indicated the highest nucleotide identity of 92.8% with a previously reported AlYVV-[IN_abalpur_A_17:LC316182] strain, whereas the betasatellite exhibited a 99.8% nucleotide identity with isolates of tomato leaf curl betasatellite. Thus, our findings propose a novel AlYVV Oman virus (AlYVV-OM) variant, emphasizing the need for additional epidemiological surveillance to understand its prevalence and significance in Oman and the broader region. To effectively manage the spread of AlYVV-OM and minimize its potential harm to (agro)ecosystems, future research should focus on elucidating the genetic diversity of AlYVV-OM and its interactions with other begomoviruses.

Specific Nucleotides in the Common Region of the Begomovirus Tomato Rugose Mosaic Virus (ToRMV) Are Responsible for the Negative Interference over Tomato Severe Rugose Virus (ToSRV) in Mixed Infection.

Mixed infection between two or more begomoviruses is commonly found in tomato fields and can affect disease outcomes by increasing symptom severity and viral accumulation compared with single infection. Viruses that affect tomato include tomato severe rugose virus (ToSRV) and tomato rugose mosaic virus (ToRMV). Previous work showed that in mixed infection, ToRMV negatively affects the infectivity and accumulation of ToSRV. ToSRV and ToRMV share a high degree of sequence identity, including cis-elements in the common region (CR) and their specific recognition sites (iteron-related domain, IRD) within the Rep gene. Here, we investigated if divergent sites in the CR and IRD are involved in the interaction between these two begomoviruses. ToSRV clones were constructed containing the same nucleotides as ToRMV in the CR (ToSRV-A(ToR:CR)), IRD (ToSRV-A(ToR:IRD)) and in both regions (ToSRV-A(ToR:CR+IRD)). When plants were co-inoculated with ToRMV and ToSRV-A(ToR:IRD), the infectivity and accumulation of ToSRV were negatively affected. In mixed inoculation of ToRMV with ToSRV-A(ToR:CR), high infectivity of both viruses and high DNA accumulation of ToSRV-A(ToR:CR) were observed. A decrease in viral accumulation was observed in plants inoculated with ToSRV-A(ToR:CR+IRD). These results indicate that differences in the CR, but not the IRD, are responsible for the negative interference of ToRMV on ToSRV.

Exogenous delivery of dsRNA for management of mungbean yellow mosaic virus on blackgram.

MAIN CONCLUSION: Exogenous application of dsRNA molecules targeting MYMV genes offers a promising approach to effectively mitigate yellow mosaic disease in blackgram, demonstrating potential for sustainable plant viral disease management. The exogenous application of double-stranded RNA (dsRNA) molecules to control plant viral diseases is gaining traction due to its advantages over conventional methods, such as target specificity, non-polluting nature, and absence of residue formation. Furthermore, this approach does not involve genome modification. In this study, dsRNA molecules targeting the coat protein gene (dsCP) and replication initiator protein gene (dsRep) of mungbean yellow mosaic virus (MYMV) were synthesised using an in vitro transcription method. To evaluate the effectiveness of dsRNA treatment, blackgram plants exhibiting MYMV symptoms at the first trifoliate stage were subjected to exogenous application of dsRNA. Second, third, and fourth trifoliate leaves, which emerged at 7, 15, and 21 days after dsRNA application, respectively, were monitored for MYMV symptoms. Remarkably, a significant reduction in yellow mosaic disease (YMD) symptoms was observed in the newly emerged trifoliate leaves of MYMV-infected blackgram plants after treatment with dsRNA targeting both gene regions. This reduction was evident as a decrease in the intensity of yellow mosaic coverage on the leaf lamina compared to control. dsCP effectively reduced the MYMV titre in the treated plants for up to 15 days. However, dsRep demonstrated greater efficiency in conferring resistance to MYMV at 15 days post-application. These findings were supported by quantitative real-time PCR analysis, where the observed Ct values for DNA extracted from dsRep-treated plants were significantly higher compared to the Ct values of DNA from dsCP-treated plants at 15 days post-application. Similarly, higher viral copy numbers were observed in dsCP-treated plants 15 days after dsRNA treatment, in contrast to plants treated with dsRep.

Size Restriction Is Required for Proper Functioning of a Bipartite Begomovirus AC4 Protein.

Many geminiviruses, including members of the genus Begomovirus, produce a protein known as C4 or AC4. Whereas C4/AC4 typically consists of more than 80 amino acid residues, a few are much shorter. The significance of these shorter C4/AC4 proteins in viral infection and why the virus maintains their abbreviated length is not yet understood. The AC4 of the begomovirus Tomato leaf curl Hsinchu virus contains only 65 amino acids, but it extends to 96 amino acids when the natural termination codon is replaced with a normal codon. We discovered that both interrupting and extending AC4 were harmful to tomato leaf curl Hsinchu virus (ToLCHsV). The extended AC4 (EAC4) also showed a reduced ability to promote the infection of the heterologous virus Potato virus X than the wild-type AC4. When the wild-type AC4 was fused with yellow fluorescent protein (AC4-YFP), it was predominantly found in chloroplasts, whereas EAC4-YFP was mainly localized to the cell periphery. These results suggest that ToLCHsV's AC4 protein is important for viral infection, and the virus may benefit from the abbreviated length, because it may lead to chloroplast localization. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Association of a novel begomovirus species with fenugreek yellow vein disease in India.

BACKGROUND: Fenugreek (Trigonella foenum-graecum L.) is an annual medicinal and spice crop belonging to the family Fabaceae. The occurrence of a yellow vein disease was recorded in fenugreek in Jodhpur (India) in 2022. The infection of begomoviruses in legume crops results in significant yield loss and major economic loss. The current study reports an association of a novel begomovirus species associated with yellow vein disease in Fenugreek. METHODS AND RESULTS: In symptomatic fenugreek plants, geminivirus-like particles were visible under a transmission electron microscope. Further, nucleotide sequence analysis of the rolling circle amplified product revealed 2743 nucleotide DNA-A genome with close relatedness to French bean leaf curl virus (88.21%) and Senna leaf curl virus (87.63%). It was proposed as a new begomovirus species, Fenugreek yellow vein Rajasthan virus. The genome organization suggested the presence of a typical nonanucleotide sequence along with 7 ORFs in DNA-A. A possible recombination event took place in the coat protein (V1) region with Pedilanthus leaf curl virus and Chilli leaf curl virus as major and minor parents. The recombinant virus poses possible threats to several other legume crops. To the best of our knowledge, this is the first report of the association of FeYVRaV with fenugreek yellow vein disease from northwestern India. CONCLUSIONS: In conclusion, the presence of a novel begomovirus species associated with yellow vein disease in fenugreek is alarming and needs further studies on its infectivity to prevent its spread to legume crops.

Tomato golden net virus and tomato yellow net virus: two novel New World begomoviruses with monopartite genomes.

Two novel tomato-infecting begomoviruses were discovered via high-throughput sequencing in Brazil. Both viruses were also Sanger-sequenced and displayed DNA-A components phylogenetically related to New World bipartite begomoviruses. The names tomato golden net virus (ToGNV) and tomato yellow net virus (ToYNV) were proposed. The majority of the New World begomoviruses has bipartite genomes. However, extensive analyses revealed that ToGNV and ToYNV have monopartite genomes, because no cognate DNA-B components were detected. Hence, they may comprise a unique group of monopartite New World begomoviruses, which have enormous biological, molecular, and plant breeding interest.

Constant ratio between the genomic components of bipartite begomoviruses during infection and transmission.

The genomic components of multipartite viruses are encapsidated in separate virus particles, and the frequencies of genomic components represent one of the key genetic features. Many begomoviruses of economic significance are bipartite, and the details of the association between their genomic components remain largely unexplored. We first analyzed the temporal dynamics of the quantities of DNA-A and DNA-B and the B/A ratio of the squash leaf curl China virus (SLCCNV) in plants and found that while the quantities of DNA-A and DNA-B varied significantly during infection, the B/A ratio remained constant. We then found that changes in the B/A ratio in agrobacteria inoculum may significantly alter the B/A ratio in plants at 6 days post inoculation, but the differences disappeared shortly thereafter. We next showed that while the quantities of DNA-A and DNA-B among plants infected by agrobacteria, sap transmission and whitefly-mediated transmission differed significantly, the B/A ratios were similar. Further analysis of gene expression revealed that the ratio of the expression of genes encoded by DNA-A and DNA-B varied significantly during infection. Finally, we monitored the temporal dynamics of the quantities of DNA-A and DNA-B and the B/A ratio of another bipartite begomovirus, and a constant B/A ratio was similarly observed. Our findings highlight the maintenance of a constant ratio between the two genomic components of bipartite begomoviruses during infection and transmission, and provide new insights into the biology of begomoviruses.

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.

Begomovirus-Host Interactions: Viral Proteins Orchestrating Intra and Intercellular Transport of Viral DNA While Suppressing Host Defense Mechanisms.

Begomoviruses, which belong to the Geminiviridae family, are intracellular parasites transmitted by whiteflies to dicotyledonous plants thatsignificantly damage agronomically relevant crops. These nucleus-replicating DNA viruses move intracellularly from the nucleus to the cytoplasm and then, like other plant viruses, cause disease by spreading systemically throughout the plant. The transport proteins of begomoviruses play a crucial role in recruiting host components for the movement of viral DNA within and between cells, while exhibiting functions that suppress the host's immune defense. Pioneering studies on species of the Begomovirus genus have identified specific viral transport proteins involved in intracellular transport, cell-to-cell movement, and systemic spread. Recent research has primarily focused on viral movement proteins and their interactions with the cellular host transport machinery, which has significantly expanded understanding on viral infection pathways. This review focuses on three components within this context: (i) the role of viral transport proteins, specifically movement proteins (MPs) and nuclear shuttle proteins (NSPs), (ii) their ability to recruit host factors for intra- and intercellular viral movement, and (iii) the suppression of antiviral immunity, with a particular emphasis on bipartite begomoviral movement proteins.

Differential expression of genes during recovery of Nicotiana tabacum from tomato leaf curl Gujarat virus infection.

MAIN CONCLUSION: Nicotiana tabacum exhibits recovery response towards tomato leaf curl Gujarat virus. Transcriptome analysis revealed the differential expression of defense-related genes. Genes encoding for cysteine protease inhibitor, hormonal- and stress-related to DNA repair mechanism are found to be involved in the recovery process. Elucidating the role of host factors in response to viral infection is crucial in understanding the plant host-virus interaction. Begomovirus, a genus in the family Geminiviridae, is reported throughout the globe and is known to cause serious crop diseases. Tomato leaf curl Gujarat virus (ToLCGV) infection in Nicotiana tabacum resulted in initial symptom expression followed by a quick recovery in the systemic leaves. Transcriptome analysis using next-generation sequencing (NGS) revealed a large number of differentially expressed genes both in symptomatic as well as recovered leaves when compared to mock-inoculated plants. The virus infected N. tabacum results in alteration of various metabolic pathways, phytohormone signaling pathway, defense related protein, protease inhibitor, and DNA repair pathway. RT-qPCR results indicated that Germin-like protein subfamily T member 2 (NtGLPST), Cysteine protease inhibitor 1-like (NtCPI), Thaumatin-like protein (NtTLP), Kirola-like (NtKL), and Ethylene-responsive transcription factor ERF109-like (NtERTFL) were down-regulated in symptomatic leaves when compared to recovered leaves of ToLCGV-infected plants. In contrast, the Auxin-responsive protein SAUR71-like (NtARPSL) was found to be differentially down-regulated in recovered leaves when compared to symptomatic leaves and the mock-inoculated plants. Lastly, Histone 2X protein like (NtHH2L) gene was found to be down-regulated, whereas Uncharacterized (NtUNCD) was up-regulated in both symptomatic as well as recovered leaves compared to the mock-inoculated plants. Taken together, the present study suggests potential roles of the differentially expressed genes that might govern tobacco's susceptibility and/or recovery response towards ToLCGV infection.