Development of Stable Infectious cDNA Clones of Tomato Black Ring Virus Tagged with Green Fluorescent Protein.

Tomato black ring virus (TBRV) is a member of the Nepovirus genus in the Secoviridae family, which infects a wide range of important crop species worldwide. In this work, we constructed four cDNA infectious clones of the TBRV tagged with the green fluorescent protein (TBRV-GFP), which varied in (i) the length of the sequences flanking the GFP insert, (ii) the position of the GFP insert within the RNA2 polyprotein, and (iii) the addition of a self-cutting 2A protein. The presence of the GFP coding sequence in infected plants was verified by RT-PCR, while the infectivity and stability of the constructs were verified by mechanical inoculation of the host plants. The systemic spread of TBRV-GFP within plants was observed under UV light at a macroscopic level, monitoring GFP-derived fluorescence in leaves, and at a microscopic level using confocal microscopy. The obtained clones are a valuable tool for future studies of TBRV-host interactions, virus biology, and the long-term monitoring of its distribution in infected plants.

Construction and biological characterization of a cDNA infectious clone of wheat umbra-like virus in wheat and Nicotiana benthamiana.

Umbravirus-like associated RNAs (ulaRNAs) are a new group of subviral RNAs associated with plants. Little is known about the biology of ulaRNAs. We recently reported wheat umbra-like virus (WULV) from Kansas fields. In this work, we generated a full-length cDNA clone of WULV which systemically infected N. benthamiana. While agroinfiltrated leaves demonstrated severe necrosis, upper leaves were symptomless. We also showed that WULV is capable of infecting wheat in the absence of a helper virus. Furthermore, and through sap inoculation, we demonstrated that WULV is transmissible in the form of free RNA. This is the first report of a mechanically transmissible ulaRNA. Together, these findings contribute to advancing our knowledge of the biology of WULV. Moreover, the construction of the WULV infectious clone provides a valuable research tool for further investigations including the role of WULV in symptom development in interaction with other wheat viruses.

Genetic Diversity of Tomato Black Ring Virus Satellite RNAs and Their Impact on Virus Replication.

Viral satellite RNAs (satRNAs) are small subviral particles that are associated with the genomic RNA of a helper virus (HV). Their replication, encapsidation, and movement depend on the HV. In this paper, we performed a global analysis of the satRNAs associated with different isolates of tomato black ring virus (TBRV). We checked the presence of satRNAs in 42 samples infected with TBRV, performed recombination and genetic diversity analyses, and examined the selective pressure affecting the satRNAs population. We identified 18 satRNAs in total that differed in length and the presence of point mutations. Moreover, we observed a strong effect of selection operating upon the satRNA population. We also constructed infectious cDNA clones of satRNA and examined the viral load of different TBRV isolates in the presence and absence of satRNAs, as well as the accumulation of satRNA molecules on infected plants. Our data provide evidence that the presence of satRNAs significantly affects viral load; however, the magnitude of this effect differs among viral isolates and plant hosts. We also showed a positive correlation between the number of viral genomic RNAs (gRNAs) and satRNAs for two analysed TBRV isolates.

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.

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.

Occurrence of autophagy during pioneer root and stem development in Populus trichocarpa.

MAIN CONCLUSION: Autophagy is involved in developmentally programmed cell death and is identified during the early development of phloem, as well as xylem with a dual role, as both an inducer and executioner of cell death. The regulation of primary and secondary development of roots and stems is important for the establishment of root systems and for the overall survival of trees. The molecular and cellular basis of the autophagic processes, which are used at distinct moments during the growth of both organs, is crucial to understand the regulation of their development. To address this, we use Populus trichocarpa seedlings grown in a rhizotron system to examine the autophagy processes involved in root and stem development. To monitor the visual aspects of autophagy, transmission electron microscopy (TEM) and immunolocalization of AuTophaGy-related protein (ATG8) enabled observations of the phenomenon at a structural level. To gain further insight into the autophagy process at the protein and molecular level, we evaluated the expression of ATG gene transcripts and ATG protein levels. Alternations in the expression level of specific ATG genes and localization of ATG8 proteins were observed during the course of root or stem primary and secondary development. Specifically, ATG8 was present in the cells exhibiting autophagy, during the differentiation and early development of xylem and phloem tissues, including both xylary and extraxylary fibers. Ultrastructural observations revealed tonoplast invagination with the formation of autophagic-like bodies. Additionally, the accumulation of autophagosomes was identifiable during the differentiation of xylem in both organs, long before the commencement of cell death. Taken together, these results provide evidence in support of the dual role of autophagy in developmental PCD. A specific role of the controller of cell death, which is a committed step with the release of hydrolytic enzymes from the vacuole and final digestion of protoplast, from which there is no return once initiated, is only attributed to mega-autophagy.

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

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.

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.

Defective RNA particles derived from Tomato black ring virus genome interfere with the replication of parental virus.

Tomato black ring virus (TBRV) is the only member of the Nepovirus genus that is known to form defective RNA particles (D RNAs) during replication. Here, de novo generation of D RNAs was observed during prolonged passages of TBRV isolates originated from Solanum lycopersicum and Lactuca sativa in Chenopodium quinoa plants. D RNAs of about 500 nt derived by a single deletion in the RNA1 molecule and contained a portion of the 5' untranslated region and viral replicase, and almost the entire 3' non-coding region. Short regions of sequence complementarity were found at the 5' and 3' junction borders, which can facilitate formation of the D RNAs. Moreover, in this study we analyzed the effects of D RNAs on TBRV replication and symptoms development of infected plants. C. quinoa, S. lycopersicum, Nicotiana tabacum, and L. sativa were infected with the original TBRV isolates (TBRV-D RNA) and those containing additional D RNA particles (TBRV + D RNA). The viral accumulation in particular hosts was measured up to 28 days post inoculation by RT-qPCR. Statistical analyses revealed that D RNAs interfere with TBRV replication and thus should be referred to as defective interfering particles. The magnitude of the interference effect depends on the interplay between TBRV isolate and host species.

Construction of Agrobacterium tumefaciens-mediated tomato black ring virus infectious cDNA clones.

Tomato black ring virus (TBRV, genus Nepovirus) infects a wide range of economically important plants such as tomato, potato, tobacco and cucumber. Here, a successful construction of infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2) is reported for the first time. The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS). The symptoms induced on plants agroinoculated with both constructs were indistinguishable from those caused by the wild-type virus. The infectivity of obtained clones was verified by reinoculation to Nicotiana tabacum cv. Xanthi, Chenopodium quinoa and Cucumis sativus. The presence of viral particles and RNA was confirmed by electron microscopy and reverse transcription polymerase chain reaction, respectively. Constructed full-length infectious cDNA clones will serve as an excellent tool to study virus-host-vector interactions.