Transreplication Preference of the Tomato Leaf Curl Joydebpur Virus for a Noncognate Betasatellite through Iteron Resemblance on Nicotiana bethamiana.

Pepper plants (Capsicum annuum) with severe leaf curl symptoms were collected in 2013 from Bangalore, Karnataka, India. The detection results showed a co-infection between the tomato leaf curl Joydebpur virus (ToLCJoV) and tomato leaf curl Bangladesh betasatellite (ToLCBDB) through the sequencing analysis of PCR amplicons. To pinpoint the molecular mechanism of this uncommon combination, infectious clones of ToLCJoV and two different betasatellites-ToLCBDB and tomato leaf curl Joydebpur betasatellite (ToLCJoB)-were constructed and tested for their infectivity in Nicotiana benthamiana. Together, we conducted various combined agroinoculation studies to compare the interaction of ToLCJoV with non-cognate and cognate betasatellites. The natural non-cognate interaction between ToLCJoV and ToLCBDB showed severe symptoms compared to the mild symptoms of a cognate combination (ToLCJoV × ToLCJoB) in infected plants. A sequence comparison among betasatellites and their helper virus wasperformed and the iteron resemblances in ToLCBDB as well as ToLCJoB clones were processed. Mutant betasatellites that comprised iteron modifications revealed that changes in iteron sequences could disturb the transreplication process between betasatellites and their helper virus. Our study might provide an important consideration for determining the efficiency of transreplication activity between betasatellites and their helper virus.

Coat protein is responsible for tomato leaf curl New Delhi virus pathogenicity in tomato.

Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite Begomovirus belonging to the family Geminiviridae, causes severe damage to many economically important crops worldwide. In the present study, pathogenicity of Asian (ToLCNDV-In from Pakistan) and Mediterranean isolates (ToLCNDV-ES from Italy) were examined using infectious clones in tomato plants. Only ToLCNDV-In could infect the three tomato cultivars, whereas ToLCNDV-ES could not. Genome-exchange of the two ToLCNDVs revealed the ToLCNDV DNA-A segment as the main factor for ToLCNDV infectivity in tomato. In addition, serial clones with chimeric ToLCNDV-In A and ToLCNDV-ES A genome segments were generated to identify the region determining viral infectivity in tomatoes. A chimeric clone carrying the ToLCNDV-In coat protein (CP) exhibited pathogenic adaptation in tomatoes, indicating that the CP of ToLCNDV is essential for its infectivity. Analyses of infectious clones carrying a single amino acid substitution revealed that amino acid at position 143 of the CP is critical for ToLCNDV infectivity in tomatoes. To better understand the molecular basis whereby CP function in pathogenicity, a yeast two-hybrid screen of a tomato cDNA library was performed using CPs as bait. The hybrid results showed different interactions between the two CPs and Ring finger protein 44-like in the tomato genome. The relative expression levels of upstream and downstream genes and Ring finger 44-like genes were measured using quantitative reverse transcription PCR (RT-qPCR) and compared to those of control plants. This is the first study to compare the biological features of the two ToLCNDV strains related to viral pathogenicity in the same host plant. Our results provide a foundation for elucidating the molecular mechanisms underlying ToLCNDV infection in tomatoes.

Construction of an Agroinfectious Clone of a Korean Isolate of Sweet Potato Symptomless Virus 1 and Comparison of Its Infectivity According to Agrobacterium tumefaciens Strains in Nicotiana benthamiana.

Sweet potato symptomless virus 1 (SPSMV-1) is a single-stranded circular DNA virus, belonging to the genus Mastrevirus (family Geminiviridae) that was first identified on sweet potato plants in South Korea in 2012. Although SPSMV-1 does not induce distinct symptoms in sweet potato plants, its co-infection with different sweet potato viruses is highly prevalent, and thus threatens sweet potato production in South Korea. In this study, the complete genome sequence of a Korean isolate of SPSMV-1 was obtained by Sanger sequencing of polymerase chain reaction (PCR) amplicons from sweet potato plants collected in the field (Suwon). An infectious clone of SPSMV-1 (1.1-mer) was constructed, cloned into the plant expression vector pCAMBIA1303, and agro-inoculated into Nicotiana benthamiana using three Agrobacterium tumefaciens strains (GV3101, LBA4404, and EHA105). Although no visual differences were observed between the mock and infected groups, SPSMV-1 accumulation was detected in the roots, stems, and newly produced leaves through PCR. The A. tumefaciens strain LBA4404 was the most effective at transferring the SPSMV-1 genome to N. benthamiana. We confirmed the viral replication in N. benthamiana samples through strand-specific amplification using virion-sense- and complementary-sense-specific primer sets.

Insights into the Differential Composition of Stem-Loop Structures of Nanoviruses and Their Impacts.

Multipartite viruses package their genomic segments independently and mainly infect plants; few of them target animals. Nanoviridae is a family of multipartite single-stranded DNA (ssDNA) plant viruses that individually encapsidate ssDNAs of ~1 kb and transmit them through aphids without replication in aphid vectors, thereby causing important diseases in host plants, mainly leguminous crops. All of these components constitute an open reading frame to perform a specific role in nanovirus infection. All segments contain conserved inverted repeat sequences, potentially forming a stem-loop structure and a conserved nonanucleotide, TAGTATTAC, within a common region. This study investigated the variations in the stem-loop structure of nanovirus segments and their impact using molecular dynamics (MD) simulations and wet lab approaches. Although the accuracy of MD simulations is limited by force field approximations and simulation time scale, explicit solvent MD simulations were successfully used to analyze the important aspects of the stem-loop structure. This study involves the mutants' design, based on the variations in the stem-loop region and construction of infectious clones, followed by their inoculation and expression analysis, based on nanosecond dynamics of the stem-loop structure. The original stem-loop structures showed more conformational stability than mutant stem-loop structures. The mutant structures were expected to alter the neck region of the stem-loop by adding and switching nucleotides. Changes in conformational stability are suggested expression variations of the stem-loop structures found in host plants with nanovirus infection. However, our results can be a starting point for further structural and functional analysis of nanovirus infection. IMPORTANCE Nanoviruses comprise multiple segments, each with a single open reading frame to perform a specific function and an intergenic region with a conserved stem-loop region. The genome expression of a nanovirus has been an intriguing area but is still poorly understood. We attempted to investigate the variations in the stem-loop structure of nanovirus segments and their impact on viral expression. Our results show that the stem-loop composition is essential in controlling the virus segments' expression level.

An Effective Integrated Machine Learning Framework for Identifying Severity of Tomato Yellow Leaf Curl Virus and Their Experimental Validation.

Tomato yellow leaf curl virus (TYLCV) dispersed across different countries, specifically to subtropical regions, associated with more severe symptoms. Since TYLCV was first isolated in 1931, it has been a menace to tomato industrial production worldwide over the past century. Three groups were newly isolated from TYLCV-resistant tomatoes in 2022; however, their functions are unknown. The development of machine learning (ML)-based models using characterized sequences and evaluating blind predictions is one of the major challenges in interdisciplinary research. The purpose of this study was to develop an integrated computational framework for the accurate identification of symptoms (mild or severe) based on TYLCV sequences (isolated in Korea). For the development of the framework, we first extracted 11 different feature encodings and hybrid features from the training data and then explored 8 different classifiers and developed their respective prediction models by using randomized 10-fold cross-validation. Subsequently, we carried out a systematic evaluation of these 96 developed models and selected the top 90 models, whose predicted class labels were combined and considered as reduced features. On the basis of these features, a multilayer perceptron was applied and developed the final prediction model (IML-TYLCVs). We conducted blind prediction on 3 groups using IML-TYLCVs, and the results indicated that 2 groups were severe and 1 group was mild. Furthermore, we confirmed the prediction with virus-challenging experiments of tomato plant phenotypes using infectious clones from 3 groups. Plant virologists and plant breeding professionals can access the user-friendly online IML-TYLCVs web server at https://balalab-skku.org/IML-TYLCVs, which can guide them in developing new protection strategies for newly emerging viruses.

Transcriptional Analysis of the Differences between ToLCNDV-India and ToLCNDV-ES Leading to Contrary Symptom Development in Cucumber.

Tomato leaf curl New Delhi virus-ES (ToLCNDV-ES), a high threat to cucurbits in the Mediterranean Basin, is listed as a different strain from the Asian ToLCNDV isolates. In this study, the infectivity of two clones previously isolated from Italy and Pakistan were compared in cucumbers, which resulted in the opposite symptom appearance. The swapping subgenome was processed; however, the mechanisms related to the disease phenotype remain unclear. To identify the disease-associated genes that could contribute to symptom development under the two ToLCNDV infections, the transcriptomes of ToLCNDV-infected and mock-inoculated cucumber plants were compared 21 days postinoculation. The number of differentially expressed genes in ToLCNDV-India-infected plants was 10 times higher than in ToLCNDV-ES-infected samples. The gene ontology (GO) and pathway enrichment were analyzed using the Cucurbits Genomics Database. The flavonoid pathway-related genes were upregulated in ToLCNDV-ES, but some were downregulated in ToLCNDV-India infection, suggesting their role in resistance to the two ToLCNDV infections. The relative expression levels of the selected candidate genes were validated by qRT-PCR under two ToLCNDV-infected conditions. Our results reveal the different infectivity of the two ToLCNDVs in cucumber and also provide primary information based on RNA-seq for further analysis related to different ToLCNDV infections.

Interspecies Recombination-Led Speciation of a Novel Geminivirus in Pakistan.

Recombination between isolates of different virus species has been known to be one of the sources of speciation. Weeds serve as mixing vessels for begomoviruses, infecting a wide range of economically important plants, thereby facilitating recombination. Chenopodium album is an economically important weed spread worldwide. Here, we present the molecular characterization of a novel recombinant begomovirus identified from C. album in Lahore, Pakistan. The complete DNA- A genome of the virus associated with the leaf distortion occurred in the infected C. album plants was cloned and sequenced. DNA sequence analysis showed that the nucleotide sequence of the virus shared 93% identity with those of the rose leaf curl virus and the duranta leaf curl virus. Interestingly, this newly identified virus is composed of open reading frames (ORFs) from different origins. Phylogenetic networks and complementary recombination detection methods revealed extensive recombination among the sequences. The infectious clone of the newly detected virus was found to be fully infectious in C. album and Nicotiana benthamiana as the viral DNA was successfully reconstituted from systemically infected tissues of inoculated plants, thus fulfilling Koch's postulates. Our study reveals a new speciation of an emergent ssDNA plant virus associated with C. album through recombination and therefore, proposed the tentative name 'Chenopodium leaf distortion virus' (CLDV).

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.

Different Infectivity of Mediterranean and Southern Asian Tomato Leaf Curl New Delhi Virus Isolates in Cucurbit Crops.

Tomato leaf curl New Delhi virus (ToLCNDV) became an alerting virus in Europe from 2017 to 2020 because of its significant damage to Cucurbitaceae cultivation. Until now, just some cucurbit crops including sponge gourd, melon, pumpkin, and cucumber were reported to be resistant to ToLCNDV, but no commercial cultivars are available. In this study, a new isolate of ToLCNDV was identified in Pakistan and analyzed together with ToLCNDV-ES which was previously isolated in Italy. Furthermore, infectious clones of two ToLCNDV isolates were constructed and agroinoculated into different cucurbit crops to verify their infectivity. Results showed that both isolates exhibited severe infection on all tested cucurbit (>70%) except watermelon. Thus, those cultivars may be good candidates in the first step of screening genetic resources for resistance on both Southeast Asian and Mediterranean ToLCNDV isolates. Additional, comparison pathogenicity of different geographical ToLCNDV isolates will be aided to understand viral characterization as such knowledge could facilitate breeding resistance to this virus.

Identification of a Novel Geminivirus in Fraxinus rhynchophylla in Korea.

Fraxinus rhynchophylla, common name ash, belongs to the family Oleaceae and is found in China, Korea, North America, the Indian subcontinent, and eastern Russia. It has been used as a traditional herbal medicine in Korea and various parts of the world due to its chemical constituents. During a field survey in March 2019, mild vein thickening (almost negligible) was observed in a few ash trees. High-throughput sequencing of libraries of total DNA from ash trees, rolling-circle amplification (RCA), and polymerase chain reaction (PCR) allowed the identification of a Fraxinus symptomless virus. This virus has five confirmed open reading frames along with a possible sixth open reading frame that encodes the movement protein and is almost 2.7 kb in size, with a nonanucleotide and stem loop structure identical to begomoviruses. In terms of its size and structure, this virus strongly resembles begomoviruses, but does not show any significant sequence identity with them. To confirm movement of the virus within the trees, different parts of infected trees were examined, and viral movement was successfully observed. No satellite molecules or DNA B were identified. Two-step PCR confirmed the virion and complementary strands during replication in both freshly collected infected samples of ash tree and Nicotiana benthamiana samples agro-inoculated with infectious clones. This taxon is so distantly grouped from other known geminiviruses that it likely represents a new geminivirus genus.

Adaptation and Codon-Usage Preference of Apple and Pear-Infecting Apple Stem Grooving Viruses.

Apple stem grooving virus (ASGV; genus Capillovirus) is an economically important virus. It has an approx. 6.5 kb, monopartite, linear, positive-sense, single-stranded RNA genome. The present study includes identification of 24 isolates-13 isolates from apple (Pyrus malus L.) and 11 isolates from pear (Pyrus communis L.)-from different agricultural fields in South Korea. The coat protein (CP) gene of the corresponding 23 isolates were amplified, sequenced, and analyzed. The CP sequences showed phylogenetic separation based on their host species, and not on the geography, indicating host adaptation. Further analysis showed that the ASGV isolated in this study followed host adaptation influenced and preferred by the host codon-usage.

Nanovirus Disease Complexes: An Emerging Threat in the Modern Era.

Multipartite viruses package their genomic segments independently and mainly infect plants; few target animals. Nanoviridae is a family of multipartite single-stranded DNA plant viruses that individually encapsidate single-stranded DNAs of approximately 1 kb and transmit them through aphids without replication in the aphid vectors, thereby causing important diseases of leguminous crops and banana. Significant findings regarding nanoviruses have recently been made on important features, such as their multicellular way of life, the transmission of distinct encapsidated genome segments through the vector body, evolutionary ambiguities, mode of infection, host range and geographical distribution. This review deals with all the above-mentioned features in view of recent advances with special emphasis on the emergence of new species and recognition of new host range of nanoviruses and aims to shed light on the evolutionary linkages, the potentially devastating impact on the world economy, and the future challenges imposed by nanoviruses.

Biotic stress triggered small RNA and RNAi defense response in plants.

The yield of crops is largely affected by different types of biotic stresses. To minimize the damage, crop plants adapted themselves to overcome the stress conditions through gene expression reprogramming at transcriptional and post-transcriptional levels. With a better knowledge of plants' responses in adverse environments, new methodologies and strategies have been applied to develop better stress-tolerant plants. In this manner, small RNAs (micro RNA and small-interfering RNA) are reported to play a central role to combat biotic stresses in plants. Depending upon the stress stimuli, these small RNAs can up or down regulate the genes expression, that indicate their potential role in overcoming the stress. These stress-induced small RNAs may reduce the expression of the target gene(s) that might negatively influence plants' response to the adverse conditions. Contrariwise, miRNA, a class of small RNA, can downregulate its expression to upregulate the expression of the target gene(s), which might positively aid to the stress adaptation. Along with this, benefits of RNA interference (RNAi) have also been stated in functional genomic research on insects, fungi and plant pathogens. RNAi is involved in the safe transport of dsRNA to the targeted mRNA(s) in the biotic stress-causing agents (for example fungi and insects) and saves the plant from damage, which is a safer approach compared to use of chemical pesticides. The current review summarizes the role of small RNAs and the use of RNAi to save the plants from biotic stress conditions.

Seed Transmission of Tomato Leaf Curl New Delhi Virus from Zucchini Squash in Italy.

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus affecting tomato cultivation on the Indian subcontinent. Recently, however, a new strain of the virus, named ToLCNDV-ES, has spread to Mediterranean countries such as Spain, Italy, and Tunisia, and occurred in Cucurbita crops, causing economic damage. Although ToLCNDV is spread by the sweet potato whitefly (Bemisia tabaci), like other begomoviruses, it has not been clear how ToLCNDV suddenly spread from the Indian subcontinent to the Mediterranean region. In 2017, ToLCNDV was diagnosed in young seedlings germinated naturally from fruits fallen in the prior year on a farm located in Giugliano in Campania, Naples, Italy, suggesting a possible role of the seeds in vertical transmission of the virus. Because sweet potato whiteflies were widespread naturally in that region, it was necessary to verify that in an artificial insect vector-free condition. Seeds were harvested from two ToLCNDV-infected zucchini squash cultivars in Naples in 2017 and 2018 to examine whether ToLCNDV can be transmitted from zucchini squash seeds to young plants. Viral DNA was amplified from these seeds and 1- to 3-week-old seedlings germinated from them with a ToLCNDV-specific primer set. According to PCR results, viral contamination was confirmed from all harvested seeds and dissemination was proven from 61.36% of tested seedling samples. Mechanical transmission from seed-borne virus-infected seedlings to healthy zucchini squash plants was also succesful, demonstrating that seedlings from ToLCNDV-infected seeds did act as inoculum. This is the first report demonstrating that ToLCNDV is a seed-transmissible virus in zucchini squash plants in Italy.

The threat of seed-transmissible pepper yellow leaf curl Indonesia virus in chili pepper.

Recently, chili pepper (Capsicum annuum) plants in Indonesia have been devastated by a notorious bipartite begomovirus infection named Pepper yellow leaf curl Indonesia virus (PepYLCIV), which causes a distinct decrease in chili pepper production. Pepper yellow diseases have been known since early 2000; however, the spread of this virus thus far is distressing. These diseases can reduce chili yields by 20-100% in Indonesia. As previously known, begomovirus can be transmitted through whitefly to several host plants from the families Solanaceae, Compositae, and Leguminosae. In the field, a single plant was observed with severe symptoms of pepper yellow leaf curl disease, while other plants in the same field were asymptomatic and healthy. The observation leads to the possibility that the virus can be transmitted from previously infected chili pepper plants through seeds, as begomovirus transmission through seeds has been reported before. This study was conducted using seeds from chili peppers infected with viruses from different places in Indonesia. Whole seeds, embryos, and seedlings from PepYLCIV infected seeds were investigated in this study by performing viral genome DNA extraction, uracil DNA glycosylase-PCR, and sequencing analysis. Results revealed that both DNA-A and DNA-B of PepYLCIV in seeds and embryos of infected chili pepper plants were detected. The results also showed that 25-67% of PepYLCIV DNA-A and 50-100% of DNA-B were detected from seedlings grown from infected chili pepper seed collected from different location, thus confirming PepYLCIV as a seed-transmissible virus in chili pepper plants.