A guide to the contained use of plant virus infectious clones

Plant virus infectious clones are important tools with wide-ranging applications in different areas of biology and medicine. Their uses in plant pathology include the study of plant–virus interactions, and screening of germplasm as part of prebreeding programmes for virus resistance. They can also be modified to induce transient plant gene silencing (Virus Induced Gene Silencing – VIGS) and as expression vectors for plant or exogenous proteins, with applications in both plant pathology and more generally for the study of plant gene function. Plant viruses are also increasingly being investigated as expression vectors for in planta production of pharmaceutical products, known as molecular farming. However, plant virus infectious clones may pose a risk to the environment due to their ability to reconstitute fully functional, transmissible viruses. These risks arise from both their inherent pathogenicity and the effect of any introduced genetic modifications. Effective containment measures are therefore required. There has been no single comprehensive review of the biosafety considerations for the contained use of genetically modified plant viruses, despite their increasing importance across many biological fields. This review therefore explores the biosafety considerations for working with genetically modified plant viruses in contained environments, with focus on plant growth facilities. It includes regulatory frameworks, risk assessment, assignment of biosafety levels, facility features and working practices. The review is based on international guidance together with information provided by plant virus researchers

Detection of infectious tobamoviruses in irrigation and drainage canals in Greater Poland

Water samples were collected from irrigation ditches and drainage canals surrounding fields in southern Greater Poland. Initially, the samples were subjected to low and highspeed centrifugation and obtained pellets were used to perform biological assays. Viral identification involved biological, electron microscopic as well as molecular methods. The occurrence of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) was demonstrated in 12 of the 17 examined water sources. The molecular analysis results showed TMV and ToMV co-infections in the analysed water samples. To our knowledge, this is the first report of tobamoviruses being found in environmental water in Poland

Detection of RNA1 and RNA2 of Soil-borne wheat mosaic virus in winter wheat grown from infected seeds

A Polish isolate of Soil-borne wheat mosaic virus (SBWMV-Pol1) was characterized by limited pathogenicity and a low concentration of virus particles in infected plant tissues. The aim of this research was to consider the possibility of seed-transmission dissemination of the virus. Seeds of winter wheat cv. Muszelka served as material for the studies. Two methods were involved in the diagnostics of seedlings grown from potentially infected seeds: enzyme-linked immunosorbent assay (ELISA), as the screening assay and immuno-capture-reverse transcription-polymerase chain reaction (IC-RT-PCR) for molecular confirmation of the infection. RNA1 and RNA2 of SBWMV-Pol1 were detected in 6 out of 1,410 plants submitted to diagnostic procedures. The possibility of seed transmission of SBWMV-Pol1 was discussed

Genetic diversity of Tomato black ring virus isolates collected from different hosts

Trabajo presentado a la Conferencia Virus Genomics and Evolution, celebrada en Hinxton (UK) del 18 al 20 de junio de 2018.This work was supported by the project no. 2015/17/B/NZ8/02407 from National Science Centre, Poland.Peer reviewe

Defective RNA particles of Tomato black ring virus: origin, structure and biological effect

Resumen del trabajo presentado a la Conferencia Power of Viruses, celebrada en Poreč (Croatia) del 16 al 18 de mayo de 2018.Tomato black ring virus (TBRV) infects a wide range of economically important plants, and is distributed worldwide. The genome of TBRV consists of two single stranded RNAs of positive polarity. Both RNAs contain a small protein VPg at their S' ends and have polyadenylated 3' ends. RNA1 is responsible for viral replication and polyproteins' processing and RNA2 for encapsidation and movement in plant. The TBRV infection can be accompanied by subviral particles such as satellite or defective RNAs. Defective RNA particles are deletion/and/or rearrangement variants of the viral genomes created during replication. The presence of subviral RNAs might have a great impact on viral replication, accumulation and symptoms observed on infected plants. D RNAs which are referred to interfere with multiplication of their helper viruses are called defective interfering RNAs (DI RNAs). TBRV is the only member of the Nepovirus genus that is known to form defective RNA particles (D RNAs) during replication. In this study, we analyzed the origin and structure of D RNAs generated de novo during prolonged passages of TBRV isolates in Chenopodium quinoa plants. Moreover, the effect of D RNAs on helper virus replication and symptoms observed on infected plants was estimated. Two TBRV isolates, originated from Solanum lycopersicum and Lactuca sativa, were serially passaged in C. quinoa and after 1S passages the purified viral preparations were obtained. Viral RNA was isolated using phenol-chloroform method. The analysis of RNA profile revealed the presence of short, D RNA molecules. D RNAs were amplified and sequenced, and obtained sequences were compared with the helper virvs genome. C. quinoa, S. lycopersicum and Nicotiana tabacum were infected.with TBRV+D RNA and TBRV-D RNA. The symptoms and viral accumulation were manito red 7, 14, 21, and 28 dpi. The accumulation level of TBRV in each host was measured by RT-qPCR and statistical analyses were performed. The analysis revealed that D RNAs derived by interna! deletion in the RNA1 molecule of TBRV and contained a portian of S' non-coding region, a fragment of polymerase gene and almost entire 3' non-coding region. Plants infected with TBRV+D RNA displayed milder symptoms in comparison to those infected with TBRV-D RNA. Statistical analyses confirmed that D RNAs interfere with TBRV replication and thus should be referred to as defective interfering particles.Peer reviewe

Accumulation of 24 nucleotide transgene‐derived siRNAs is associated with crinivirus immunity in transgenic plants

RNA silencing is a conserved antiviral defence mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing-mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harbouring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RNA-dependent RNA polymerase (RdRp) sequence exhibited immunity to systemic LIYV infection. Deep sequencing analysis was performed to characterize virus-derived small interfering RNAs (vsiRNAs) generated on systemic LIYV infection in non-transgenic N. benthamiana plants as well as transgene-derived siRNAs (t-siRNAs) derived from the immune-transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t-siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants, except for a significant increase in t-siRNAs of 24 nucleotides in length, which was consistent with small RNA northern blot results that showed the abundance of t-siRNAs of 21, 22 and 24 nucleotides in length. The accumulated 24-nucleotide sequences have not yet been reported in transgenic plants partially resistant to criniviruses, and thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24-nucleotide t-siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24-nucleotide t-siRNAs is associated with crinivirus immunity in transgenic plants

High-Throughput Sequencing Facilitates Discovery of New Plant Viruses in Poland

Viruses cause epidemics on all major crops of agronomic importance, and a timely and accurate identification is essential for control. High throughput sequencing (HTS) is a technology that allows the identification of all viruses without prior knowledge on the targeted pathogens. In this paper, we used HTS technique for the detection and identification of different viral species occurring in single and mixed infections in plants in Poland. We analysed various host plants representing different families. Within the 20 tested samples, we identified a total of 13 different virus species, including those whose presence has not been reported in Poland before: clover yellow mosaic virus (ClYMV) and melandrium yellow fleck virus (MYFV). Due to this new finding, the obtained sequences were compared with others retrieved from GenBank. In addition, cucurbit aphid-borne yellows virus (CABYV) was also detected, and due to the recent occurrence of this virus in Poland, a phylogenetic analysis of these new isolates was performed. The analysis revealed that CABYV population is highly diverse and the Polish isolates of CABYV belong to two different phylogenetic groups. Our results showed that HTS-based technology is a valuable diagnostic tool for the identification of different virus species originating from variable hosts, and can provide rapid information about the spectrum of plant viruses previously not detected in a region

Autophagy counteracts instantaneous cell death during seasonal senescence of the fine roots and leaves in Populus trichocarpa

Abstract Background Senescence, despite its destructive character, is a process that is precisely-regulated. The control of senescence is required to achieve remobilization of resources, a principle aspect of senescence. Remobilization allows plants to recapture valuable resources that would otherwise be lost to the environment with the senescing organ. Autophagy is one of the critical processes that is switched on during senescence. This evolutionarily conserved process plays dual, antagonistic roles. On the one hand, it counteracts instantaneous cell death and allows the process of remobilization to be set in motion, while on the other hand, it participates in the degradation of cellular components. Autophagy has been demonstrated to occur in many plant species during the senescence of leaves and flower petals. Little is known, however, about the senescence process in other ephemeral organs, such as fine roots, whose lifespan is also relatively short. We hypothesized that, like the case of seasonal leaf senescence, autophagy also plays a role in the senescence of fine roots, and that both processes are synchronized in their timing. Results We evaluated which morphological and cytological symptoms are universal or unique in the senescence of fine roots and leaves. The results of our study confirmed that autophagy plays a key role in the senescence of fine roots, and is associated also with the process of cellular components degradation. In both organs, structures related to autophagy were observed, such as autophagic bodies and autophagosomes. The role of autophagy in the senescence of these plant organs was further confirmed by an analysis of ATG gene expression and protein detection. Conclusions The present study is the first one to examine molecular mechanisms associated with the senescence of fine roots, and provide evidence that can be used to determine whether senescence of fine roots can be treated as another example of developmentally programmed cell death (dPCD). Our results indicate that there is a strong similarity between the senescence of fine roots and other ephemeral organs, suggesting that this process occurs by the same autophagy-related mechanisms in all plant ephemeral organs

Occurrence, Genetic Variability of Tomato Yellow Ring Orthotospovirus Population and the Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Its Rapid Detection

Tomato-infecting viruses have been considered as a serious threat to tomato crops in Poland. Therefore, during 2014–2021, 234 tomato samples delivered directly by greenhouse tomato growers to Plant Disease Clinic of IPP-NRI were tested. Eight virus species: pepino mosaic virus (PepMV), tomato yellow ring orthotospovirus (TYRV), tomato spotted wilt orthotospovirus (TSWV), potato virus Y (PVY), cucumber mosaic virus (CMV), tomato black ring virus (TBRV) and tomato mosaic virus (ToMV) were detected in single or mixed infection in 89 samples. The presence of TYRV was established for the first time in Poland in 2014. Since then, its presence has been observed in single and mixed infection with TSWV and CMV. Here, we analysed the genetic variability of TYRV population based on complete nucleocapsid (N) protein gene sequence of 55 TYRV isolates. Maximum-likelihood reconstruction revealed the presence of three distinct, well-supported phylogroups. Moreover, the effect of host species on virus diversity was confirmed. Therefore, RT-LAMP assay was developed for the rapid and efficient detection of TYRV isolates that can be implemented in field and greenhouse conditions