Plant Viral Genomes Research Articles

Real time portable genome sequencing for global food security

Crop losses due to viral diseases and pests are major constraints on food security and income for millions of households in sub-Saharan Africa (SSA). Such losses can be reduced if plant diseases and pests are correctly diagnosed and identified early. Currently, accurate diagnosis for definitive identification of plant viruses and their vectors in SSA mostly relies on standard PCR and next generation sequencing technologies (NGS). However, it can take up to 6 months before results generated using these approaches are available. The long time taken to detect or identify viruses impedes quick, within-season decision-making necessary for early action, crop protection advice and disease control measures by farmers. This ultimately compounds the magnitude of crop losses and food shortages suffered by farmers. The MinION portable pocket DNA sequencer was used, to our knowledge globally for the first time, to sequence whole plant virus genomes. We used this technology to identify the begomoviruses causing the devastating cassava mosaic virus, which is ravaging smallholder farmers’ crops in sub-Saharan Africa.

Non-replicative Integral Membrane Proteins Encoded by Plant Alpha-Like Viruses: Emergence of Diverse Orphan ORFs and Movement Protein Genes.

Fast accumulation of sequencing data on plant virus genomes and plant transcriptomes demands periodic re-evaluation of current views on the genome evolution of viruses. Here, we substantiate and further detail our previously mostly speculative model on the origin and evolution of triple gene block (TGB) encoding plant virus movement proteins TGB1, TGB2, and TGB3. Recent experimental data on functional competence of transport gene modules consisting of two proteins related to TGB1 and TGB2, as well as sequence analysis data on similarity of TGB2 and TGB3 encoded by a viral genome and virus-like RNAs identified in a plant transcriptomes, suggest that TGB evolution involved events of gene duplication and gene transfer between viruses. In addition, our analysis identified that plant RNA-seq data assembled into RNA virus-like contigs encode a significant variety of hydrophobic proteins. Functions of these orphan proteins are still obscure; however, some of them are obviously related to hydrophobic virion proteins of recently sequenced invertebrate (mostly insect) viruses, therefore supporting the current view on a common origin for many groups of plant and insect RNA-containing viruses. Moreover, these findings may suggest that the function of at least some orphan hydrophobic proteins is to provide plant viruses with the ability to infect insect hosts. In general, our observations emphasize that comparison of RNA virus sequences in a large variety of land plants and algae isolated geographically and ecologically may lead to experimental confirmation of previously purely speculative schemes of evolution of single genes, gene modules, and whole genomes.

First report of Maize yellow mosaic virus infecting maize in Ecuador

Maize yellow mosaic virus (MaYMV) was first described in maize (Zea mays) from China and proposed as a new virus in the genus Polerovirus (Chen et al., 1). Recently, MaYMV was also detected in symptomatic maize plants in Burkina Faso (Palanga et al., 3) indicating a global distribution for the virus. In May 2016 maize plants showing leaf chlorosis and downward curling of leaf tips were collected from a large production area (c. 125,000 hectares in maize cultivation) in the Los Rios Province in Ecuador. It has been estimated that the disease causes yield losses of 10-30%. Total RNA from symptomatic leaf samples was extracted using the RNeasy Plant Mini Kit (Qiagen, USA) and subjected to deep sequencing analysis using the Illumina platform. Quality filtered paired end reads (8,777,028) were aligned with reference genomes of known plant viruses using standard bioinformatic tools. The transcriptome was highly enriched with viral RNA reads (>5%) showing significant similarity with Sugarcane mosaic virus (SCMV) and other MaYMV sequences. These short reads were assembled into contigs and full-length and nearly full-length genome sequences of SCMV (9632 nt; Genbank Accession No. K006657) and MaYMV (5488 nt; KY052793) were identified. Alignments and phylogenetic trees were performed using SeaView software (Gouy et al., 2). The Ecuadorian MaYMV isolate had the highest nucleotide identity (96%) with a Chinese isolate (KU291103). MaYMV has a typical Polerovirus organisation with six open reading frames (ORFs; 0-5) including ORF3a. ORF3a is translated from a non-standard start codon (in this case AUA) and encodes a protein responsible for long distance movement of the virus in plants (Smirnova et al., 5). ORF3a (nt 3389-3523) shows a single amino acid substitution compared with the Chinese isolate (KU291103). ORF0 (nt 42-836) shows 97% amino acid identity with MaYMV isolate KU291100 (Table 1) and harbours an essential F-box like element (Pazhouhandeh et al., 4). The phylogenetic tree (Fig. 1) constructed from ORF0 amino acid sequences indicates significant similarity to the Chinese MaYMV isolate and high variability between different species of the genus Polerovirus. Phylogenetic analysis of ORF3 from selected species of the genera Enamovirus, Luteovirus and Polerovirus places the Ecuadorian isolate within a group of poleroviruses that infect a wide range of host plants (Fig. 2). MaMYV has been reported to be generally present in mixed infections with other viruses, including Maize chlorotic mottle virus, Southern rice black-streaked dwarf virus or SCMV, but has also been found in single infections in China (Chen et al., 1) displaying characteristic disease symptoms. The significance of mixed infections with SCMV in disease pathology in Ecuador is unclear and requires further investigation. The authors are thankful to SENESCYT Ecuador (Prometeo Project) and Agrocalidad, Ecuador for their financial support.

Discovering and sequencing new plant viral genomes by next‐generation sequencing: description of a practical pipeline

AbstractSmall‐scale sequencing has improved substantially in recent decades, culminating in the development of next‐generation sequencing (NGS) technologies. Modern NGS methods have helped the discovery of many new plant viruses. Nevertheless, there is still a need to establish solid assembly pipelines targeting small genomes characterised by low identities to known viral sequences. Here, we describe and discuss the fundamental steps required for discovering and sequencing new plant viral genomes by NGS. A practical pipeline and standard alternative tools used in NGS analysis are presented.

Sequence‐independent Amplification with Genome Multiplexing to Establish Complete Genome of Multipartite RNA Viruses: Cucumber mosaic virus as a Case Study

AbstractReverse transcription–polymerase chain reaction (RT‐PCR) is considered one of the accurate and reliable techniques for plant virus detection and genome characterization. Environmental conditions, error prone replication and biological competition lead to mutations and sequence variation. This results in generation of various strains/variants. These conditions present difficulties in genome amplification, especially with techniques which utilize prior sequence information, such as RT‐PCR. Keeping in view this difficulty, in this study, we used replicating forms (RFs) from (Cucumber mosaic virus) infected Nicotiana tabacum var. Xanthi as a precursor for ligation of self‐primed anchor primer at 3′‐hydroxyl end and subsequently used it for first strand cDNA synthesis. This cDNA was amplified further by sequence‐independent single‐primer amplification (SISPA) for genome multiplexing of CMV, a tripartite ssRNA positive‐sense virus. It was also observed that the presence of anchor primer aids towards cloning of genome segments. These results demonstrate genome multiplexing for complete genome characterization of single‐stranded, multipartite plant RNA viruses as the new application of SISPA.

Next-Generation Sequencing and Genome Editing in Plant Virology.

Next-generation sequencing (NGS) has been applied to plant virology since 2009. NGS provides highly efficient, rapid, low cost DNA, or RNA high-throughput sequencing of the genomes of plant viruses and viroids and of the specific small RNAs generated during the infection process. These small RNAs, which cover frequently the whole genome of the infectious agent, are 21–24 nt long and are known as vsRNAs for viruses and vd-sRNAs for viroids. NGS has been used in a number of studies in plant virology including, but not limited to, discovery of novel viruses and viroids as well as detection and identification of those pathogens already known, analysis of genome diversity and evolution, and study of pathogen epidemiology. The genome engineering editing method, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been successfully used recently to engineer resistance to DNA geminiviruses (family, Geminiviridae) by targeting different viral genome sequences in infected Nicotiana benthamiana or Arabidopsis plants. The DNA viruses targeted include tomato yellow leaf curl virus and merremia mosaic virus (begomovirus); beet curly top virus and beet severe curly top virus (curtovirus); and bean yellow dwarf virus (mastrevirus). The technique has also been used against the RNA viruses zucchini yellow mosaic virus, papaya ringspot virus and turnip mosaic virus (potyvirus) and cucumber vein yellowing virus (ipomovirus, family, Potyviridae) by targeting the translation initiation genes eIF4E in cucumber or Arabidopsis plants. From these recent advances of major importance, it is expected that NGS and CRISPR-Cas technologies will play a significant role in the very near future in advancing the field of plant virology and connecting it with other related fields of biology.

Noncoding RNAs of Plant Viruses and Viroids: Sponges of Host Translation and RNA Interference Machinery.

Noncoding sequences in plant viral genomes are well-known to control viral replication and gene expression in cis. However, plant viral and viroid noncoding (nc)RNA sequences can also regulate gene expression acting in trans, often acting like 'sponges' that bind and sequester host cellular machinery to favor viral infection. Noncoding sequences of small subgenomic (sg)RNAs of Barley yellow dwarf virus (BYDV) and Red clover necrotic mosaic virus (RCNMV) contain a cap-independent translation element that binds translation initiation factor eIF4G. We provide new evidence that a sgRNA of BYDV can globally attenuate host translation, probably by sponging eIF4G. Subgenomic ncRNA of RCNMV is generated via 5' to 3' degradation by a host exonuclease. The similar noncoding subgenomic flavivirus (sf)RNA, inhibits the innate immune response, enhancing viral pathogenesis. Cauliflower mosaic virus transcribes massive amounts of a 600-nt ncRNA, which is processed into small RNAs that overwhelm the host's RNA interference (RNAi) system. Viroids use the host RNAi machinery to generate viroid-derived ncRNAs that inhibit expression of host defense genes by mimicking a microRNA. More examples of plant viral and viroid ncRNAs are likely to be discovered, revealing fascinating new weaponry in the host-virus arms race.

Interaction network of tobacco etch potyvirus NIa protein with the host proteome during infection.

BackgroundThe genomes of plant viruses have limited coding capacity, and to complete their infectious cycles, viral factors must target, direct or indirectly, many host elements. However, the interaction networks between viruses and host factors are poorly understood. The genus Potyvirus is the largest group of plus-strand RNA viruses infecting plants. Potyviral nuclear inclusion a (NIa) plays many roles during infection. NIa is a polyprotein consisting of two domains, viral protein genome-linked (VPg) and protease (NIaPro), separated by an inefficiently utilized self-proteolytic site. To gain insights about the interaction between potyviral NIa and the host cell during infection, we constructed Tobacco etch virus (TEV, genus Potyvirus) infectious clones in which the VPg or the NIaPro domains of NIa were tagged with the affinity polypeptide Twin-Strep-tag and identified the host proteins targeted by the viral proteins by affinity purification followed by mass spectrometry analysis (AP-MS).ResultsWe identified 232 different Arabidopsis thaliana proteins forming part of complexes in which TEV NIa products were also involved. VPg and NIaPro specifically targeted 89 and 76 of these proteins, respectively, whereas 67 proteins were targeted by both domains and considered full-length NIa targets. Taking advantage of the currently known A. thaliana interactome, we constructed a protein interaction network between TEV NIa domains and 516 host proteins. The most connected elements specifically targeted by VPg were G-box regulating factor 6 and mitochondrial ATP synthase δ subunit; those specifically targeted by NIaPro were plasma membrane aquaporin PIP2;7 and actin 7, whereas those targeted by full-length NIa were heat shock protein 70–1 and photosystem protein LHCA3. Moreover, a contextualization in the global A. thaliana interactome showed that NIa targets are not more connected with other host proteins than expected by chance, but are in a position that allows them to connect with other host proteins in shorter paths. Further analysis of NIa-targeted host proteins revealed that they are mainly involved in response to stress, metabolism, photosynthesis, and localization. Many of these proteins are connected with the phytohormone ethylene.ConclusionsPotyviral NIa targets many host elements during infection, establishing a network in which information is efficiently transmitted.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2394-y) contains supplementary material, which is available to authorized users.

A Case Study on Discovery of Novel Citrus Leprosis Virus Cytoplasmic Type 2 Utilizing Small RNA Libraries by Next Generation Sequencing and Bioinformatic Analyses

The advent of innovative sequencing technology referred to as “Next-Generation” Sequencing (NGS), provides a new approach to identify the ‘unknown known’ and ‘unknown unknown’ viral pathogens without a priori knowledge. The genomes of plant viruses can be rapidly determined even when occurring at extremely low titers in the infected host. The method is based on massively parallel sequencing of the population of small RNA molecules 18-35 nucleotides in length produced by RNA silencing host defense. Improvements in chemistries, bioinformatic tools and advances in engineering has reduced the costs of NGS, increased its accessibility, and enabled its application in the field of plant virology. In this review, we discuss the utilization of the Illumina GA IIX platform combined with the application of molecular biology and bioinformatic tools for the discovery of a novel cytoplasmic Citrus leprosis virus (CiLV). This new virus produced symptoms typical of CiLV but was not detected with either serological or PCR-based assays for the previously described virus. The new viral genome was also present in low titer in sweet orange (Citrus sinensis), an important horticultural crop with incomplete genomic resources. This is a common situation in horticultural research and provides an example of the broader utility of this approach. In addition to the discovery of novel viruses, the sequence data may be useful for studies of viral evolution and ecology and the interactions between viral and host transcriptomes.

A model for intracellular movement of Cauliflower mosaic virus: the concept of the mobile virion factory.

The genomes of many plant viruses have a coding capacity limited to <10 proteins, yet it is becoming increasingly clear that individual plant virus proteins may interact with several targets in the host for establishment of infection. As new functions are uncovered for individual viral proteins, virologists have realized that the apparent simplicity of the virus genome is an illusion that belies the true impact that plant viruses have on host physiology. In this review, we discuss our evolving understanding of the function of the P6 protein of Cauliflower mosaic virus (CaMV), a process that was initiated nearly 35 years ago when the CaMV P6 protein was first described as the 'major inclusion body protein' (IB) present in infected plants. P6 is now referred to in most articles as the transactivator (TAV)/viroplasmin protein, because the first viral function to be characterized for the Caulimovirus P6 protein beyond its role as an inclusion body protein (the viroplasmin) was its role in translational transactivation (the TAV function). This review will discuss the currently accepted functions for P6 and then present the evidence for an entirely new function for P6 in intracellular movement.

Translation of the shallot virus X TGB3 gene depends on non-AUG initiation and leaky scanning.

Triple gene block (TGB), a conserved gene module found in the genomes of many filamentous and rod-shaped plant viruses, encodes three proteins, TGB1, TGB2 and TGB3, required for viral cell-to-cell movement through plasmodesmata and systemic transport via the phloem. The genome of Shallot virus X, the type species of the genus Allexivirus, includes TGB1 and TGB2 genes, but contains no canonical ORF for TGB3 protein. However, a TGB3-like protein-encoding sequence lacking an AUG initiator codon has been found in the shallot virus X (ShVX) genome in a position typical for TGB3 genes. This putative TGB3 gene is conserved in all allexiviruses. Here, we carried out sequence analysis to predict possible non-AUG initiator codons in the ShVX TGB3-encoding sequence. We further used an agroinfiltration assay in Nicotiana benthamiana to confirm this prediction. Site-directed mutagenesis was used to demonstrate that the ShVX TGB3 could be translated on a bicistronic mRNA template via a leaky scanning mechanism.

Phylogenetic relationship of some "accessory" helicases of plant positive-stranded RNA viruses: toward understanding the evolution of triple gene block.

Recently, we hypothesized that silencing suppression activity gained by a viral replicative helicase led to the emergence of the second helicase possessing activity of the viral silencing suppressor and/or movement protein (MP). Our hypothesis accounted for the evolutionary origin of the specialized ‘triple gene block’ (TGB) in plant virus genomes encoding the MPs TGB1, TGB2, and TGB3 required for viral cell-to-cell transport through plasmodesmata. Here, we used public transcriptome databases to identify previously unrecognized viruses. The analysis of novel viral genomes further supported the previously proposed scenario of TGB origin and evolution, which included the following steps. First, the accessory helicase gene could have been acquired by horizontal gene transfer (HGT) presumably occured independently in different virus groups. Second, the TGB2 gene evolved by HGT or autonomization of the C-terminal transmembrane domain found in at least one TGB1 helicase. Third, the TGB3 gene has most likely emerged in the genomic block consisting of the TGB1 and TGB2 genes.

Conserved motifs in a tombusvirus polymerase modulate genome replication, subgenomic transcription, and amplification of defective interfering RNAs.

The replication of plus-strand RNA virus genomes is mediated by virally encoded RNA-dependent RNA polymerases (RdRps). We have investigated the role of the C-proximal region in the RdRp of tomato bushy stunt virus (TBSV) in mediating viral RNA synthesis. TBSV is the prototype species in the genus Tombusvirus, family Tombusviridae, and its RdRp is responsible for replicating the viral genome, transcribing two subgenomic mRNAs, and supporting replication of defective interfering RNAs. Comparative sequence analysis of the RdRps of tombusvirids identified three highly conserved motifs in their C-proximal regions, and these sequences were subsequently targeted for mutational analysis in TBSV. The results revealed that these motifs are important for (i) synthesizing viral genomic RNA and subgenomic mRNAs, (ii) facilitating plus- and/or minus-strand synthesis, and (iii) modulating trans-replication of a defective interfering RNA. These motifs were also found to be conserved in other plant viruses as well as in a fungal and insect virus. The collective findings are discussed in relation to viral RNA synthesis and taxonomy. Little is currently known about the structure and function of the viral polymerases that replicate the genomes of RNA plant viruses. Tombusviruses, the prototype of the tombusvirids, have been used as model plus-strand RNA plant viruses for understanding many of the steps in the infectious process; however, their polymerases remain poorly characterized. To help address this issue, the function of the C-terminal region of the polymerase of a tombusvirus was investigated. Three conserved motifs were identified and targeted for mutational analysis. The results revealed that these polymerase motifs are important for determining what type of viral RNA is produced, facilitating different steps in viral RNA production, and amplifying subgenomic RNA replicons. Accordingly, the C-terminal region of the tombusvirus polymerase is needed for a variety of fundamental activities. Furthermore, as these motifs are also present in distantly related viruses, the significance of these results extends beyond tombusvirids.

Plant virology and next generation sequencing: experiences with a Potyvirus.

Next generation sequencing is quickly emerging as the go-to tool for plant virologists when sequencing whole virus genomes, and undertaking plant metagenomic studies for new virus discoveries. This study aims to compare the genomic and biological properties of Bean yellow mosaic virus (BYMV) (genus Potyvirus), isolates from Lupinus angustifolius plants with black pod syndrome (BPS), systemic necrosis or non-necrotic symptoms, and from two other plant species. When one Clover yellow vein virus (ClYVV) (genus Potyvirus) and 22 BYMV isolates were sequenced on the Illumina HiSeq2000, one new ClYVV and 23 new BYMV sequences were obtained. When the 23 new BYMV genomes were compared with 17 other BYMV genomes available on Genbank, phylogenetic analysis provided strong support for existence of nine phylogenetic groupings. Biological studies involving seven isolates of BYMV and one of ClYVV gave no symptoms or reactions that could be used to distinguish BYMV isolates from L. angustifolius plants with black pod syndrome from other isolates. Here, we propose that the current system of nomenclature based on biological properties be replaced by numbered groups (I–IX). This is because use of whole genomes revealed that the previous phylogenetic grouping system based on partial sequences of virus genomes and original isolation hosts was unsustainable. This study also demonstrated that, where next generation sequencing is used to obtain complete plant virus genomes, consideration needs to be given to issues regarding sample preparation, adequate levels of coverage across a genome and methods of assembly. It also provided important lessons that will be helpful to other plant virologists using next generation sequencing in the future.

Scrutinizing Virus Genome Termini by High-Throughput Sequencing

Analysis of genomic terminal sequences has been a major step in studies on viral DNA replication and packaging mechanisms. However, traditional methods to study genome termini are challenging due to the time-consuming protocols and their inefficiency where critical details are lost easily. Recent advances in next generation sequencing (NGS) have enabled it to be a powerful tool to study genome termini. In this study, using NGS we sequenced one iridovirus genome and twenty phage genomes and confirmed for the first time that the high frequency sequences (HFSs) found in the NGS reads are indeed the terminal sequences of viral genomes. Further, we established a criterion to distinguish the type of termini and the viral packaging mode. We also obtained additional terminal details such as terminal repeats, multi-termini, asymmetric termini. With this approach, we were able to simultaneously detect details of the genome termini as well as obtain the complete sequence of bacteriophage genomes. Theoretically, this application can be further extended to analyze larger and more complicated genomes of plant and animal viruses. This study proposed a novel and efficient method for research on viral replication, packaging, terminase activity, transcription regulation, and metabolism of the host cell.

Occurrence and analysis of imperfect microsatellites in diverse potyvirus genomes

Simple sequence repeats (SSRs) or microsatellites are known to exhibit ubiquitous across all kingdoms of life including viruses. However, imperfections in simple sequence repeats have been analyzed in genomes of human, Escherichia coli and Human Immunodeficiency virus. The assessment of compound microsatellites in plant viral genomes is yet to be studied. Potyviruses severely affect crop plant growth and reduce economic yield in diverse cropping systems worldwide. Hence, we analyze the nature and distribution of compound microsatellites present in complete genome of 45 potyvirus species. The results indicate that compound microsatellites accounted for about 0% to 15.15% of all microsatellites and have low complexity as compared to that of prokaryotic genomes. Overall, 14% of compound microsatellites were of similar motifs and such motif duplications were observed for CA, TA and AG repeats. Among all 45 potyvirus genomes analyzed, SSR couple (AG)-x-(AC) was found to be the most abundant one. Hence it is apparent that in contrast to eukaryotes, majority of compound microsatellites in potyviruses were composed of variant motifs. We also highlight the relative frequency of different classes of compound microsatellites as well as their patterns of distribution and correlate with biology of potyviruses. Further characterization of such variation is important for elucidating the origin, mutational processes, and structure of these widely used, but incompletely understood sequences.

Identification of a Plant Viral RNA Genome in the Nucleus

Viruses contain either DNA or RNA as genomes. DNA viruses replicate within nucleus, while most RNA viruses, especially (+)-sense single-stranded RNA, replicate and are present within cytoplasm. We proposed a new thought that is contrary to the common notion that (+)-sense single-stranded RNA viruses are present only in the cytoplasm. In this study, we question whether the genome of a plant RNA virus (non-retroviral) is present in the nucleus of infected cells? Hibiscus chlorotic ringspot virus (HCRSV) RNA was detected in the nucleus of infected cells, as shown by fluorescent in situ hybridization. Western blot using anti-histone 3 and anti-phosphoenolpyruvate carboxylase showed that nuclei were highly purified from mock and HCRSV-infected kenaf (Hibiscus cannabilis L.) leaves, respectively. The p23 and HCRSV coat protein (CP) coding regions were both amplified from total RNA extracted from isolated nuclei. Viral RNA in the nucleus may be used to generate viral microRNAs (vir-miRNAs), as five putative vir-miRNAs were predicted from HCRSV using the vir-miRNAs prediction database. The vir-miRNA (hcrsv-miR-H1-5p) was detected using TaqMan® stem-loop real-time PCR, and by northern blot using DIG-end labeled probe in HCRSV-infected kenaf leaves. Finally, a novel nuclear localization signal (NLS) was discovered in p23 of HCRSV. The NLS interacts with importin α and facilitates viral RNA genome to enter nucleus. We demonstrate the presence of a (+)-sense single-stranded viral RNA within nucleus.

Crystal Structure of the Superfamily 1 Helicase from Tomato Mosaic Virus

The genomes of the Tomato mosaic virus and many other plant and animal positive-strand RNA viruses of agronomic and medical importance encode superfamily 1 helicases. Although helicases play important roles in viral replication, the crystal structures of viral superfamily 1 helicases have not been determined. Here, we report the crystal structure of a fragment (S666 to Q1116) of the replication protein from Tomato mosaic virus. The structure reveals a novel N-terminal domain tightly associated with a helicase core. The helicase core contains two RecA-like α/β domains without any of the accessory domain insertions that are found in other superfamily 1 helicases. The N-terminal domain contains a flexible loop, a long α-helix, and an antiparallel six-stranded β-sheet. On the basis of the structure, we constructed deletion mutants of the S666-to-Q1116 fragment and performed split-ubiquitin-based interaction assays in Saccharomyces cerevisiae with TOM1 and ARL8, host proteins that are essential for tomato mosaic virus RNA replication. The results suggested that both TOM1 and ARL8 interact with the long α-helix in the N-terminal domain and that TOM1 also interacts with the helicase core. Prediction of secondary structures in other viral superfamily 1 helicases and comparison of those structures with the S666-to-Q1116 structure suggested that these helicases have a similar fold. Our results provide a structural basis of viral superfamily 1 helicases.

Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis

Insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. Their numerous advantages make fluorescent proteins the markers of choice in most studies. However, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. Here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. The reporter system is based on expression of Antirrhinum majus MYB-related Rosea1 (Ros1) transcription factor (220 amino acids; 25.7 kD) that activates a series of biosynthetic genes leading to accumulation of colored anthocyanins. Using two different tobacco etch potyvirus recombinant clones tagged with Ros1, we show that infected tobacco (Nicotiana tabacum) tissues turn bright red, demonstrating that in this context, the sole expression of Ros1 is sufficient to induce pigment accumulation to a level readily detectable to the naked eye. This marker system also reports viral load qualitatively and quantitatively by means of a very simple extraction process. The Ros1 marker remained stable within the potyvirus genome through successive infectious passages from plant to plant. The main limitation of this marker system is that color output will depend on each particular plant host-virus combination and must be previously tested. However, our experiments demonstrate accurate tracking of turnip mosaic potyvirus infecting Arabidopsis (Arabidopsis thaliana) and either tobacco mosaic virus or potato X virus infecting Nicotiana benthamiana, stressing the general applicability of the method.

Rapid Genetic Diversification and High Fitness Penalties Associated with Pathogenicity Evolution in a Plant Virus

Under the gene-for-gene model of host-pathogen coevolution, recognition of pathogen avirulence factors by host resistance factors triggers host defenses and limits infection. Theory predicts that the evolution of higher levels of pathogenicity will be associated with fitness penalties and that the cost of higher pathogenicity must be much smaller than that of not infecting the host. The analysis of pathogenicity costs is of academic and applied relevance, as these are determinants for the success of resistance genes bred into crops for disease control. However, most previous attempts of addressing this issue in plant pathogens yielded conflicting and inconclusive results. We have analyzed the costs of pathogenicity in pepper-infecting tobamoviruses defined by their ability to infect pepper plants with different alleles at the resistance locus L. We provide conclusive evidence of pathogenicity-associated costs by comparison of pathotype frequency with the fraction of the crop carrying the various resistance alleles, by timescaled phylogenies, and by temporal analyses of population dynamics and selection pressures using nucleotide sequences. In addition, experimental estimates of relative fitness under controlled conditions also provided evidence of high pathogenicity costs. These high pathogenicity costs may reflect intrinsic properties of plant virus genomes and should be considered in future models of host-parasite coevolution.