Assessment of the Efficacy and Mode of Action of Benzo(1,2,3)-Thiadiazole-7-Carbothioic Acid S-Methyl Ester (BTH) and Its Derivatives in Plant Protection Against Viral Disease.

Systemic acquired resistance (SAR) induction is one of the primary defence mechanisms of plants against a broad range of pathogens. It can be induced by infectious agents or by synthetic molecules, such as benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH). SAR induction is associated with increases in salicylic acid (SA) accumulation and expression of defence marker genes (e.g., phenylalanine ammonia-lyase (PAL), the pathogenesis-related (PR) protein family, and non-expressor of PR genes (NPR1)). Various types of pathogens and pests induce plant responses by activating signalling pathways associated with SA, jasmonic acid (JA) and ethylene (ET). This work presents an analysis of the influence of BTH and its derivatives as resistance inducers in healthy and virus-infected plants by determining the expression levels of selected resistance markers associated with the SA, JA, and ET pathways. The phytotoxic effects of these compounds and their influence on the course of viral infection were also studied. Based on the results obtained, the best-performing BTH derivatives and their optimal concentration for plant performance were selected, and their mode of action was suggested. It was shown that application of BTH and its derivatives induces increased expression of marker genes of both the SA- and JA-mediated pathways.

Rapid detection of genetically diverse tomato black ring virus isolates using reverse transcription loop-mediated isothermal amplification.

A reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) has been developed for detection of tomato black ring virus (TBRV) isolates collected from different hosts. One-step RT-LAMP was performed with a set of four primers, the design of which was based on the coat protein gene. Results of RT-LAMP were visualized by direct staining of products with fluorescent dyes, agarose gel electrophoresis, and analysis of amplification curves. The sensitivity of RT-LAMP was 100-fold greater than that of RT-PCR. The RT-LAMP assay developed here is a useful and practical method for diagnosis of TBRV.

Two types of defective RNAs arising from the tomato black ring virus genome.

Short defective RNAs (D-RNAs) associated with tomato black ring virus (TBRV) were isolated, cloned and sequenced. As a result, two types of D-RNAs associated with different TBRV isolates were identified. Both types were derived from RNA1. The first one contained sequences from the 5' and 3' untranslated regions (UTR) and from the 5' region of a single large open reading frame. The second one included a portion of the coding region for the RNA-dependent RNA polymerase flanked by a short fragment of the 5' UTR and the entire 3' UTR. The possible nature and origin of these RNA species is discussed.

Synthesis, characterization and biological activity of bifunctional ionic liquids based on dodine ion.

BACKGROUND: Development of new plant protection strategies has become an urgent matter in modern agriculture, in view of the evidently proved negative effect of currently used active ingredients of pesticides. In recent years, much effort has been made to eliminate the use of pesticides established to be toxic to pollinators. RESULTS: In this study, we present a group of new bifunctional ionic liquids based on dodine (N-dodecylguanidine) cation whose physical and biological properties have been modified relative to those of the commercially available N-dodecylguanidine acetate. The decreased level of residue of active substances in plant tissues reduces their availability to pollinators, which increases the safety of their use. Moreover, lower environmental impact in combination with high antifungal activity and an additional biological function, that is the systemic acquired resistance induction, are in line with the goals of sustainable agriculture. CONCLUSION: The presented approach shows the possibility of derivatization of commonly used fungicide into the form of bifunctional salts whose physical and biological properties can be easily modified. The paper reports successful design and synthesis of new sustainable and green chemicals for the modern agriculture, being less toxic to the environment and human health but still effective against pathogens. © 2021 Society of Chemical Industry.

Tridimensional model structure and patterns of molecular evolution of Pepino mosaic virus TGBp3 protein.

BACKGROUND: Pepino mosaic virus (PepMV) is considered one of the most dangerous pathogens infecting tomatoes worldwide. The virus is highly diverse and four distinct genotypes, as well as inter-strain recombinants, have already been described. The isolates display a wide range on symptoms on infected plant species, ranging from mild mosaic to severe necrosis. However, little is known about the mechanisms and pattern of PepMV molecular evolution and about the role of individual proteins in host-pathogen interactions. METHODS: The nucleotide sequences of the triple gene block 3 (TGB3) from PepMV isolates varying in symptomatology and geographic origin have been analyzed. The modes and patterns of molecular evolution of the TGBp3 protein were investigated by evaluating the selective constraints to which particular amino acid residues have been subjected during the course of diversification. The tridimensional structure of TGBp3 protein has been modeled de novo using the Rosetta algorithm. The correlation between symptoms development and location of specific amino acids residues was analyzed. RESULTS: The results have shown that TGBp3 has been evolving mainly under the action of purifying selection operating on several amino acid sites, thus highlighting its functional role during PepMV infection. Interestingly, amino acid 67, which has been previously shown to be a necrosis determinant, was found to be under positive selection. CONCLUSIONS: Identification of diverse selection events in TGB3p3 will help unraveling its biological functions and is essential to an understanding of the evolutionary constraints exerted on the Potexvirus genome. The estimated tridimensional structure of TGBp3 will serve as a platform for further sequence, structural and function analysis and will stimulate new experimental advances.

Quasispecies nature of Pepino mosaic virus and its evolutionary dynamics.

Genetic variability is an essential feature of RNA viruses. It allows them to adapt to the ever-changing environmental conditions. Important biological properties of the viruses, their infectivity, adaptability, and host range, may also depend on the level of quasispecies diversity. Here, we present the analysis of the genetic polymorphism of Pepino mosaic virus (PepMV). The examined populations were isolated from the naturally infected tomato plants (Solanum lycopersicum). In order to determine the complexity of the PepMV populations, the number of different viral variants and their genetic diversity was established. Moreover, phylogenetic trees were created to depict relations between the identified variants. For the first time we have shown that the PepMV exists as a quasispecies. The observed level of genetic variability allows PepMV for a quick and flexible adaptation to different hosts. Our results suggest that the level of PepMV variability possibly influences the course of infection.

Infectious RNA transcripts derived from cloned cDNA of a pepino mosaic virus isolate.

For the first time, a full-length cDNA clone of the RNA genome of pepino mosaic virus (PepMV) was constructed. RNA was extracted from purified virions of isolate PepMV-Pa and used for cDNA synthesis. The full-length cDNA was produced as one 6.4-kb fragment representing the entire PepMV genome. This fragment was ligated into the pCR-XL-TOPO vector downstream of T7 RNA polymerase promoter, which was included in the 5' primer sequence used for RT-PCR. The PepMV-Pa RNA transcripts obtained were infectious in different host plants, causing symptoms indistinguishable from those of the wild-type isolate. The presence and authenticity of the progeny virus were verified by ELISA, RT-PCR and nucleotide sequencing.

Complete nucleotide sequence of a Polish strain of Peanut stunt virus (PSV-P) that is related to but not a typical member of subgroup I.

Peanut stunt virus (PSV) is a common legume pathogen present worldwide. It is also infectious for many other plants including peanut and some vegetables. Viruses of this species are classified at present into three subgroups based on their serology and nucleotide homology. Some of them may also carry an additional subviral element - satellite RNA. Analysis of the full genome sequence of a Polish strain - PSV-P - associated with satRNA was performed and showed that it may be classified as a derivative of the subgroup I sharing 83.9-87.9% nucleotide homology with other members of this subgroup. A comparative study of sequenced PSV strains indicates that PSV-P shows the highest identity level with PSV-ER or PSV-J depending on the region used for analysis. Phylogenetic analyses, on the other hand, have revealed that PSV-P is related to representatives of the subgroup I to the same degree, with the exception of the coat protein coding sequence where PSV-P is clustered together with PSV-ER.

The Defense Response of Nicotiana benthamiana to Peanut Stunt Virus Infection in the Presence of Symptom Exacerbating Satellite RNA.

Peanut stunt virus (PSV) is a widespread disease infecting legumes. The PSV strains are classified into four subgroups and some are defined by the association of satellite RNAs (satRNAs). In the case of PSV, the presence of satRNAs alters the symptoms of disease in infected plants. In this study, we elucidated the plant response to PSV-G strain, which occurs in natural conditions without satRNA. However, it was found that it might easily acquire satRNA, which exacerbated pathogenesis in Nicotiana benthamiana. To explain the mechanisms underlying PSV infection and symptoms exacerbation caused by satRNA, we carried out transcriptome profiling of N. benthamiana challenged by PSV-G and satRNA using species-specific microarrays. Co-infection of plants with PSV-G + satRNA increased the number of identified differentially expressed genes (DEGs) compared with the number identified in PSV-G-infected plants. In both treatments, the majority of up-regulated DEGs were engaged in translation, ribosome biogenesis, RNA metabolism, and response to stimuli, while the down-regulated DEGs were required for photosynthesis. The presence of satRNA in PSV-G-infected plants caused different trends in expression of DEGs associated with phosphorylation, ATP binding, and plasma membrane.

Leadzyme formed in vivo interferes with tobacco mosaic virus infection in Nicotiana tabacum.

We developed a new method for inhibiting tobacco mosaic virus infection in tobacco plants based on specific RNA hydrolysis induced by a leadzyme. We identified a leadzyme substrate target sequence in genomic tobacco mosaic virus RNA and designed a 16-mer oligoribonucleotide capable of forming a specific leadzyme motif with a five-nucleotide catalytic loop. The synthetic 16-mer RNA was applied with nontoxic, catalytic amount of lead to infected tobacco leaves. We observed inhibition of tobacco mosaic virus infection in tobacco leaves in vivo due to specific tobacco mosaic virus RNA cleavage effected by leadzyme. A significant reduction in tobacco mosaic virus accumulation was observed even when the leadzyme was applied up to 2 h after inoculation of leaves with tobacco mosaic virus. This process, called leadzyme interference, is determined by specific recognition and cleavage of the target site by the RNA catalytic strand in the presence of Pb(2+).

[Subviral molecules of RNA associated with plant ss(+)RNA viruses]. / Subwirusowe czasteczki RNA zwiazane z wirusami roslinnymi typu ss(+)RNA.

Plant ss(+)RNA viruses besides their genome RNAs often are associated with additional subviral RNA molecules which occur naturally or are generated de novo during infection. There are such molecules like: satellite, defective, defective interfering and chimeric RNAs. Subviral RNAs can not replicate and encapsidate by oneself. Helper viruses supply the protein complexes that are necessary to these processes. The subviral molecules are characterized by small size. Recombination, deletion and accumulation of mutation are the main ways of arising subviral elements, although the origin of satRNAs is unknown. The unique feature of subviral RNAs is their ability to modify of infection progress caused by helper virus. They can attenuate or enhance the intensity of disease symptoms. The overall influence on disease development depends on three-component complex consisting of: plant host-virus' strain--subviral RNA. This article is a synthetic review of information concerning subviral RNA molecules of plant viruses, their structure, functions and origin.

Ultrastructural insights into tomato infections caused by three different pathotypes of Pepino mosaic virus and immunolocalization of viral coat proteins.

This paper presents studies on an ultrastructural analysis of plant tissue infected with different pathotypes of Pepino mosaic virus (PepMV) and the immunolocalization of viral coat proteins. Because the PepMV virus replicates with a high mutation rate and exhibits significant genetic diversity, therefore, isolates of PepMV display a wide range of symptoms on infected plants. In this work, tomato plants of the Beta Lux cultivar were inoculated mechanically with three pathotypes representing the Chilean 2 (CH2) genotype: mild (PepMV-P22), necrotic (PepMV-P19) and yellowing (PepMV-P5-IY). The presence of viral particles in all infected plants in the different compartments of various cell types (i.e. spongy and palisade mesophyll, sieve elements and xylem vessels) was revealed via ultrastructural analyses. For the first time, it was possible to demonstrate the presence of crystalline inclusions, composed of virus-like particles. In the later stage of PepMV infection (14 dpi) various pathotype-dependent changes in the structure of the individual organelles (i.e. mitochondria, chloroplasts) were found. The strongest immunogold labeling of the viral coat proteins was also observed in plants infected by necrotic isolates. A large number of viral coat proteins were marked in the plant conductive elements, both xylem and phloem.

Restriction analysis of genetic variability of Polish isolates of Tomato black ring virus.

Several different isolates of Tomato black ring virus (TBRV) have been collected in Poland from cucumber, tomato, potato and black locust plants. Biological tests showed some differences in the range of infected plants and the type of symptoms, which was the basis for selection of seven the most biologically different TBRV isolates. According to the sequence of TBRV-MJ, several primer pairs were designed and almost the entire sequence of both genomic RNAs was amplified. The RT-PCR products derived from all tested TBRV isolates were digested by restriction enzymes. On the basis of the restriction patterns, the variable and the conserved regions of the TBRV genome were defined and the relationships between the Polish TBRV isolates established.

Molecular characterisation of the full-length genome of olive latent virus 1 isolated from tomato.

Olive latent virus 1 (OLV-1) is a species of the Necrovirus genus. So far, it has been reported to infect olive, citrus tree and tulip. Here, we determined and analysed the complete genomic sequence of an isolate designated as CM1, which was collected from tomato plant in the Wielkopolska region of Poland and represents the prevalent isolate of OLV-1. The CM1 genome consists of monopartite single-stranded positive-sense RNA genome sized 3,699 nt with five open reading frames (ORFs) and small inter-cistronic regions. ORF1 encodes a polypeptide with a molecular weight of 23 kDa and the read-through (RT) of its amber stop codon results in ORF1 RT that encodes the virus RNA-dependent RNA polymerase. ORF2 and ORF3 encode two peptides, with 8 kDa and 6 kDa, respectively, which appear to be involved in cell-to-cell movement. ORF4 is located in the 3' terminal and encodes a protein with 30 kDa identified as the viral coat protein (CP). The differences in CP region of four OLV-1 isolates whose sequences have been deposited in GenBank were observed. Nucleotide sequence identities of the CP of tomato CM1 isolate with those of olive, citrus and tulip isolates were 91.8%, 89.5% and 92.5%, respectively. In contrast to other OLV-1 isolates, CM1 induced necrotic spots on tomato plants and elicited necrotic local lesions on Nicotiana benthamiana, followed by systemic infection. This is the third complete genomic sequence of OLV-1 reported and the first one from tomato.

Single mutation converts mild pathotype of the Pepino mosaic virus into necrotic one.

Pepino mosaic virus (PepMV) is a member of the Flexiviridae family, genus Potexvirus. PepMV isolates from the same genotype differ in the symptoms they induce in Datura inoxia and Solanum lycopersicum. Necrotic isolates cause necrosis on these plants whereas mild isolates are very often asymptomatic or induce mild mosaic. Sequence analysis of the genomes of mild (PepMV-P22) and necrotic (PepMV-P19) strains revealed that they differ by several nonsynonymous mutations. Eleven mutations are located in the RdRp coding region and one mutation is placed in the TGB3 coding region. To investigate whether these mutations affect the symptom induction, both PepMV-P22 and PepMV-P19 were mutagenized. As a result we found that the genetic determinant responsible for necrosis induction was amino acid 67 of TGB3. This showed that one point mutation is sufficient to alter the virus from mild to aggressive in tomato and D. inoxia.

Evidence for RNA recombination between distinct isolates of Pepino mosaic virus.

Genetic recombination plays an important role in the evolution of virus genomes. In this study we analyzed publicly available genomic sequences of Pepino mosaic virus (PepMV) for recombination events using several bioinformatics tools. The genome-wide analyses not only confirm the presence of previously found recombination events in PepMV but also provide the first evidence for double recombinant origin of the US2 isolate.

Full length genome sequence of Polish isolate of Beet soil-borne virus confirms low level of genetic diversity.

The complete nucleotide sequence of a Polish isolate of Beet soil-borne virus was determined for the first time. The genome organization was identical with those previously established for isolates from Germany and China. A comparison of the Polish isolate with others deposited in GenBank revealed high level of nucleotide identity, about 98-100%, throughout the genome analyzed. The ratio between non-synonymous and synonymous substitutions was rather low suggesting a negative selective pressure. The non-synonymous mutations were particulary frequent in triple gene block.

A new and efficient method for inhibition of RNA viruses by DNA interference.

We report here a new method for inhibition of RNA viruses induced by dsDNA. We demonstrated that both long dsDNA molecules and short interfering DNA with a sequence complementary to that of viral RNA inhibited tobacco mosaic virus expression and prevented virus spread. Also, the expression of the HIV-1 gp41 gene in HeLa cells was inhibited by complementary short interfering DNA. We showed that Dicer processed dsDNA, which suggests activation of the cellular machinery involved in silencing of RNA. For the silencing of viral RNA effected with dsDNA, we coined the term DNA interference technology.

Complete genomic RNA sequence of the Polish Pepino mosaic virus isolate belonging to the US2 strain.

We have determined the complete nucleotide and amino acid sequences of the Polish Pepino mosaic virus (PepMV) isolate marked as PepMV-PK. The PepMV-PK genome consists of a single positive-sense RNA strand of 6412-nucleotide-long that contains five open reading frames (ORFs). ORF1 encodes the putative viral polymerase (RdRp), ORFs 2-4 the triple gene block (TGB 1-3), and ORF5-coat protein CP. Two short untranslated regions flank the coding ones and there is a poly (A) tail at the 3' end of the genomic RNA. Thus, the genome organization of PepMV-PK is that of a typical member of the genus Potexvirus. Phylogenetic analysis based on full-length genomes of PepMV sequences showed that PepMV-PK was most closely related to the Ch2 isolate from Chile. Comparison of PepMV-PK and Ch2 showed the following nucleotide identities: 98% for the RdRp, 99% for the CP genes, and 98, 99, and 98% for the TGB1, TGB2, and TBG3, respectively. This high level of nucleotide sequence identity between the Chilean and Polish PepMV-PK isolates suggest their common origin.

The nucleotide sequence of a Polish isolate of Tomato torrado virus.

A new virus was isolated from greenhouse tomato plants showing symptoms of leaf and apex necrosis in Wielkopolska province in Poland in 2003. The observed symptoms and the virus morphology resembled viruses previously reported in Spain called Tomato torrado virus (ToTV) and that in Mexico called Tomato marchitez virus (ToMarV). The complete genome of a Polish isolate Wal'03 was determined using RT-PCR amplification using oligonucleotide primers developed against the ToTV sequences deposited in Genbank, followed by cloning, sequencing, and comparison with the sequence of the type isolate. Phylogenetic analyses, performed on the basis of fragments of polyproteins sequences, established the relationship of Polish isolate Wal'03 with Spanish ToTV and Mexican ToMarV, as well as with other viruses from Sequivirus, Sadwavirus, and Cheravirus genera, reported to be the most similar to the new tomato viruses. Wal'03 genome strands has the same organization and very high homology with the ToTV type isolate, showing only some nucleotide and deduced amino acid changes, in contrast to ToMarV, which was significantly different. The phylogenetic tree clustered aforementioned viruses to the same group, indicating that they have a common origin.