Functional role of geminivirus encoded proteins in the host: Past and present.

During plant-pathogen interaction, plant exhibits a strong defense system utilizing diverse groups of proteins to suppress the infection and subsequent establishment of the pathogen. However, in response, pathogens trigger an anti-silencing mechanism to overcome the host defense machinery. Among plant viruses, geminiviruses are the second largest virus family with a worldwide distribution and continue to be production constraints to food, feed, and fiber crops. These viruses are spread by a diverse group of insects, predominantly by whiteflies, and are characterized by a single-stranded DNA (ssDNA) genome coding for four to eight proteins that facilitate viral infection. The most effective means to managing these viruses is through an integrated disease management strategy that includes virus-resistant cultivars, vector management, and cultural practices. Dynamic changes in this virus family enable the species to manipulate their genome organization to respond to external changes in the environment. Therefore, the evolutionary nature of geminiviruses leads to new and novel approaches for developing virus-resistant cultivars and it is essential to study molecular ecology and evolution of geminiviruses. This review summarizes the multifunctionality of each geminivirus-encoded protein. These protein-based interactions trigger the abrupt changes in the host methyl cycle and signaling pathways that turn over protein normal production and impair the plant antiviral defense system. Studying these geminivirus interactions localized at cytoplasm-nucleus could reveal a more clear picture of host-pathogen relation. Data collected from this antagonistic relationship among geminivirus, vector, and its host, will provide extensive knowledge on their virulence mode and diversity with climate change.

Current status, breeding strategies and future prospects for managing chilli leaf curl virus disease and associated begomoviruses in Chilli (Capsicum spp.).

Chilli leaf curl virus disease caused by begomoviruses, has emerged as a major threat to global chilli production, causing severe yield losses and economic harm. Begomoviruses are a highly successful and emerging group of plant viruses that are primarily transmitted by whiteflies belonging to the Bemisia tabaci complex. The most effective method for mitigating chilli leaf curl virus disease losses is breeding for host resistance to Begomovirus. This review highlights the current situation of chilli leaf curl virus disease and associated begomoviruses in chilli production, stressing the significant issues that breeders and growers confront. In addition, the various breeding methods used to generate begomovirus resistant chilli cultivars, and also the complicated connections between the host plant, vector and the virus are discussed. This review highlights the importance of resistance breeding, emphasising the importance of multidisciplinary approaches that combine the best of traditional breeding with cutting-edge genomic technologies. subsequently, the article highlights the challenges that must be overcome in order to effectively deploy begomovirus resistant chilli varieties across diverse agroecological zones and farming systems, as well as understanding the pathogen thus providing the opportunities for improving the sustainability and profitability of chilli production.

REPercussions: how geminiviruses recruit host factors for replication.

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

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.

Development and application of crude sap-based recombinase polymerase amplification assay for the detection and occurrence of grapevine geminivirus A in Indian grapevine cultivars.

Geminiviruses are known to infect several fields and horticultural crops around the globe. Grapevine geminivirus A (GGVA) was reported in the United States in 2017, and since then, it has been reported in several countries. The complete genome recovered through high-throughput sequencing (HTS)-based virome analysis in Indian grapevine cultivars had all of the six open reading frames (ORFs) and a conserved nonanucleotide sequence 5'-TAATATTAC-3' similar to all other geminiviruses. Recombinase polymerase amplification (RPA), an isothermal amplification technique, was developed for the detection of GGVA in grapevine samples employing crude sap lysed in 0.5 M NaOH solution and compared with purified DNA/cDNA as a template. One of the key advantages of this assay is that it does not require any purification or isolation of the viral DNA and can be performed in a wide range of temperatures (18°C-46°C) and periods (10-40 min), which makes it a rapid and cost-effective method for the detection of GGVA in grapevine. The developed assay has a sensitivity up to 0.1 fg µl-1 using crude plant sap as a template and detected GGVA in several grapevine cultivars of a major grapevine-growing area. Because of its simplicity and rapidity, it can be replicated for other DNA viruses infecting grapevine and will be a very useful technique for certification and surveillance in different grapevine-growing regions of the country.

Occurrence of Tomato Leaf Curl New Delhi Virus in Cucurbit Plants in China.

In August 2022, melon (Cucumis melo), cucumber (Cucumis sativus) and luffa (Luffa aegyptiaca) plants showed virus-like symptoms characteristic of geminiviruses (yellowish green, mosaic patterns and severe curling of leaves, short internodes, and stunting) in 10 greenhouses in Fengxian district and 20 greenhoues in Jiading district of Shanghai, China. Fifty symptomatic leaf samples were randomly collected: 28 from melon, 17 from cucumber, and 5 from luffa. To investigate the etiology of the observed disease, total DNA and RNA was extracted via a DNA extraction kit (Tiangen, Not: DP350) and TRIzol reagent (Sigma-Aldrich, Not: T9424), respectively. Healthy melon plants grown in a growth chamber served as negative control. The DNA and RNA samples were screened for the presence of geminiviruses, Cucurbit chlorotic yellow virus (CCYV), Melon yellow spot virus (MYSV), Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV) and Cucumber green mottle mosaic virus (CGMMV) through PCR or RT‒PCR with geminiviruses (Deng et al. 1994), CCYV, MYSV, CMV, ZYMV and CGMMV (Zeng et al. 2011, 2019) primers. The PCR results showed that 28 melon leaves and 17 cucumber leaves were positive for geminivirus and CCYV, respectively, 5 luffa samples were infected with only geminivirus, and virus was not detected from the healthy plants. These results indicate that these two viruses are widely distributed throughout cucurbit crops in Shanghai, China. All the geminiviruses sequences (approximately 510 bp) were quite similar to each other and were most similar (99.4%) to the Tomato leaf curl New Delhi virus (ToLCNDV) sequence (GenBank Accession No. OP356207) (Li et al. 2022). To confirm the presence of geminiviruses, the segments of DNA-A and DNA-B were amplified by PCR with 4 ToLCNDV-specific primer sets (Mizutani et al. 2011) and sequenced from 10 samples (4 melon, 4 cucumber and 2 luffa). Both DNA-A and DNA-B of the ToLCNDV sequences and features were deposited in GenBank under the accession numbers OQ190939-OQ190948 (DNA-A, 2739 nt) and OQ190949-OQ190958 (DNA-B, 2693 nt). BLASTn analysis of Shanghai isolates of ToLCNDV (DNA-A and DNA-B) showed that the sequences shared nucleotide identities ranged from 99.3% to 100% among them and with values of more than 99.4% nucleotide identity with ToLCNDV isolates from tomato in China (OP356207 and OP356208) (Li et al. 2022). To confirm the virus infection, we have successfully constructed an infectious clone for 0823-1 isolate in the binary plasmid and inoculated melon with and without an infectious clone. The melon plants inoculated with ToLCNDV 15 dpi showed the high accumulation of the virus and displayed symptoms similar to viruses in greenhouse. Based on the complete sequences, results of the molecular phylogenetic analysis (Fig. 2) and infectious clone, these geminiviruses were identified as ToLCNDV. ToLCNDV has been reported to occur and spread by the whitefly (Bemisia tabaci) in many Asian countries (Sohrab et al. 2003; Sohrab et al. 2011; Aamir et al. 2020) and Europe (Juárez et al. 2014; Ruiz et al. 2015; Luigi et al. 2019). Large populations of whiteflies were also present in all our surveyed areas. However, to our knowledge, this is the first report of the occurrence of ToLCNDV in cucurbit plants in China. The presence of ToLCNDV and CCYV can cause severe losses in crop yields or even crop failure. In addition to TYLCV, ToLCNDV is another major geminivirus-induced disease threatening cucurbit and other vegetable production in China.

Plant Defense and Viral Counter-Defense during Plant-Geminivirus Interactions.

Geminiviruses are the largest family of plant viruses that cause severe diseases and devastating yield losses of economically important crops worldwide. In response to geminivirus infection, plants have evolved ingenious defense mechanisms to diminish or eliminate invading viral pathogens. However, increasing evidence shows that geminiviruses can interfere with plant defense response and create a suitable cell environment by hijacking host plant machinery to achieve successful infections. In this review, we discuss recent findings about plant defense and viral counter-defense during plant-geminivirus interactions.

The Role of Extensive Recombination in the Evolution of Geminiviruses.

Mutation, recombination and pseudo-recombination are the major forces driving the evolution of viruses by the generation of variants upon which natural selection, genetic drift and gene flow can act to shape the genetic structure of viral populations. Recombination between related virus genomes co-infecting the same cell usually occurs via template swapping during the replication process and produces a chimeric genome. The family Geminiviridae shows the highest evolutionary success among plant virus families, and the common presence of recombination signatures in their genomes reveals a key role in their evolution. This review describes the general characteristics of members of the family Geminiviridae and associated DNA satellites, as well as the extensive occurrence of recombination at all taxonomic levels, from strain to family. The review also presents an overview of the recombination patterns observed in nature that provide some clues regarding the mechanisms involved in the generation and emergence of recombinant genomes. Moreover, the results of experimental evolution studies that support some of the conclusions obtained in descriptive or in silico works are summarized. Finally, the review uses a number of case studies to illustrate those recombination events with evolutionary and pathological implications as well as recombination events in which DNA satellites are involved.

Manipulation of plant RNA biology by geminiviruses.

Viruses are intracellular parasites that have evolved to effectively manipulate the cells they infect. As a result of the viral infection, multiple cellular processes are altered, suppressed, or redirected, partially due to the viral co-option of the host's molecular machinery. RNA biology plays a central role in virus-host interactions, since it is at the basis of viral gene expression, splicing of viral transcripts, anti-viral RNA silencing, and-at least in the case of RNA viruses-genome replication, and therefore is heavily targeted by viruses. The plant DNA geminiviruses, causal agents of devasting diseases in crops worldwide, are no exception, and RNA processing is tightly entrenched in their infection cycle. In this review, we will discuss the relevance of the manipulation of RNA biology by geminiviruses for a successful viral infection and the underlying molecular mechanisms, and suggest some of the multiple remaining open questions in this field.

The rising threat of geminiviruses: molecular insights into the disease mechanism and mitigation strategies.

BACKGROUND: Geminiviruses are among the most threatening emerging plant viruses, accountable for a huge loss to agricultural production worldwide. These viruses have been responsible for some serious outbreaks during the last few decades across different parts of the world. Sincere efforts have been made to regulate the disease incidence by incorporating a multi-dimensional approach, and this process has been facilitated greatly by the advent of molecular techniques. But, the mixed infection due to the polyphagous nature of vectors results in viral recombination followed by the emergence of novel viral strains which thus renders the existing mitigation strategies ineffective. Hence, a multifaceted insight into the molecular mechanism of the disease is really needed to understand the regulatory points; much has been done in this direction during the last few years. The present review aims to explore all the latest developments made so far and to organize the information in a comprehensive manner so that some novel hypotheses for controlling the disease may be generated. METHODS AND RESULTS: Starting with the background information, diverse genera of geminiviruses are listed along with their pathological and economic impacts. A comprehensive and detailed mechanism of infection is elaborated to study the interactions between vector, host, and virus at different stages in the life cycle of geminiviruses. Finally, an effort isalso made to analyze the progress made at the molecular level for the development of various mitigation strategies and suggest more effective and better approaches for controlling the disease. CONCLUSION: The study has provided a thorough understanding of molecular mechanism of geminivirus infection.

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.

Cassava mosaic disease and its whitefly vector in Cameroon: Incidence, severity and whitefly numbers from field surveys.

Cassava plays a key role in the food security and economy of Cameroon, but its production is constrained by cassava mosaic disease (CMD). However, comprehensive surveys of CMD in Cameroon have been lacking. This study aimed at evaluating the current status of CMD and its whitefly vector. Field surveys were conducted in 2020 using a sampling, diagnostics and data storage protocol that has been harmonized across 10 West and Central African countries for ease of comparison. Thirty plants per field were assessed for CMD severity, whitefly abundance and source of infection. Surveys were conducted in 343 fields and confirmed the presence of CMD in all 10 regions of Cameroon. Among the 10,057 assessed plants, 33.07% were deemed healthy (asymptomatic). At the field level, only 6.7% fields were found to be healthy. The mean CMD incidence across the country was 66.93%, and the mean severity score was 2.28. The main mode of infection was likely through contaminated cuttings. The mean whitefly count per plant was 5.78. This study is the first countrywide survey of CMD in Cameroon and provides insights that can be useful for improving the country's CMD intervention and management strategies.

Occurrence and distribution of geminiviruses in China.

Geminiviruses are a group of plant viruses that cause severe diseases in many economically important crops worldwide, leading to devastating losses to agricultural production. Here we summarize the occurrence and distribution of geminiviruses in China, which provides valuable information for further epidemiological studies and supports the development of effective disease management strategies.

Identification of Crucial Amino Acids in Begomovirus C4 Proteins Involved in the Modulation of the Severity of Leaf Curling Symptoms.

Begomoviruses frequently inflict upward or downward leaf curling symptoms on infected plants, leading to severe economic damages. Knowledge of the underlying mechanism controlling the leaf curling severity may facilitate the development of alternative disease management strategies. In this study, through genomic recombination between Ageratum yellow vein virus Nan-Tou strain (AYVV-NT) and Tomato leaf curl virus Tai-Chung Strain (TLCV-TC), which caused upward and downward leaf curling on Nicotiana benthamiana, respectively, it was found that the coding region of C4 protein might be involved in the determination of leaf curling directions. Sequence comparison and mutational analysis revealed that the cysteine and glycine at position 8 and 14 of AYVV-TC C4 protein, respectively, are involved in the modulation of leaf curling symptoms. Cross-protection assays further demonstrated that N. benthamiana inoculated with AYVV-carrying mutations of the aforementioned amino acids exhibited attenuated leaf curling symptoms under the challenge of wild-type AYVV-NT. Together, these findings revealed a new function of begomovirus C4 proteins involved in the modulation of leaf curling severity during symptom formation and suggested potential applications for managing viral diseases through manipulating the symptoms.

Geminiviruses boost active DNA demethylation for counter-defense.

DNA methylation regulates gene expression under abiotic and biotic stresses. Recently, Gui et al. discovered that geminiviruses subverted DNA methylation-mediated defense through boosting the active DNA demethylation mediated by host DNA glycosylases to promote viral virulence. Their findings reveal a distinctive counter-defense strategy exploited by invading pathogens to achieve successful infection.

Geminiviruses also encode small proteins with specific functions.

Geminiviruses are small DNA viruses with single-stranded circular genomes that encode for few known proteins. Recently, Gong et al. reconsidered their protein identification criteria and identified open reading frames that encode small proteins with specific cellular localizations and virulence functions. Their data warrants exploration of such small proteins in other geminiviruses.

Twenty years of evolution and diversification of digitaria streak virus in Digitaria setigera.

Within the family Geminiviridae, the emergence of new species results from their high mutation and recombination rates. In this study, we report the variability and evolution of digitaria streak virus (DSV), a mastrevirus isolated in 1986 from the grass Digitaria setigera in an island of the Vanuatu archipelago. Viral DNA of DSV samples was amplified from D. setigera specimens, derived from the naturally infected original plant, which were propagated in different laboratories in France and Italy for more than 20 years. From the consensus sequences, the nucleotide substitution rate was estimated for the period between a sample and the original sequence published in 1987, as well as for the period between samples. In addition, the intra-host genetic complexity and diversity of 8 DSV populations with a total of 165 sequenced haplotypes was characterized. The evolutionary rate of DSV was estimated to be between 1.13 × 10-4 and 9.87 × 10-4 substitutions/site/year, within the ranges observed in other single-stranded DNA viruses and RNA viruses. Bioinformatic analyses revealed high variability and heterogeneity in DSV populations, which confirmed that mutant spectra are continuously generated and are organized as quasispecies. The analysis of polymorphisms revealed nucleotide substitution biases in viral genomes towards deamination and oxidation of single-stranded DNA. The differences in variability in each of the genomic regions reflected a dynamic and modular evolution in the mutant spectra that was not reflected in the consensus sequences. Strikingly, the most variable region of the DSV genome, encoding the movement protein, showed rapid fixation of the mutations in the consensus sequence and a concomitant dN/dS ratio of 6.130, which suggests strong positive selection in this region. Phylogenetic analyses revealed a possible divergence in three genetic lineages from the original Vanuatu DSV isolate.

No Evidence for Seed Transmission of Tomato Yellow Leaf Curl Sardinia Virus in Tomato.

Seed transmission is an important factor in the epidemiology of plant pathogens. Geminiviruses are serious pests spread in tropical and subtropical regions. They are transmitted by hemipteran insects, but a few cases of transmission through seeds were recently reported. Here, we investigated the tomato seed transmissibility of the begomovirus tomato yellow leaf curl Sardinia virus (TYLCSV), one of the agents inducing the tomato yellow leaf curl disease, heavily affecting tomato crops in the Mediterranean area. None of the 180 seedlings originating from TYLCSV-infected plants showed any phenotypic alteration typical of virus infection. Moreover, whole viral genomic molecules could not be detected in their cotyledons and true leaves, neither by membrane hybridization nor by rolling-circle amplification followed by PCR, indicating that TYLCSV is not a seed-transmissible pathogen for tomato. Examining the localization of TYLCSV DNA in progenitor plants, we detected the virus genome by PCR in all vegetative and reproductive tissues, but viral genomic and replicative forms were found only in leaves, flowers and fruit flesh, not in seeds and embryos. Closer investigations allowed us to discover for the first time that these embryos were superficially contaminated by TYLCSV DNA but whole genomic molecules were not detectable. Therefore, the inability of TYLCSV genomic molecules to colonize tomato embryos during infection justifies the lack of seed transmissibility observed in this host.