The Identification of Viral Pathogens in a Physostegia virginiana Plant Using High-Throughput RNA Sequencing.

Physostegia virginiana is an important ornamental and cut-flower plant in China. Its commonly used method of clonal propagation leads to virus accumulation in this plant. However, which viruses can infect the Physostegia virginiana plant remains to be illuminated. In this work, five viral pathogens in a Physostegia virginiana plant with virus-like symptoms of yellow, shriveled, and curled leaves were identified using RNA-seq, bioinformatics, and molecular biological techniques. These techniques allowed us to identify five viruses comprising one known alfalfa mosaic virus (AMV) and four novel viruses. The novel viruses include a virus belonging to the genus Fabavirus, temporarily named Physostegia virginiana crinkle-associated virus 1 (PVCaV1); two viruses belonging to the genus Caulimovirus, temporarily named Physostegia virginiana caulimovirus 1 and 2 (PVCV1 and PVCV2); and a virus belonging to the genus Fijivirus, temporarily named Physostegia virginiana fijivirus (PVFV). The genome sequences of PVCaV1, PVCV1, and PVCV2, and the partial genome sequence of PVFV were identified. Genome organizations and genetic evolutionary relationships of all four novel viruses were analyzed. PVCaV1 has a relatively close evolutionary relationship with five analyzed fabiviruses. PVCV1 and PVCV2 have separately a closest evolutionary relationship with lamium leaf distortion-associated virus (LLDAV) and figwort mosaic virus (FMV), and PVFV has a close evolutionary relationship with the five analyzed fijiviruses. Additionally, PVCaV1 can infect Nicotiana benthamiana plants via friction inoculation. The findings enrich our understanding of Physostegia virginiana viruses and contribute to the prevention and control of Physostegia virginiana viral diseases.

Comparative Plant Transcriptome Profiling of Arabidopsis thaliana Col-0 and Camelina sativa var. Celine Infested with Myzus persicae Aphids Acquiring Circulative and Noncirculative Viruses Reveals Virus- and Plant-Specific Alterations Relevant to Aphid Feeding Behavior and Transmission.

Evidence is accumulating that plant viruses alter host plant traits in ways that modify their insect vectors' behavior. These alterations often enhance virus transmission, which has led to the hypothesis that these effects are manipulations caused by viral adaptation. However, we lack a mechanistic understanding of the genetic basis of these indirect, plant-mediated effects on vectors, their dependence on the plant host, and their relation to the mode of virus transmission. Transcriptome profiling of Arabidopsis thaliana and Camelina sativa plants infected with turnip yellows virus (TuYV) or cauliflower mosaic virus (CaMV) and infested with the common aphid vector Myzus persicae revealed strong virus- and host-specific differences in gene expression patterns. CaMV infection caused more severe effects on the phenotype of both plant hosts than did TuYV infection, and the severity of symptoms correlated strongly with the proportion of differentially expressed genes, especially photosynthesis genes. Accordingly, CaMV infection modified aphid behavior and fecundity more strongly than did infection with TuYV. Overall, infection with CaMV, relying on the noncirculative transmission mode, tends to have effects on metabolic pathways, with strong potential implications for insect vector-plant host interactions (e.g., photosynthesis, jasmonic acid, ethylene, and glucosinolate biosynthetic processes), while TuYV, using the circulative transmission mode, alters these pathways only weakly. These virus-induced deregulations of genes that are related to plant physiology and defense responses might impact both aphid probing and feeding behavior on infected host plants, with potentially distinct effects on virus transmission. IMPORTANCE Plant viruses change the phenotype of their plant hosts. Some of the changes impact interactions of the plant with insects that feed on the plants and transmit these viruses. These modifications may result in better virus transmission. We examine here the transcriptomes of two plant species infected with two viruses with different transmission modes to work out whether there are plant species-specific and transmission mode-specific transcriptome changes. Our results show that both are the case.

Synthetic Salicylic acid inducible recombinant promoter for translational research.

In the present study, we have developed an inter-molecularly shuffled caulimoviral promoter for protein over-expression by placing the Upstream Activation Sequence (UAS) of Figwort Mosaic Virus (FMV; -249 to -54) at the 5'-end of the Cassava Vein Mosaic Virus (CsVMV) promoter fragment 8 (CsVMV8; -215 to +166) to design a hybrid promoter; FUASCsV8CP. The FUASCsV8CP promoter exhibited approximately 2.1 and 2.0 times higher GUS-activities than that obtained from the CaMV35S promoter, in tobacco (Xanthi Brad) protoplasts and in Agroinfiltration assays respectively. Hereto, when FUASCsV8CP was assayed using transgenic tobacco plants (T2- generation), it showed 2.0 times stronger activity than CaMV35S promoter and almost equivalent activity to that of CaMV35S2 promoter. The promoter displayed Salicylic acid (SA) inducibility and hence can also be used for ensuring effective gene expression in plants under constitutive as well as specific inducible conditions. Furthermore, FUASCsV8CP was used to drive the expression of victoviral Vin gene (encoding Victoriocin) transiently in tobacco. The recombinant Victoriocin could be successfully detected by western blotting three days post infiltration. Also, the in vitro Agar-based killing zone assays employing plant-derived Victoriocin-His (obtained from transient expression of Vin) revealed enhanced antifungal activity of Victoriocin against hemi-biotrophic pathogen Phoma exigua Desm. var. exigua.

Diverse and variable virus communities in wild plant populations revealed by metagenomic tools.

Wild plant populations may harbour a myriad of unknown viruses. As the majority of research efforts have targeted economically important plant species, the diversity and prevalence of viruses in the wild has remained largely unknown. However, the recent shift towards metagenomics-based sequencing methodologies, especially those targeting small RNAs, is finally enabling virus discovery from wild hosts. Understanding this diversity of potentially pathogenic microbes in the wild can offer insights into the components of natural biodiversity that promotes long-term coexistence between hosts and parasites in nature, and help predict when and where risks of disease emergence are highest. Here, we used small RNA deep sequencing to identify viruses in Plantago lanceolata populations, and to understand the variation in their prevalence and distribution across the Åland Islands, South-West Finland. By subsequent design of PCR primers, we screened the five most common viruses from two sets of P. lanceolata plants: 164 plants collected from 12 populations irrespective of symptoms, and 90 plants collected from five populations showing conspicuous viral symptoms. In addition to the previously reported species Plantago lanceolata latent virus (PlLV), we found four potentially novel virus species belonging to Caulimovirus, Betapartitivirus, Enamovirus, and Closterovirus genera. Our results show that virus prevalence and diversity varied among the sampled host populations. In six of the virus infected populations only a single virus species was detected, while five of the populations supported between two to five of the studied virus species. In 20% of the infected plants, viruses occurred as coinfections. When the relationship between conspicuous viral symptoms and virus infection was investigated, we found that plants showing symptoms were usually infected (84%), but virus infections were also detected from asymptomatic plants (44%). Jointly, these results reveal a diverse virus community with newly developed tools and protocols that offer exciting opportunities for future studies on the eco-evolutionary dynamics of viruses infecting plants in the wild.

Exploring the role of cellular homologous of the 30K-superfamily of plant virus movement proteins.

Genes orthologous to the 30K-superfamily of movement proteins (MP) from plant viruses have been recently discovered by bioinformatics analyses as integrated elements in the genome of most vascular plants. However, their functional relevance for plants is still unclear. Here, we undertake some preliminary steps into the functional characterization of one of these putative MP genes found in Arabidopsis thaliana. We found that the AtMP gene is expressed at different stages of the plant development, with accumulation being highest in flowers but lowest in mature siliques. We also found down-regulation of the gene may result in a small delay in plant development and in an exacerbation of the negative effect of salinity in germination efficiency. We have also explored whether changes in expression of the endogenous AtMP have any effect on susceptibility to infection with several viruses, and found that the infectivity of tobacco rattle tobravirus was strongly dependent on the expression of the endogenous AtMP. Finally, we have cloned the endogenous MP from four different plant species into an expression vector that allows for specifically assessing their activity as cell-to-cell movement proteins and have shown that though some may still retain the ancestral activity, they do so in a quite inefficient manner, thus suggesting they have acquired a novel function during adaptation to the host genome.

Recombinant Promoter (MUASCsV8CP) Driven Totiviral Killer Protein 4 (KP4) Imparts Resistance Against Fungal Pathogens in Transgenic Tobacco.

Development of disease-resistant plant varieties achieved by engineering anti-microbial transgenes under the control of strong promoters can suffice the inhibition of pathogen growth and simultaneously ensure enhanced crop production. For evaluating the prospect of such strong promoters, we comprehensively characterized the full-length transcript promoter of Cassava Vein Mosaic Virus (CsVMV; -565 to +166) and identified CsVMV8 (-215 to +166) as the highest expressing fragment in both transient and transgenic assays. Further, we designed a new chimeric promoter 'MUASCsV8CP' through inter-molecular hybridization among the upstream activation sequence (UAS) of Mirabilis Mosaic Virus (MMV; -297 to -38) and CsVMV8, as the core promoter (CP). The MUASCsV8CP was found to be ∼2.2 and ∼2.4 times stronger than the CsVMV8 and CaMV35S promoters, respectively, while its activity was found to be equivalent to that of the CaMV35S2 promoter. Furthermore, we generated transgenic tobacco plants expressing the totiviral 'Killer protein KP4' (KP4) under the control of the MUASCsV8CP promoter. Recombinant KP4 was found to accumulate both in the cytoplasm and apoplast of plant cells. The agar-based killing zone assays revealed enhanced resistance of plant-derived KP4 against two deuteromycetous foliar pathogenic fungi viz. Alternaria alternata and Phoma exigua var. exigua. Also, transgenic plants expressing KP4 inhibited the growth progression of these fungi and conferred significant fungal resistance in detached-leaf and whole plant assays. Taken together, we establish the potential of engineering "in-built" fungal stress-tolerance in plants by expressing KP4 under a novel chimeric caulimoviral promoter in a transgenic approach.

RETRACTED: WRKY71 and TGA1a physically interact and synergistically regulate the activity of a novel promoter isolated from Petunia vein-clearing virus.

Caulimoviral promoters have become excellent tools for efficient transgene expression in plants. However, the transcriptional framework controlling their systematic regulation is poorly understood. To understand this regulatory mechanism, we extensively studied a novel caulimoviral promoter, PV8 (-163 to +138, 301 bp), isolated from Petunia vein-clearing virus (PVCV). PVCV was found to be Salicylic acid (SA)-inducible and 2.5-3.0 times stronger than the widely used CaMV35S promoter. In silico analysis of the PV8 sequence revealed a unique clustering of two stress-responsive cis-elements, namely, as-11 and W-box1-2, located within a span of 31 bp (-74 to -47) that bound to the TGA1a and WRKY71 plant transcription factors (TFs), respectively. We found that as-1 (TTACG) and W-box (TGAC) elements occupied both TGA1a and WRKY71 on the PV8 backbone. Mutational studies demonstrated that the combinatorial influence of as-1 (-57) and W-box1-2 (-74 and -47) on the PV8 promoter sequence largely modulated its activity. TGA1a and WRKY71 physically interacted and cooperatively enhanced the transcriptional activity of the PV8 promoter. Biotic stress stimuli induced PV8 promoter activity by ~1.5 times. We also established the possible pathogen-elicitor function of AtWRKY71 and NtabWRKY71 TFs. Altogether, this study elucidates the interplay between TFs, biotic stress and caulimoviral promoter function.

Structural characterization of a novel full-length transcript promoter from Horseradish Latent Virus (HRLV) and its transcriptional regulation by multiple stress responsive transcription factors.

KEY MESSAGE: The promoter fragment described in this study can be employed for strong transgene expression under both biotic and abiotic stress conditions. Plant-infecting Caulimoviruses have evolved multiple regulatory mechanisms to address various environmental stimuli during the course of evolution. One such mechanism involves the retention of discrete stress responsive cis-elements which are required for their survival and host-specificity. Here we describe the characterization of a novel Caulimoviral promoter isolated from Horseradish Latent Virus (HRLV) and its regulation by multiple stress responsive Transcription factors (TFs) namely DREB1, AREB1 and TGA1a. The activity of full length transcript (Flt-) promoter from HRLV (- 677 to + 283) was investigated in both transient and transgenic assays where we identified H12 (- 427 to + 73) as the highest expressing fragment having ~ 2.5-fold stronger activity than the CaMV35S promoter. The H12 promoter was highly active and near-constitutive in the vegetative and reproductive parts of both Tobacco and Arabidopsis transgenic plants. Interestingly, H12 contains a distinct cluster of cis-elements like dehydration-responsive element (DRE-core; GCCGAC), an ABA-responsive element (ABRE; ACGTGTC) and as-1 element (TGACG) which are known to be induced by cold, drought and pathogen/SA respectively. The specific binding of DREB1, AREB1 and TGA1a to DRE, ABRE and as-1 elements respectively were confirmed by the gel-binding assays using H12 promoter-specific probes. Detailed mutational analysis of the H12 promoter suggested that the presence of DRE-core and as-1 element was indispensable for its activity which was further confirmed by the transactivation assays. Our studies imply that H12 could be a valuable genetic tool for regulated transgene expression under diverse environmental conditions.

Complete genome sequence of a putative new caulimovirus which exists as endogenous pararetroviral sequences in Angelica dahurica.

A virus isolate designated Angelica bushy stunt virus (AnBSV), provisionally representing a new species in the genus Caulimovirus, was discovered in the medicinal plant Angelica dahurica. The complete 8,300-nt genomic DNA of AnBSV had seven putative open reading frames containing conserved domains/motifs, which are typical features of caulimoviruses, and showed the greatest nucleotide sequence identity (74% identity and 27% query coverage) to a lamium leaf distortion virus isolate. Interestingly, the new caulimovirus exists as endogenous pararetroviral sequences in the host plant and is considered to have multiple defective plant genome-integrated copies that may lead to the generation of subgenomic DNA species.

Genome-wide analysis of EgEVE_1, a transcriptionally active endogenous viral element associated to small RNAs in Eucalyptus genomes

Abstract Endogenous viral elements (EVEs) are the result of heritable horizontal gene transfer from viruses to hosts. In the last years, several EVE integration events were reported in plants by the exponential availability of sequenced genomes. Eucalyptus grandis is a forest tree species with a sequenced genome that is poorly studied in terms of evolution and mobile genetic elements composition. Here we report the characterization of E. grandis endogenous viral element 1 (EgEVE_1), a transcriptionally active EVE with a size of 5,664 bp. Phylogenetic analysis and genomic distribution demonstrated that EgEVE_1 is a newly described member of the Caulimoviridae family, distinct from the recently characterized plant Florendoviruses. Genomic distribution of EgEVE_1 and Florendovirus is also distinct. EgEVE_1 qPCR quantification in Eucalyptus urophylla suggests that this genome has more EgEVE_1 copies than E. grandis. EgEVE_1 transcriptional activity was demonstrated by RT-qPCR in five Eucalyptus species and one intrageneric hybrid. We also identified that Eucalyptus EVEs can generate small RNAs (sRNAs),that might be involved in de novo DNA methylation and virus resistance. Our data suggest that EVE families in Eucalyptus have distinct properties, and we provide the first comparative analysis of EVEs in Eucalyptus genomes.

Novel Synthetic Promoters from the Cestrum Yellow Leaf Curling Virus.

Constitutive promoters direct gene expression uniformly in most tissues and cells at all stages of plant growth and development; they confer steady levels of transgene expression in plant cells and hence their demand is high in plant biology. The gene silencing due to promoter homology can be avoided by either using diverse promoters isolated from different plant and viral genomes or by designing synthetic promoters. The aim of this chapter was to describe the basic protocols needed to develop and analyze novel, synthetic, nearly constitutive promoters from Cestrum yellow leaf curling virus (CmYLCV) through promoter/leader deletion and activating cis-sequence analysis. We also describe the methods to evaluate the strength of the promoters efficiently in various transient expression systems like agroinfiltration assay, gene-gun method, and assay in tobacco protoplasts. Besides, the detailed methods for developing transgenic plants (tobacco and Arabidopsis) for evaluation of the promoter using the GUS reporter gene are also described. The detailed procedure for electrophoretic mobility shift assay (EMSA) coupled with super-shift EMSA analysis are also described for showing the binding of tobacco transcription factor, TGA1a to cis-elements in the CmYLCV distal promoter region.

RNA based viral silencing suppression in plant pararetroviruses.

The 35S promoter of cauliflower mosaic virus and that of other plant pararetroviruses gives rise to an RNA, which is both a pre-genome and a polycistronic mRNA. The 600 nucleotide long very structured leader of this RNA is also transcribed separately. The resulting 8S RNA is then converted to a double strand giving rise to a huge set of siRNAs, which suppress silencing. In this Mini-Review I discuss how this versatile stretch of 600 nts constitutes a masterpiece of evolution.