Complete genome sequencing and characterization of a potential new genotype of Citrus tristeza virus in Iran.

Citrus tristeza virus (CTV) is one of the economically destructive viruses affecting citrus trees worldwide, causing significant losses in fruit production. Comparative genomic studies have shown genetic diversity in various regions of the genome of CTV isolates, which has classified the virus into several genotypes. In recent years, some orange citrumelo-tolerant rootstocks showed yellowing, decline, and vein clearing in northern Iran (Mazandaran province, Sari). We confirmed the presence of CTV in the symptomatic trees by reverse transcription PCR (RT-PCR). The complete genome of a Sari isolate of CTV (Sari isolate) was sequenced using next-generation sequencing (NGS) technology. In addition, phylogenetic analysis, differential gene expression of the virus and identification of its variants in a population were studied. We obtained the final contigs of the virus (nt) and annotated all genomes to viral ORFs, untranslated regions (UTRs), intergenic regions, and 5' and 3' ends of the genome. Phylogenetic analysis of the Sari isolate and other genotypes of CTV showed that the Sari isolates were placed in a distinct cluster without a sister group. Based on the number of specific transcripts (TPM) in CTV RNA -Seq, P13 was the most highly expressed gene related to the host range of the virus and its systemic infection. The ORFs of the polyprotein, P33, and P18 showed variation in a single population of the sari isolate. The CTV has a potential for variation in a population in a host, and these variations may contribute to the best fit of the CTV in different situations. In Iran, whole genome sequencing of the CTV was performed for the first time, and we gained new insights into CTV variation in a population.

Gene network modeling and pathway analysis of maize transcriptomes in response to Maize Iranian mosaic virus.

Maize Iranian mosaic virus (MIMV, family Rhabdoviridae) is one of the factors limiting cereal production in Iran. In the present study, we sought to find critical genes and key pathways involved in MIMV infection and analyzed gene networks, pathways and promoters using transcriptome data. We determined the hub genes involved in pathways related to the proteasome and ubiquitin. The results showed the important role of the cellular endoplasmic reticulum in MIMV infection. Network cluster analysis confirmed the result of GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The discovered miRNAs belonged to miR166, miR167, miR169, miR395, miR399, miR408 and miR482 families, which are involved in various pathogenicity or resistance processes against MIMV or other viruses. The results of this study provide a list of hub genes, important pathways and new insights for the future development of virus-resistant transgenic crops and clarify the basic mechanism of plant response.

Regulatory non-coding RNA: The core defense mechanism against plant pathogens.

Plant pathogens damage crops and threaten global food security. Plants have evolved complex defense networks against pathogens, using crosstalk among various signaling pathways. Key regulators conferring plant immunity through signaling pathways include protein-coding genes and non-coding RNAs (ncRNAs). The discovery of ncRNAs in plant transcriptomes was first considered "transcriptional noise". Recent reviews have highlighted the importance of non-coding RNAs. However, understanding interactions among different types of noncoding RNAs requires additional research. This review attempts to consider how long-ncRNAs, small-ncRNAs and circular RNAs interact in response to pathogenic diseases within different plant species. Developments within genomics and bioinformatics could lead to the further discovery of plant ncRNAs, knowledge of their biological roles, as well as an understanding of their importance in exploiting the recent molecular-based technologies for crop protection.

Network for network concept offers new insights into host- SARS-CoV-2 protein interactions and potential novel targets for developing antiviral drugs.

SARS-CoV-2, the causal agent of COVID-19, is primarily a pulmonary virus that can directly or indirectly infect several organs. Despite many studies carried out during the current COVID-19 pandemic, some pathological features of SARS-CoV-2 have remained unclear. It has been recently attempted to address the current knowledge gaps on the viral pathogenicity and pathological mechanisms via cellular-level tropism of SARS-CoV-2 using human proteomics, visualization of virus-host protein-protein interactions (PPIs), and enrichment analysis of experimental results. The synergistic use of models and methods that rely on graph theory has enabled the visualization and analysis of the molecular context of virus/host PPIs. We review current knowledge on the SARS-COV-2/host interactome cascade involved in the viral pathogenicity through the graph theory concept and highlight the hub proteins in the intra-viral network that create a subnet with a small number of host central proteins, leading to cell disintegration and infectivity. Then we discuss the putative principle of the "gene-for-gene and "network for network" concepts as platforms for future directions toward designing efficient anti-viral therapies.

Characterization of maize miRNAs responsive to maize Iranian mosaic virus infection.

MicroRNAs (miRNAs) play key regulatory roles in the plant's response to biotic and abiotic stresses and have fundamental functions in plant-virus interactions. The study of changes in miRNAs in response to virus infection can provide molecular details for a better understanding of virus-host interactions. Maize Iranian mosaic virus (MIMV) infects maize and certain other poaceous plants but miRNA changes in response to MIMV infection are unknown. In the present study, we compared the miRNA profiles of MIMV-infected and uninfected maize and characterized their predicted roles in response to the virus. Small RNA sequencing of maize identified 257 conserved miRNAs of 26 conserved families in uninfected and MIMV-infected maize libraries. Among them, miR395, miR166 and miR156 family members were highly represented. Small RNA data were confirmed using RT-qPCR. In addition, 33 potential novel miRNAs were predicted. The data show that 13 miRNAs were up-regulated and 113 were down-regulated in response to MIMV infection. Several of those miRNAs are known to be important in the response to plant pathogens. To determine the potential roles of individual miRNAs in response to MIMV, miRNA targets, predicted interactions with circular RNAs and comparative transcriptome data were analyzed. The expression profiles of different miRNAs in response to MIMV provide novel insights into the roles of miRNAs in the interaction between MIMV and maize plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03134-1.

Highlight of potential impact of new viral genotypes of SARS-CoV-2 on vaccines and anti-viral therapeutics.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of the coronavirus disease (COVID-19) pandemic, has infected millions of people globally. Genetic variation and selective pressures lead to the accumulation of single nucleotide polymorphism (SNP) within the viral genome that may affect virulence, transmission rate, viral recognition and the efficacy of prophylactic and interventional measures. To address these concerns at the genomic level, we assessed the phylogeny and SNPs of the SARS-CoV-2 mutant population collected to date in Iran in relation to globally reported variants. Phylogenetic analysis of mutant strains revealed the occurrence of the variants known as B.1.1.7 (Alpha), B.1.525 (Eta), and B.1.617 (Delta) that appear to have delineated independently in Iran. SNP analysis of the Iranian sequences revealed that the mutations were predominantly positioned within the S protein-coding region, with most SNPs localizing to the S1 subunit. Seventeen S1-localizing SNPs occurred in the RNA binding domain that interacts with ACE2 of the host cell. Importantly, many of these SNPs are predicted to influence the binding of antibodies and anti-viral therapeutics, indicating that the adaptive host response appears to be imposing a selective pressure that is driving the evolution of the virus in this closed population through enhancing virulence. The SNPs detected within these mutant cohorts are addressed with respect to current prophylactic measures and therapeutic interventions.

Comparative phylogenetic analysis of SARS-CoV-2 spike protein-possibility effect on virus spillover.

Coronavirus disease 2019 has developed into a dramatic pandemic with tremendous global impact. The receptor-binding motif (RBM) region of the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), binds to host angiotensin-converting enzyme 2 (ACE2) receptors for infection. As ACE2 receptors are highly conserved within vertebrate species, SARS-CoV-2 can infect significant animal species as well as human populations. An analysis of SARS-CoV-2 genotypes isolated from human and significant animal species was conducted to compare and identify mutation and adaptation patterns across different animal species. The phylogenetic data revealed seven distinct phylogenetic clades with no significant relationship between the clades and geographical locations. A high rate of variation within SARS-CoV-2 mink isolates implies that mink populations were infected before human populations. Positions of most single-nucleotide polymorphisms (SNPs) within the spike (S) protein of SARS-CoV-2 genotypes from the different hosts are mostly accumulated in the RBM region and highlight the pronounced accumulation of variants with mutations in the RBM region in comparison with other variants. These SNPs play a crucial role in viral transmission and pathogenicity and are keys in identifying other animal species as potential intermediate hosts of SARS-CoV-2. The possible roles in the emergence of new viral strains and the possible implications of these changes, in compromising vaccine effectiveness, deserve urgent considerations.

A short overview of CRISPR-Cas technology and its application in viral disease control.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) together with CRISPR-associated (Cas) proteins have catalysed a revolution in genetic engineering. Native CRISPR-Cas systems exist in many bacteria and archaea where they provide an adaptive immune response through sequence-specific degradation of an invading pathogen's genome. This system has been reconfigured for use in genome editing, drug development, gene expression regulation, diagnostics, the prevention and treatment of cancers, and the treatment of genetic and infectious diseases. In recent years, CRISPR-Cas systems have been used in the diagnosis and control of viral diseases, for example, CRISPR-Cas12/13 coupled with new amplification techniques to improve the specificity of sequence-specific fluorescent probe detection. Importantly, CRISPR applications are both sensitive and specific and usually only require commonly available lab equipment. Unlike the canonical Cas9 which is guided to double-stranded DNA sites of interest, Cas13 systems target RNA sequences and thus can be employed in strategies directed against RNA viruses or for transcriptional silencing. Many challenges remain for these approach, including issues with specificity and the requirement for better mammalian delivery systems. In this review, we summarize the applications of CRISPR-Cas systems in controlling mammalian viral infections. Following necessary improvements, it is expected that CRISPR-Cas systems will be used effectively for such applications in the future.

Quasi-species nature and differential gene expression of severe acute respiratory syndrome coronavirus 2 and phylogenetic analysis of a novel Iranian strain.

A novel coronavirus related to severe acute respiratory syndrome virus, (SARS-CoV-2) is the causal agent of the COVID-19 pandemic. Despite the genetic mutations across the SARS-CoV-2 genome being recently investigated, its transcriptomic genetic polymorphisms at inter-host level and the viral gene expression level based on each Open Reading Frame (ORF) remains unclear. Using available High Throughput Sequencing (HTS) data and based on SARS-CoV-2 infected human transcriptomic data, this study presents a high-resolution map of SARS-CoV-2 single nucleotide polymorphism (SNP) hotspots in a viral population at inter-host level. Four throat swab samples from COVID-19 infected patients were pooled, with RNA-Seq read retrieved from SRA NCBI to detect 21 SNPs and a replacement across the SARS-CoV-2 genomic population. Twenty-two RNA modification sites on viral transcripts were identified that may cause inter-host genetic diversity of this virus. In addition, the canonical genomic RNAs of N ORF showed higher expression in transcriptomic data and reverse transcriptase quantitative PCR compared to other SARS-CoV-2 ORFs, indicating the importance of this ORF in virus replication or other major functions in virus cycle. Phylogenetic and ancestral sequence analyses based on the entire genome revealed that SARS-CoV-2 is possibly derived from a recombination event between SARS-CoV and Bat SARS-like CoV. Ancestor analysis of the isolates from different locations including Iran suggest shared Chinese ancestry. These results propose the importance of potential inter-host level genetic variations to the evolution of SARS-COV-2, and the formation of viral quasi-species. The RNA modifications discovered in this study may cause amino acid sequence changes in polyprotein, spike protein, product of ORF8 and nucleocapsid (N) protein, suggesting further insights to understanding the functional impacts of mutations in the life cycle and pathogenicity of SARS-CoV-2.

Maize Iranian mosaic virus (family Rhabdoviridae) improves biological traits of its vector Laodelphax striatellus.

Plant viruses can alter the behavior or performance of their arthropod vectors, either indirectly (through effects of virus infection on the host plant) or directly (from virus acquisition by the vector). Given the diversity of plant viruses and their arthropod vectors, the effects for any specific system are not possible to predict. Here, we present experimental evidence that acquisition of maize Iranian mosaic virus (MIMV, genus Nucleorhabdovirus, family Rhabdoviridae) modifies the biological traits of its insect vector, the small brown planthopper (SBPH) Laodelphax striatellus. MIMV is an economically important virus of maize and several other grass species. It is transmitted by SBPHs in a persistent-propagative manner. We evaluated the effects of MIMV acquisition by SBPH on its life history when reared on healthy barley plants (Hordeum vulgare). We conclude that 1) MIMV acquisition by SBPHs increases female fecundity, duration of the nymph stage, adult longevity, and survival of SBPHs, (2) the mortality rate and female-to-male sex ratio are reduced in MIMV-infected planthoppers, and (3) MIMV infection increases the concentration of some biochemical components of the infected plants, including carbohydrates, some amino acids, and total protein, which might influence the life traits of its insect vector. The results indicate the potential of MIMV to improve the ecological fitness of its vector, SBPH, through direct or indirect effects, with the potential to increase the spread of the virus.

Evolutionary timescale and geographical movement of cucumber mosaic virus, with focus on Iranian strains.

Cucumber mosaic virus (CMV) is a geographically widespread plant virus with a very broad host range. The virus has been detected in diverse crops all over Iran. In this study, we estimated the timescale of the evolution of CMV subgroup I and the geographical movement of the virus with a focus on Iranian strains. Analyses using the MP and CP genes and their concatenation revealed that the CMV population within subgroup I had a single ancestor dating back to about 450-550 years ago. The Iranian strains formed three clusters in a maximum-clade-credibility phylogenetic tree. It was found that the most recent common ancestor of the Iranian strains within each cluster dates back to less than 100 years ago. Our results also suggest that both short- and long-distance migration of Iranian CMV strains has occurred in the last 100 years.

Enhanced anti-tumor efficacy and reduced cardiotoxicity of doxorubicin delivered in a novel plant virus nanoparticle.

Plant viruses are one of the newly applied nanoparticles as drug delivery vehicles. Here, we investigated drug delivery performance of Johnson grass chlorotic stripe mosaic virus (JgCSMV) conjugated to folic acid (FA) for targeted delivery of doxorubicin (Dox). The FA-JgCSMV-Dox complex was synthesized and characterized using spectrophotometry, native and denaturing gel electrophoresis and transmission electron microscopy, which disclosed that JgCSMV virions encapsulated Dox and showed comparable size and morphology to the native particles. The JgCSMV nanoparticles loaded with Dox showed a sustained drug release profile in tumor tissue and improved the uptake of Dox in breast cancer cells, leading to enhanced tumor homing. Lastly, we demonstrated that FA-JgCSMV-Dox reduced the tumor growth and cardiotoxicity of athymic mice bearing human breast cancer xenografts in comparison to free Dox. This study is the first report on applicability of JgCSMV for Dox delivery with superior benefits over generally marketed formulations of doxorubicin.

Detection and profiling of circular RNAs in uninfected and maize Iranian mosaic virus-infected maize.

Circular RNAs (circRNAs) are covalently closed non-coding RNAs that are usually derived from exonic regions of genes, but can also arise from intronic and intergenic regions. Studies of circRNAs in humans, animals and several plant species have shown an altered population of circRNAs in response to abiotic and biotic stress. Recently it was shown that circRNAs also occur in maize, but it is unknown if maize circRNAs are responsive to stress. Maize Iranian mosaic virus (MIMV, genus Nucleorhabdovirus, family Rhabdoviridae) causes an economically important disease in maize and other gramineous crops in Iran. In this study, we used data from RNA-Seq of MIMV-infected maize and uninfected controls to identify differentially expressed circRNAs. Such circRNAs were confirmed by two-dimensional polyacrylamide gel electrophoresis, northern blot, RT-qPCR and sequencing. A total of 1443 circRNAs were identified in MIMV-infected maize and 1165 circRNAs in uninfected maize. Two hundred and one circRNAs were in common between MIMV-infected and uninfected samples. Of these, 155 circRNAs were up-regulated and 5 down-regulated in MIMV infected plants, compared to the uninfected control. This study for the first time identified and profiled circRNA expression in maize in response to virus infection. Moreover, we predict that 33 circRNAs may bind 23 maize miRNAs, possibly affecting plant metabolism and development. Our data suggest a role for circRNAs in plant cell regulation and response to biotic stress such as virus infection, and give new insights into the complexity of plant-microbe interactions.

Changes in maize transcriptome in response to maize Iranian mosaic virus infection.

BACKGROUND: Maize Iranian mosaic virus (MIMV, genus Nucleorhabdovirus, family Rhabdoviridae) causes an economically important disease in maize and other gramineous crops in Iran. MIMV negative-sense RNA genome sequence of 12,426 nucleotides has recently been completed. Maize Genetics and Genomics database shows that 39,498 coding genes and 4,976 non-coding genes of maize have been determined, but still some transcripts could not be annotated. The molecular host cell responses of maize to MIMV infection including differential gene expression have so far not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Complementary DNA libraries were prepared from total RNA of MIMV-infected and mock-inoculated maize leaves and sequenced using Illumina HiSeq 2500. Cleaned raw transcript reads from MIMV-infected maize were mapped to reads from uninfected maize and to a maize reference genome. Differentially expressed transcripts were characterized by gene ontology and biochemical pathway analyses. Transcriptome data for selected genes were validated by real-time quantitative PCR. CONCLUSION/SIGNIFICANCE: Approximately 42 million clean reads for each treatment were obtained. In MIMV-infected maize compared to uninfected plants, 1689 transcripts were up-regulated and 213 transcripts were down-regulated. In response to MIMV infection, several pathways were activated in maize including immune receptor signaling, metabolic pathways, RNA silencing, hormone-mediated pathways, protein degradation, protein kinase and ATP binding activity, and fatty acid metabolism. Also, several transcripts including those encoding hydrophobic protein RCI2B, adenosylmethionine decarboxylase NAC transcription factor and nucleic acid binding, leucine-rich repeat, heat shock protein, 26S proteasome, oxidoreductases and endonuclease activity protein were up-regulated. These data will contribute to the identification of genes and pathways involved in plant-virus interactions that may serve as future targets for improved disease control.

Gene expression and population polymorphism of maize Iranian mosaic virus in Zea mays, and intracellular localization and interactions of viral N, P, and M proteins in Nicotiana benthamiana.

Maize Iranian mosaic virus (MIMV; Mononegavirales, Rhabdoviridae, Nucleorhabdovirus) infects maize and several other poaceous plants. MIMV encodes six proteins, i.e., nucleocapsid protein (N), polymerase cofactor phosphoprotein (P), putative movement protein (P3), matrix protein (M), glycoprotein (G), and large RNA-dependent RNA polymerase (L). In the present study, MIMV gene expression and genetic polymorphism of an MIMV population in maize were determined. N, P, P3, and M protein genes were more highly expressed than the 5' terminal G and L genes. Twelve single nucleotide polymorphisms were identified across the genome within a MIMV population in maize from RNA-Seq read data pooled from three infected plants indicating genomic variations of potential importance to evolution of the virus. MIMV N, P, and M proteins that are known to be involved in rhabdovirus replication and transcription were characterized as to their intracellular localization and interactions. N protein accumulated exclusively in the nucleus and interacted with itself and with P protein. P protein accumulated in both the nucleus and cell periphery and interacted with itself, N and M proteins in the nucleus. M protein was localized in the cell periphery and on endomembranes, and interacted with P protein in the nucleus. MIMV proteins show a distinctive combination of intracellular localizations and interactions.

Completed sequence and corrected annotation of the genome of maize Iranian mosaic virus.

Maize Iranian mosaic virus (MIMV) is a negative-sense single-stranded RNA virus that is classified in the genus Nucleorhabdovirus, family Rhabdoviridae. The MIMV genome contains six open reading frames (ORFs) that encode in 3΄ to 5΄ order the nucleocapsid protein (N), phosphoprotein (P), putative movement protein (P3), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). In this study, we determined the first complete genome sequence of MIMV using Illumina RNA-Seq and 3'/5' RACE. MIMV genome ('Fars' isolate) is 12,426 nucleotides in length. Unexpectedly, the predicted N gene ORF of this isolate and of four other Iranian isolates is 143 nucleotides shorter than that of the MIMV coding-complete reference isolate 'Shiraz 1' (Genbank NC_011542), possibly due to a minor error in the previous sequence. Genetic variability among the N, P, P3 and G ORFs of Iranian MIMV isolates was limited, but highest in the G gene ORF. Phylogenetic analysis of complete nucleorhabdovirus genomes demonstrated a close evolutionary relationship between MIMV, maize mosaic virus and taro vein chlorosis virus.

Effects of potato spindle tuber viroid infection on tomato metabolic profile.

Viroids are the smallest plant pathogens consisting of a single stranded circular RNA molecule with a strong secondary structure, lacking a coat protein or any other proteins. The mechanism of viroid pathogenicity has remained unclear. Recent advances in instrumentation and data mining have made it possible to study the effects of various stresses on primary and secondary metabolisms. Here, we have utilized metabolic profiling approach to show how PSTVd infection alters tomato metabolic profile and the related pathways. Three terminal leaflets of third true leaf of 20-day-old tolerant tomato cultivar 'Moneymaker' were mechanically inoculated by PSTVd intermediate variant cDNAs and samples were taken from eighth leaf, 19days post-inoculation. Metabolites were extracted and analyzed by gas chromatography/mass spectrometry (GC/MS) and subjected to statistical data analysis. Affected pathways were identified by Pathway Tools program and were compared with microarray data previously reported. The study showed that 79 metabolites changed significantly and 23 pathways were identified in relation to these metabolites. Fourteen of these pathways were similar to those reported in other works. The altered pathways in PSTVd infected tomato leaves included, eight cutin and wax biosynthesis, seven pathways that produce defense related compounds, two energy generator pathways, three hormone biosynthesis pathways, two signal transduction pathways, and one nucleotide biosynthesis pathway. Our data on up/down-regulation of pathways supported the data produced on their corresponding gene(s) up/down-regulation.

Isolation and Partial Characterization of a Novel Cytorhabdovirus from Citrus Trees Showing Foliar Symptoms in Iran.

Citrus ringspot is a graft-transmissible disease, and at least two taxonomically distinct viral species are associated with this syndrome: Citrus psorosis virus (CPsV) and Indian citrus ringspot virus (ICRSV). Neither of these two viruses was detected, however, by serological or molecular assays in symptomatic tissues from citrus trees in southern Iran, where the ringspot syndrome is widespread. By contrast, electron microscopy and molecular assays revealed the presence of a rhabdovirus-like virus, which was graft transmitted to several citrus species and mechanically to herbaceous hosts. Virus particles were bacilliform and resembled rhabdovirus nucleocapsids deprived of the lipoprotein envelope. Partial sequences of the viral nucleoprotein and RNA polymerase genes showed a distant genetic relatedness with cytorhabdoviruses. This virus appears to be a novel species, for which the name Iranian citrus ringspot-associated virus (IrCRSaV) is suggested.

Identification and characterization of conserved and variable regions of lime witches' broom phytoplasma genome.

Several segments (∼20  kbp) of the lime witches' broom (LWB) phytoplasma genome (16SrII group) were sequenced and analysed. A 5.7  kbp segment (LWB-C) included conserved genes whose phylogenetic tree was consistent with that generated using 16S rRNA genes. Another 6.4  kbp LWB phytoplasma genome segment (LWB-NC) was structurally similar to the putative mobile unit or sequence variable mosaic genomic region of phytoplasmas, although it represented a new arrangement of genes or pseudogenes such as phage-related protein genes and tra5 insertion sequences. Sequence- and phylogenetic-based evidence suggested that LWB-NC is a genomic region which includes horizontally transferred genes and could be regarded as a hot region to incorporate more foreign genes into the genome of LWB phytoplasma. The presence of phylogenetically related fragments of retroelements was also verified in the LWB phytoplasma genome. Putative intragenomic retrotransposition or retrohoming of these elements might have been determinant in shaping and manipulating the LWB phytoplasma genome. Altogether, the results of this study suggested that the genome of LWB phytoplasma is colonized by a variety of genes that have been acquired through horizontal gene transfer events, which may have further affected the genome through intragenomic mobility and insertion at cognate or incognate sites. Some of these genes are expected to have been involved in the development of features specific to LWB phytoplasma.

A new strategy for generating geminivirus resistant plants using a DNA betasatellite/split barnase construct.

The betasatellite DNA associated with cotton leaf curl disease contains a single ORF, ßC1, which is a pathogenicity determinant. Deletion of the ßC1 ORF showed that it was not required for betasatellite replication in the presence of Tomato leaf curl virus-Australia (TLCV-Au). A series of betasatellite/split mutant barnase gene constructs, in which a direct repeat of the Bacillus amyloliquefaciens barnase gene flanked the betasatellite, were shown to replicate in tobacco in the presence of TLCV-Au. A betasatellite/split intact barnase gene construct, with the optimal direct repeat unit of the barnase gene, was introduced into Nicotiana tabacum plants. Approximately one third of the transgenic lines containing the betasatellite/split barnase gene constructs were shown to be completely resistant to the TLCV-Au infection. The betasatellite/split intact barnase gene cassette ensures that there is no expression of the barnase in the absence of TLCV-Au, but upon infection of the cell with the virus, release of the betasatellite/split barnase cassette as a replicating molecule resulting in the reconstitution and expression of an active barnase gene and the destruction of the infected cell. This system offers the potential to provide resistance in a variety of plant species against geminiviruses that support the replication of betasatellite.