Cereal leaf beetle-associated bacteria enhance the survival of their host upon insecticide treatments and respond differently to insecticides with different modes of action.

The cereal leaf beetle (CLB, Oulema melanopus) is one of the major cereal pests. The effect of insecticides belonging to different chemical classes, with different mechanisms of action and the active substances' concentrations on the CLB bacterial microbiome, was investigated. Targeted metagenomic analysis of the V3-V4 regions of the 16S ribosomal gene was used to determine the composition of the CLB bacterial microbiome. Each of the insecticides caused a decrease in the abundance of bacteria of the genus Pantoea, and an increase in the abundance of bacteria of the genus Stenotrophomonas, Acinetobacter, compared to untreated insects. After cypermethrin application, a decrease in the relative abundance of bacteria of the genus Pseudomonas was noted. The dominant bacterial genera in cypermethrin-treated larvae were Lactococcus, Pantoea, while in insects exposed to chlorpyrifos or flonicamid it was Pseudomonas. Insecticide-treated larvae were characterized, on average, by higher biodiversity and richness of bacterial genera, compared to untreated insects. The depletion of CLB-associated bacteria resulted in a decrease in larval survival, especially after cypermethrin and chlorpyrifos treatments. The use of a metagenome-based functional prediction approach revealed a higher predicted function of bacterial acetyl-CoA C-acetyltransferase in flonicamid and chlorpyrifos-treated larvae and tRNA dimethyltransferase in cypermethrin-treated insects than in untreated insects.

Dicer-Like Protein 4 and RNA-Dependent RNA Polymerase 6 Are Involved in Tomato Torrado Virus Pathogenesis in Nicotiana benthamiana.

Tomato torrado virus (ToTV) is a type member of the Torradovirus genus in the Secoviridae family known to cause severe necrosis in susceptible tomato varieties. ToTV also infects other Solanaceae plants, including Nicotiana benthamiana, where it induces distinctive disease symptoms: plant growth drop with the emergence of spoon-like malformed systemic leaves. Virus-induced post-transcriptional gene silencing (PTGS) is significant among plant defense mechanisms activated upon virus invasion. The PTGS, however, can be counteracted by suppressors of RNA silencing commonly found in viruses, which efficiently disrupt the antiviral defense of their host. Here, we addressed the question of PTGS antiviral activity and its suppression in N. benthamiana during ToTV infection-a phenomenon not described for any representative from the Torradovirus genus so far. First, we showed that neither the Vp26-a necrosis-inducing pathogenicity determinant of ToTV-nor other structural viral proteins limited the locally induced PTGS similar to p19, a well-characterized potent suppressor of RNA silencing of tombusviruses. Moreover, by employing wild-type and transgenic lines of N. benthamiana with suppressed Dicer-like 2 (DCL2), Dicer-like 4 (DCL4), Argonaute 2 and RNA-dependent RNA polymerase 6 (RDR6) proteins, we proved their involvement in anti-ToTV defense. Additionally, we identified DCL4 as the major processor of ToTV-derived siRNA. More importantly, our results indicate the essential role of the Suppressor of Gene Silencing 3 (SGS3)/RDR6 pathway in anti-ToTV defense. Finally, we conclude that ToTV might not require a potent RNA silencing suppressor during infection of the model plant N. benthamiana.

The Effect of Benzo(1,2,3)-thiadiazole-7-carbothioic Acid S-Methyl Ester (BTH) and Its Cholinium Ionic Liquid Derivative on the Resistance Induction and Antioxidant Properties of Tomato (Solanum lycopersicum L.).

Tomatoes are one of the most important vegetables thanks to their taste attributes and nutritional value. Their cultivation is threatened by various pathogens including viruses. The application of resistance inducers (RI), such as benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) may be used to enhance plant performance against viruses. Here we aimed to compare the impact of BTH and its choline derivative (Chol-BTH) on resistance induction and antioxidant properties of healthy plants and tomato mosaic virus (ToMV)-infected ones. The response of tomato plants to treatment with BTH or Chol-BTH was manifested by increased expression of not only pathogenesis-related (PR) genes but also WRKY and Jasmonate Zim-domain protein (JAZ) genes and increased jasmonic acid (JA) levels. The effect of BTH as a resistance inducer was observed early after application, while with Chol-BTH the plant defense system reacted more strongly after 8 days. The antioxidant properties of RI-treated tomatoes are related to both glutathione content and peroxidase activity. In the case of BTH, an increase in these activities occurred early after application, while in the case of Chol-BTH, the glutathione level was particularly high in the plant early after treatment, and high peroxidase activity was observed 8 days post-treatment. Overall, the collected results indicate that Chol-BTH, due to its physicochemical parameters (e.g., good solubility) and biological activity (increased expression of lignification-related genes, supported by increases in peroxidase activity and total phenolic compounds levels), can also be a very useful agent inducing tomato resistance against viral pathogens.

Herbicide resistance status impacts the profile of non-anthocyanin polyphenolics and some phytomedical properties of edible cornflower (Centaurea cyanus L.) flowers.

To ensure sufficient food supply worldwide, plants are treated with pesticides to provide protection against pathogens and pests. Herbicides are the most commonly utilised pesticides, used to reduce the growth of weeds. However, their long-term use has resulted in the emergence of herbicide-resistant biotypes in many weed species. Cornflower (Centaurea cyanus L., Asteraceae) is one of these plants, whose biotypes resistant to herbicides from the group of acetolactate synthase (ALS) inhibitors have begun to emerge in recent years. Some plants, although undesirable in crops and considered as weeds, are of great importance in phytomedicine and food production, and characterised by a high content of health-promoting substances, including antioxidants. Our study aimed to investigate how the acquisition of herbicide resistance affects the health-promoting properties of plants on the example of cornflower, as well as how they are affected by herbicide treatment. To this end, we analysed non-anthocyanin polyphenols and antioxidant capacity in flowers of C. cyanus from herbicide-resistant and susceptible biotypes. Our results indicated significant compositional changes associated with an increase in the content of substances and activities that have health-promoting properties. High antioxidant activity and higher total phenolic and flavonoid compounds as well as reducing power were observed in resistant biotypes. The latter one increased additionally after herbicide treatment which might also suggest their role in the resistance acquisition mechanism. Overall, these results show that the herbicide resistance development, although unfavourable to crop production, may paradoxically have very positive effects for medicinal plants such as cornflower.

Transcriptome analysis indicates the involvement of herbicide-responsive and plant-pathogen interaction pathways in the development of resistance to ACCase inhibitors in Apera spica-venti.

BACKGROUND: The continuous use of the herbicides contributes to the emergence of the resistant populations of numerous weed species that are tolerant to multiple herbicides with different modes of action (multiple resistance) which is provided by non-target-site resistance mechanisms. In this study, we addressed the question of rapid acquisition of herbicide resistance to pinoxaden (acetyl CoA carboxylase inhibitor) in Apera spica-venti, which endangers winter cereal crops and has high adaptation capabilities to inhabit many rural locations. To this end, de novo transcriptome of Apera spica-venti was assembled and RNA-sequencing analysis of plants resistant and susceptible to pinoxaden treated with this herbicide was performed. RESULTS: The obtained data showed that the prime candidate genes responsible for herbicide resistance were those encoding 3-ketoacyl-CoA synthase 12-like, UDP-glycosyltransferases (UGT) including UGT75K6, UGT75E2, UGT83A1-like, and glutathione S-transferases (GSTs) such as GSTU1 and GSTU6. Also, such highly accelerated herbicide resistance emergence may result from the enhanced constitutive expression of a wide range of genes involved in detoxification already before herbicide treatment and may also influence response to biotic stresses, which was assumed by the detection of expression changes in genes encoding defence-related proteins, including receptor kinase-like Xa21. Moreover, alterations in the expression of genes associated with methylation in non-treated herbicide-resistant populations were identified. CONCLUSION: The obtained results indicated genes that may be involved in herbicide resistance. Moreover, they provide valuable insight into the possible effect of resistance on the weed interaction with the other stresses by indicating pathways associated with both abiotic and biotic stresses. © 2023 Society of Chemical Industry.

Insect Gut Bacteria Promoting the Growth of Tomato Plants (Solanum lycopersicum L.).

We investigated gut bacteria from three insect species for the presence of plant growth properties (PGP). Out of 146 bacterial strains obtained from 20 adult specimens of Scolytidae sp., 50 specimens of Oulema melanopus, and 150 specimens of Diabrotica virgifera, we selected 11 strains displaying the following: PGP, phosphate solubility, production of cellulase, siderophore, lipase, protease, and hydrogen cyanide. The strains were tested for growth promotion ability on tomato (Lycopersicon esculentum) plants. Each strain was tested individually, and all strains were tested together as a bacterial consortium. Tomato fruit yield was compared with the negative control. The plants treated with bacterial consortium showed a significant increase in fruit yield, in both number of fruits (+41%) and weight of fruits (+44%). The second highest yield was obtained for treatment with Serratia liquefaciens Dv032 strain, where the number and weight of yielded fruits increased by 35% and 30%, respectively. All selected 11 strains were obtained from Western Corn Rootworm (WCR), Diabrotica virgifera. The consortium comprised: Ewingella americana, Lactococcus garvieae, L. lactis, Pseudomonas putida, Serratia liquefaciens, and S. plymuthica. To our knowledge, this is the first successful application of D. virgifera gut bacteria for tomato plant growth stimulation that has been described.

The Impact of Oulema melanopus-Associated Bacteria on the Wheat Defense Response to the Feeding of Their Insect Hosts.

Wheat production is threatened by the destructive effects of numerous pests, including Oulema melanopus (cereal leaf beetle, CLB). Both adults and larvae of CLB damage grain crops, but the target of insecticide treatments are the larvae. Insect-associated bacteria are important for many of the insects' life processes and may also modulate plant defense responses to feeding of their insect host. The aim of our study was to elucidate the early wheat plants' reaction to this herbivore feeding and to disclose the CLB-associated bacteria modulation of the wheat-insect interactions. Transcriptome analyses were performed for the leaves wounded mechanically and by feeding of the CLB larvae as well as for the distal leaves to study both, the plant's local and systemic response. Comparative transcriptome analysis indicated that 24 h after the plant treatment, a much larger number of up-regulated DEGs in damaged leaves was noted, especially those on which larvae were fed. It may suggest that at the analysed time point, the local response was stronger than the systemic one. In the leaves on which larvae with natural bacterial flora were fed (local response), the number of up- and down-regulated differentially expressed genes (DEGs) was 7136 and 7411, respectively, in comparison to the dataset obtained for the leaves wounded by larvae with a reduced number of bacteria. In the distal leaves, 3015 up- and 2372 down-regulated DEGs were noted. CLB-associated bacteria were found to affect many aspects of the physiology of wheat plants, especially in wounded leaves, including the expression of genes related to primary metabolism, phytohormone signaling and photosynthesis. We also observed that CLB-associated bacteria mitigated numerous anti-herbivore processes and pathways associated with the synthesis of metabolites and proteins, potentially harmful to the insects. The bacteria also reversed the expression of some genes involved, inter alia, in the phosphorylation of proteins, oxidative stress, cell wall organization, and biogenesis. Understanding the role of CLB-associated bacteria in the plant's defense response will be important to the fields of pest control and herbivore and its host ecology and evolution.

Beetles as Plant Pathogen Vectors.

Herbivorous insects, likewise, other organisms, are exposed to diverse communities of microbes from the surrounding environment. Insects and microorganisms associated with them share a range of relationships, including symbiotic and pathogenic. Insects damage plants by feeding on them and delivering plant pathogens to wounded places, from where pathogens spread over the plant. Thus insects can be considered as both pests and reservoirs or vectors of plant pathogens. Although beetles are not mentioned in the first place as plant pathogen vectors, their transmission of pathogens also takes place and affects the ecosystem. Here we present an overview of beetles as vectors of plant pathogens, including viruses, bacteria, fungi, nematodes, and Oomycota, which are responsible for developing plant diseases that can have a significant impact on crop yield and quality.

The analysis of reference genes expression stability in susceptible and resistant Apera spica-venti populations under herbicide treatment.

Weed resistance to herbicides constitutes a serious problem to world crop production. One of the weeds that are significantly threatening the crops' yield and quality is Apera spica-venti. The target-site resistance (TSR) mechanism of A. spica-venti has been widely studied, though, little is known about its non-target-site resistance (NTSR) mechanisms at the molecular level. Molecular examination of NTSR is, to a great extent, based on the expression profiles of selected genes, e.g. those participating in detoxification. However, to obtain reliable results of gene expression analysis, the use of a normalizer is required. The aim of this study was to select the best reference genes in A. spica-venti plants of both populations, susceptible and resistant to ALS inhibitor, under treatment with herbicide. Eleven housekeeping genes were chosen for their expression stability assessment. The efficiency correction of raw quantification cycles (Cq) was included in the gene expression stability analyses, which resulted in indicating the TATA-box binding protein (TBP), glyceraldehyde-3-phosphate dehydrogenase, cytosolic (GAPC), and peptidyl-prolyl cis-trans isomerase CYP28 (CYP28) genes as the most stably expressed reference genes. The obtained results are of vital importance for future studies on the expression of genes associated with the non-target-site resistance mechanisms in the A. spica-venti populations susceptible and resistant to herbicides.

The structure of the cereal leaf beetle (Oulema melanopus) microbiome depends on the insect's developmental stage, host plant, and origin.

Cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae) is a serious agricultural pest that causes considerable damages to agricultural production. The aim of this study was to characterize the bacterial communities associated with larvae and imagoes of CLB collected from various cereal host species and locations. The bacterial profile was characterized by 16S rRNA gene sequencing at the V3-V4 hypervariable region. Using taxonomy-based analysis, the bacterial community of CLB containing 16 phyla, 26 classes, 49 orders, 78 families, 94 genera, and 63 species of bacteria was identified. The abundance of Wolbachia, Rickettsia, and Lactococcus genus was significantly higher in CLB imagoes than in larvae. Statistical analysis confirmed that the bacterial community of the larvae is more diverse in comparison to imagoes and that insects collected from spring barley and wheat are characterized by a much higher biodiversity level of bacterial genera and species than insects collected from other cereals. Obtained results indicated that the developmental stage, the host plant, and the insect's sampling location affected the CLB's microbiome. Additionally, the CLB core microbiome was determined. It consists of 2 genera (Wolbachia and Rickettsia) shared by at least 90% tested CLB insects, regardless of the variables analysed.

Analysis of the Role of Bradysia impatiens (Diptera: Sciaridae) as a Vector Transmitting Peanut Stunt Virus on the Model Plant Nicotiana benthamiana.

Bradysia species, commonly known as fungus gnats, are ubiquitous in greenhouses, nurseries of horticultural plants, and commercial mushroom houses, causing significant economic losses. Moreover, the insects from the Bradysia genus have a well-documented role in plant pathogenic fungi transmission. Here, a study on the potential of Bradysia impatiens to acquire and transmit the peanut stunt virus (PSV) from plant to plant was undertaken. Four-day-old larvae of B. impatiens were exposed to PSV-P strain by feeding on virus-infected leaves of Nicotiana benthamiana and then transferred to healthy plants in laboratory conditions. Using the reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR (RT-qPCR), and digital droplet PCR (RT-ddPCR), the PSV RNAs in the larva, pupa, and imago of B. impatiens were detected and quantified. The presence of PSV genomic RNA strands as well as viral coat protein in N. benthamiana, on which the viruliferous larvae were feeding, was also confirmed at the molecular level, even though the characteristic symptoms of PSV infection were not observed. The results have shown that larvae of B. impatiens could acquire the virus and transmit it to healthy plants. Moreover, it has been proven that PSV might persist in the insect body transstadially. Although the molecular mechanisms of virion acquisition and retention during insect development need further studies, this is the first report on B. impatiens playing a potential role in plant virus transmission.

Multiple cellular compartments engagement in Nicotiana benthamiana-peanut stunt virus-satRNA interactions revealed by systems biology approach.

KEY MESSAGE: PSV infection changed the abundance of host plant's transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and cytosol, affecting photosynthesis, translation, transcription, and splicing. Virus infection is a process resulting in numerous molecular, cellular, and physiological changes, a wide range of which can be analyzed due to development of many high-throughput techniques. Plant RNA viruses are known to replicate in the cytoplasm; however, the roles of chloroplasts and other cellular structures in the viral replication cycle and in plant antiviral defense have been recently emphasized. Therefore, the aim of this study was to analyze the small RNAs, transcripts, proteins, and phosphoproteins affected during peanut stunt virus strain P (PSV-P)-Nicotiana benthamiana interactions with or without satellite RNA (satRNA) in the context of their cellular localization or functional connections with particular cellular compartments to elucidate the compartments most affected during pathogenesis at the early stages of infection. Moreover, the processes associated with particular cell compartments were determined. The 'omic' results were subjected to comparative data analyses. Transcriptomic and small RNA (sRNA)-seq data were obtained to provide new insights into PSV-P-satRNA-plant interactions, whereas previously obtained proteomic and phosphoproteomic data were used to broaden the analysis to terms associated with cellular compartments affected by virus infection. Based on the collected results, infection with PSV-P contributed to changes in the abundance of transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and the cytosol, and the most affected processes were photosynthesis, translation, transcription, and mRNA splicing. Furthermore, sRNA-seq and phosphoproteomic analyses indicated that kinase regulation resulted in decreases in phosphorylation levels. The kinases were associated with the membrane, cytoplasm, and nucleus components.

Pantoea ananatis, A New Bacterial Pathogen Affecting Wheat Plants (Triticum L.) in Poland.

Wheat (Triticum aestivum) is one of the most economically important crops in the world. During the routine monitoring of wheat pest, the cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae), in the Greater Poland region, it was observed that some leaves wounded by CLB also displayed brownish lesions with clear margins and yellow halo, disease symptoms resembling a bacterial infection. The aim of this study was therefore to investigate those symptoms to establish a causal agent of the disease. The identification based on the results of the Biolog's Gen III system, 16S rRNA, and gyrB genes sequencing, revealed the presence of eight strains of Pantoea ananatis bacteria. Four strains were derived from wheat leaves (Ta024, Ta027, Ta030, Ta046), and four from the CLB's oral secretion (OUC1, OUD2, OUF2, and OUG1). They shared the nucleotide identity ranging from 99 to 100% to P. ananatis strains deposited in the GenBank database. Additionally, the multi-locus sequence analysis (MLSA) of concatenated sequences of partial atpD, fusA, gyrB, rplB, and rpoB genes was performed. All P. ananatis strains isolated in Poland, grouped into one cluster supported with high bootstrap value. Pathogenicity tests performed on four varieties of wheat plants have identified P. ananatis strains as a causal agent of wheat disease. To our knowledge, this is the first report of P. ananatis affecting wheat plants.

Evaluation of the expression stability of reference genes in Apis mellifera under pyrethroid treatment.

Honeybees (Apis mellifera L.), which unquestionably play an economically important role in pollination and agricultural production, are at risk of decline. To study changes in gene expression in insects upon exposure to pesticides or other external stimuli, appropriate reference genes are required for data normalization. Since there is no such gene that is absolutely invariable under all experimental conditions, the aim of this study was to identify the most stable targets suitable for subsequent normalization in quantitative experiments based on real-time polymerase chain reaction in honeybee research. Here, we evaluated the expression of fifteen candidate housekeeping genes from three breeding lines of honeybees treated with pyrethroids to identify the most stable genes. The tested insects were exposed to deltamethrin or lambda-cyhalothrin, and then, changes in the accumulation of selected transcripts were assessed, followed by statistical analyses. We concluded that AmRPL32, AmACT and AmRPL13a were the commonly recorded most stable genes in honeybees treated with the selected pyrethroids.

Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants.

Green fluorescent protein (GFP)-tagged viruses are basic research tools widely applied in studies concerning molecular determinants of disease during virus infection. Here, we described a new generation of genetically stable infectious clones of tomato torrado virus isolate Kra (ToTVpJL-Kra) that could infect Nicotiana benthamiana and Solanum lycopersicum. Importantly, a modified variant of the viral RNA2-with inserted sGFP (forming, together with virus RNA1, into ToTVpJL-KraGFP)-was engineered as well. RNA2 of ToTVpJL-KraGFP was modified by introducing an additional open reading frame (ORF) of sGFP flanked with an amino acid-coding sequence corresponding to the putative virus protease recognition site. Our further analysis revealed that sGFP-tagged ToTV-Kra was successfully passaged by mechanical inoculation and spread systemically in plants. Therefore, the clone might be applied in studying the in vivo cellular, tissue, and organ-level localization of ToTV during infection. By performing whole-plant imaging, followed by fluorescence and confocal microscopy, the presence of the ToTVpJL-KraGFP-derived fluorescence signal was confirmed in infected plants. All this information was verified by sGFP-specific immunoprecipitation and western blot analysis. The molecular biology of the torradovirus-plant interaction is still poorly characterized; therefore, the results obtained here opened up new possibilities for further research. The application of sGFP-tagged virus infectious clones and their development method can be used for analyzing plant-virus interactions in a wide context of plant pathology.

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.

Contribution of Tomato torrado virus Vp26 coat protein subunit to systemic necrosis induction and virus infectivity in Solanum lycopersicum.

BACKGROUND: Tomato torrado virus (ToTV) infection manifests with burn-like symptoms on leaves, leaflets and upper stem parts of susceptible infected plants. The symptoms caused by ToTV may be considered as one of the most severe virus-induced forms of systemic necrosis, which spreads within the whole plant and leads to a lethal phenotype. However, to date there are no data revealing which viral genes encode for a specific pathogenicity determinant that triggers the plant necrotic response for any torradovirus. In this study we evaluated the influence of three coat protein subunits of ToTV: Vp23, Vp26 and Vp35, transiently expressed from a PVX-based vector, and checked their association with the induction of systemic necrosis in infected Solanum lycopersicum L. (cv. Beta Lux), a natural host of ToTV. METHODS: To estimate how ToTV coat protein subunits might contribute in plant response to virus infection we over-expressed the proteins from PVX-based vector in tomato and analyzed enzymatic activities related with plant defense response. By doing protein qualitative analysis performed by mass spectrometry we indicated the PR10 in protein fraction with induced ribonuclease activity. RESULTS: We observed that only the Vp26 enhanced PVX pathogenicity causing severe necrosis of the infected plant. Moreover, we indicated increased RNase and oxidative activities in plants infected with PVX-Vp26 chimeras only. Importantly, we suspected that this increased RNase activity is associated with increased accumulation of PR10 mRNA and products of its translation. CONCLUSIONS: On the basis of the obtained results, we indicated that Vp26 acts as the elicitor of hypersensitive response-like reactions of PVX-Vp26 manifesting with enhanced pathogenicity of the recombined PVX. This might be the first described suspected necrosis determinant of torradoviruses infecting tomatoes.

The Role of the Chloroplast in the Replication of Positive-Sense Single-Stranded Plant RNA Viruses.

Positive-sense single-stranded plant RNA viruses are obligate intracellular parasites that infect many agriculturally important crops. Most known plant RNA viruses are characterized by small genomes encoding a limited number of multifunctional viral proteins. Viral pathogens are considered to be absolutely dependent on their hosts, and viruses must recruit numerous host proteins and other factors for genomic RNA replication. Overall, the replication process depends on virus-plant protein-protein, RNA-protein and protein-lipid interactions. Recent publications provide strong evidence for the important role of chloroplasts in viral RNA synthesis. The chloroplast is considered to be a multifunctional organelle responsible for photosynthesis and for the generation of plant defense signaling molecules. High-throughput technologies (genomics and proteomics), and electron microscopy, including three-dimensional tomography, have revealed that several groups of plant RNA viruses utilize chloroplast membranes to assemble viral replication complexes (VRCs). Moreover, some chloroplast-related proteins reportedly interact with both viral proteins and their genomic RNAs and participate in trafficking these molecules to the chloroplast, where replication occurs. Here, we present the current knowledge on the important role of chloroplasts in the viral replication process.

Peanut Stunt Virus and Its Satellite RNA Trigger Changes in Phosphorylation in N. benthamiana Infected Plants at the Early Stage of the Infection.

Signaling in host plants is an integral part of a successful infection by pathogenic RNA viruses. Therefore, identifying early signaling events in host plants that play an important role in establishing the infection process will help our understanding of the disease process. In this context, phosphorylation constitutes one of the most important post-translational protein modifications, regulating many cellular signaling processes. In this study, we aimed to identify the processes affected by infection with Peanut stunt virus (PSV) and its satellite RNA (satRNA) in Nicotiana benthamiana at the early stage of pathogenesis. To achieve this, we performed proteome and phosphoproteome analyses on plants treated with PSV and its satRNA. The analysis of the number of differentially phosphorylated proteins showed strong down-regulation in phosphorylation in virus-treated plants (without satRNA). Moreover, proteome analysis revealed more down-regulated proteins in PSV and satRNA-treated plants, which indicated a complex dependence between proteins and their modifications. Apart from changes in photosynthesis and carbon metabolism, which are usually observed in virus-infected plants, alterations in proteins involved in RNA synthesis, transport, and turnover were observed. As a whole, this is the first community (phospho)proteome resource upon infection of N. benthamiana with a cucumovirus and its satRNA and this resource constitutes a valuable data set for future studies.

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.