1. 1-aminocyclopropane-1-carboxylate oxidase induction in tomato flower pedicel phloem and abscission related processes are differentially sensitive to ethyleneMarko Chersicola, Aleš Kladnik, Magda Tušek-Žnidarič, Tanja Mrak, Kristina Gruden, Marina Dermastia, 2017, izvirni znanstveni članek Povzetek: Ethylene has impact on several physiological plant processes, including abscission, during which plants shed both their vegetative and reproductive organs. Cell separation and programmed cell death are involved in abscission, and these have also been correlated with ethylene action. However, the detailed spatiotemporal pattern of the molecular events during abscission remains unknown. We examined the expression of two tomato ACO genes, LeACO1, and LeACO4 that encode the last enzyme in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate oxidase (ACO), together with the expression of other abscission-associated genes involved in cell separation and programmed cell death, during a period of 0–12 h after abscission induction in the tomato flower pedicel abscission zone and nearby tissues. In addition, we determined their localization in specific cell layers of the flower pedicel abscission zone and nearby tissues obtained by laser microdissection before and 8 h after abscission induction. The expression of both ACO genes was localized to the vascular tissues in the pedicel. While LeACO4 was more uniformly expressed in all examined cell layers, the main expression site of LeACO1 was in cell layers just outside the abscission zone in its proximal and distal part. We showed that after abscission induction, ACO1 protein was synthesized in phloem companion cells, in which it was localized mainly in the cytoplasm. Samples were additionally treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene actions, and analyzed 8 h after abscission induction. Cell-layer-specific changes in gene expression were observed together with the specific localization and ethylene sensitivity of the hallmarks of cell separation and programmed cell death. While treatment with 1-MCP prevented separation of cells through inhibition of the expression of polygalacturonases, which are the key enzymes involved in degradation of the middle lamella, this had less impact on the occurrence of different kinds of membrane vesicles and abscission-related programmed cell death. In the flower pedicel abscission zone, the physical progressions of cell separation and programmed cell death are perpendicular to each other and start in the vascular tissues.
Ključne besede: abscission, ACO, cell separation, ethylene, laser microdissection, programmed cell death, tomato, ultrastructure
Objavljeno v DiRROS: 24.07.2024; Ogledov: 17; Prenosov: 17
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2. High-throughput sequencing facilitates characterisation of a ʺforgottenʺ plant virus : the case of a henbane mosaic virus infecting tomatoAnja Pecman, Denis Kutnjak, Nataša Mehle, Magda Tušek-Žnidarič, Ion Gutiérrez-Aguirre, Patricija Pirnat, Ian Adams, Neil Boonham, Maja Ravnikar, 2018, izvirni znanstveni članek Povzetek: High-throughput sequencing has dramatically broadened the possibilities for plant virus research and diagnostics, enabling discovery of new or obscure viruses, and virus strains and rapid sequencing of their genomes. In this research, we employed high-throughput sequencing to discover a new virus infecting tomato, Henbane mosaic virus (Potyvirus, Potyviridae), which was first discovered at the beginning of 20th century in the United Kingdom in cultivated henbane. A field tomato plant with severe necrotic symptoms of unknown etiology was sampled in Slovenia and high-throughput sequencing analysis using small RNA and ribosomal RNA depleted total RNA approaches revealed a mixed infection with Potato virus M (Carlavirus, Betaflexiviridae), Southern tomato virus (Amalgavirus, Amalgamaviridae) and henbane mosaic virus in the sample. The complete genomic sequence of henbane mosaic virus was assembled from the sequencing reads. By re-inoculation of the infected material on selected test plants, henbane mosaic virus was isolated and a host range analysis was performed, demonstrating the virus was pathogenic on several plant species. Due to limited metadata in public repositories, the taxonomic identification of the virus isolate was initially putative. Thus, in the next step, we used small RNA sequencing to determine genomic sequences of four historic isolates of the virus, obtained from different virus collections. Phylogenetic analyses performed using this new sequence information enabled us to taxonomically position Henbane mosaic virus as a member of the Potyvirus genus within the chili veinal mottle virus phylogenetic cluster and define the relationship of the new tomato isolate with the historic ones, indicating the existence of at least four putative strains of the virus. The first detection of henbane mosaic virus in tomato and demonstration of its pathogenicity on this host is important for plant protection and commercial tomato production. Since the virus was initially present in a mixed infection, and its whole genome was not sequenced, it has probably been overlooked in routine diagnostics. This study confirms the applicability of a combination of high-throughput sequencing and classic plant virus characterization methods for identification and phylogenetic classification of obscure viruses and historical viral isolates, for which no or limited genome sequence data is available.
Ključne besede: henbane mosaic virus, Tomato, high-throughput sequencing, host range analysis, phylogeny, Potyvirus
Objavljeno v DiRROS: 24.07.2024; Ogledov: 23; Prenosov: 9
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3. Detection of four new tomato viruses in Serbia using post hoc high-throughput sequencing analysis of samples from a large-scale field surveyAna Vučurović, Denis Kutnjak, Nataša Mehle, Ivana Stanković, Anja Pecman, Aleksandra Bulajić, Branka Krstić, Maja Ravnikar, 2021, izvirni znanstveni članek Povzetek: Tomato production worldwide is affected by numerous plant virus species. The early and accurate detection of viruses is a critical step for disease control. However, the simultaneous detection of the most known tomato viruses can be difficult because of the high number and diversity of tomato-infecting viruses. Here, we have identified four new viruses in Serbia by applying target-independent small RNA high-throughput sequencing (HTS). HTS was applied on pools of samples and separate samples, in total comprising 30 tomato samples that exhibited (severe) virus-like symptoms and were collected in Serbia during three annual surveys (2011 to 2013). These samples had previously tested negative for the presence of 16 tomato viruses using targeted detection methods. Three divergent complete genome sequences of Physostegia chlorotic mottled virus were obtained from different localities, indicating for the first time that this virus is widespread in Serbia and might represent an emergent viral pathogen of tomato. The tomato torrado virus was detected at one locality with devastating yield losses. The southern tomato virus was detected at two localities, and the spinach latent virus was detected at one locality. In addition, we detected the presence of one already-known virus in Serbia, the tomato spotted wilt orthotospovirus. All the HTS results were subsequently confirmed by targeted detection methods. In this study, the successful application of post hoc HTS testing of a limited number of pooled samples resulted in the discovery of new viruses. Thus, our results encourage the use of HTS in research and diagnostic laboratories, including laboratories that have limited resources to resolve disease etiology.
Ključne besede: viruses, tomato, detection, identification
Objavljeno v DiRROS: 19.07.2024; Ogledov: 50; Prenosov: 27
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4. Development and validation of a one-step reverse transcription real-time PCR assay for simultaneous detection and identification of tomato mottle mosaic virus and tomato brown rugose fruit virusAntonio Tiberini, Ariana Manglli, Anna Taglienti, Ana Vučurović, Jakob Brodarič, Luca Ferretti, Marta Luigi, Andrea Gentili, Nataša Mehle, 2022, izvirni znanstveni članek Povzetek: Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and because they are seed borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the genus, tomato mottle mosaic virus, has recently gained attention due to reports in different continents and its common features with tomato brown rugose fruit virus. In this study, a new real-time RT-PCR detection system was developed for tomato brown rugose fruit virus and tomato mottle mosaic virus on tomato leaves and seeds using TaqMan chemistry. This test was designed to detect tomato mottle mosaic virus by amplifying the movement protein gene in a duplex assay with the tomato brown rugose fruit virus target on the CP-3’NTR region, which was previously validated as a single assay. The performance of this test was evaluated, displaying analytical sensitivity 10−5–10−6-fold dilution for seeds and leaves, respectively, and good analytical specificity, repeatability, and reproducibility. Using the newly developed and validated test, tomato brown rugose fruit virus detection was 100% concordant with previously performed analyses on 106 official samples collected in 2021 from different continents.
Ključne besede: real-time PCR, tomato mottle mosaic virus, tomato brown rugose fruit virus, leaves detection, seeds detections, performance criteria
Objavljeno v DiRROS: 16.07.2024; Ogledov: 71; Prenosov: 40
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5. Transcriptional and epigenetic changes during tomato yellow leaf curl virus infection in tomatoBeatriz Romero-Rodríguez, Marko Petek, Chen Jiao, Maja Križnik, Maja Zagorščak, Zhangjun Fei, Eduardo Rodríguez Bejarano, Kristina Gruden, Araceli G. Castillo, 2023, izvirni znanstveni članek Povzetek: Background Geminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV). Results We showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes. Conclusion We have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato.
Ključne besede: geminivirus, TYLCV, tomato, transcriptome, miRNA
Objavljeno v DiRROS: 15.07.2024; Ogledov: 98; Prenosov: 42
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6. Tomato brown rugose fruit virus in aqueous environments : survival and significance of water-mediated transmissionNataša Mehle, Katarina Bačnik, Irena Bajde, Jakob Brodarič, Adrian Fox, Ion Gutiérrez-Aguirre, Miha Kitek, Denis Kutnjak, Yue Lin Loh, Olivera Maksimović, Maja Ravnikar, Elise Vogel, Christine Vos, Ana Vučurović, 2023, izvirni znanstveni članek Povzetek: Tomato brown rugose fruit virus (ToBRFV) has recently emerged as a major disease of tomatoes and peppers. ToBRFV is a seed- and contact-transmitted virus. In Slovenia, ToBRFV RNA was detected in samples of wastewater, river, and water used to irrigate plants. Even though the source of detected RNA could not be clearly established, this raised the question of the significance of the detection of ToBRFV in water samples and experimental studies were performed to address this question. The data presented here confirm that the release of virus particles from the roots of infected plants is a source of infectious ToBRFV particles in water and that the virus can remain infective up to four weeks in water stored at room temperature, while its RNA can be detected for much longer. These data also indicate that irrigation with ToBRFV-contaminated water can lead to plant infection. In addition, it has been shown that ToBRFV circulated in drain water in commercial tomato greenhouses from other European countries and that an outbreak of ToBRFV can be detected by regular monitoring of drain water. A simple method for concentrating ToBRFV from water samples and a comparison of the sensitivity of different methods, including the determination of the highest ToBRFV dilution still capable of infecting test plants, were also investigated. The results of our studies fill the knowledge gaps in the epidemiology and diagnosis of ToBRFV, by studying the role of water-mediated transmission, and provide a reliable risk assessment to identify critical points for monitoring and control.
Ključne besede: tomato brown rugose fruit virus, tomato, hydroponics, water-linked epidemiology, survival
Objavljeno v DiRROS: 12.07.2024; Ogledov: 73; Prenosov: 40
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7. In-depth study of tomato and weed viromes reveals undiscovered plant virus diversity in an agroecosystemMark Paul Selda Rivarez, Anja Pecman, Katarina Bačnik, Olivera Maksimović, Ana Vučurović, Gabrijel Seljak, Nataša Mehle, Ion Gutiérrez-Aguirre, Maja Ravnikar, Denis Kutnjak, 2023, izvirni znanstveni članek Povzetek: Background: In agroecosystems, viruses are well known to influence crop health and some cause phytosanitary and economic problems, but their diversity in non-crop plants and role outside the disease perspective is less known. Extensive virome explorations that include both crop and diverse weed plants are therefore needed to better understand roles of viruses in agroecosystems. Such unbiased exploration is available through viromics, which could generate biological and ecological insights from immense high-throughput sequencing (HTS) data. Results: Here, we implemented HTS-based viromics to explore viral diversity in tomatoes and weeds in farming areas at a nation-wide scale. We detected 125 viruses, including 79 novel species, wherein 65 were found exclusively in weeds. This spanned 21 higher-level plant virus taxa dominated by Potyviridae, Rhabdoviridae, and Tombusviridae, and four non-plant virus families. We detected viruses of non-plant hosts and viroid-like sequences and demonstrated infectivity of a novel tobamovirus in plants of Solanaceae family. Diversities of predominant tomato viruses were variable, in some cases, comparable to that of global isolates of the same species. We phylogenetically classified novel viruses and showed links between a subgroup of phylogenetically related rhabdoviruses to their taxonomically related host plants. Ten classified viruses detected in tomatoes were also detected in weeds, which might indicate possible role of weeds as their reservoirs and that these viruses could be exchanged between the two compartments. Conclusions: We showed that even in relatively well studied agroecosystems, such as tomato farms, a large part of very diverse plant viromes can still be unknown and is mostly present in understudied non-crop plants. The overlapping presence of viruses in tomatoes and weeds implicate possible presence of virus reservoir and possible exchange between the weed and crop compartments, which may influence weed management decisions. The observed variability and widespread presence of predominant tomato viruses and the infectivity of a novel tobamovirus in solanaceous plants, provided foundation for further investigation of virus disease dynamics and their effect on tomato health. The extensive insights we generated from such in-depth agroecosystem virome exploration will be valuable in anticipating possible emergences of plant virus diseases and would serve as baseline for further post-discovery characterization studies.
Ključne besede: tomato, weed, virus, viroid, virome, virus discovery, virus diversity, phylogenetics, metagenomics, viromics
Objavljeno v DiRROS: 13.04.2023; Ogledov: 775; Prenosov: 177
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8. Discrimination between abiotic and biotic drought stress in tomatoes using hyperspectral imagingNik Susič, Uroš Žibrat, Saša Širca, Polona Strajnar, Jaka Razinger, Matej Knapič, Andrej Vončina, Gregor Urek, Barbara Gerič Stare, izvirni znanstveni članek Povzetek: Crop plants are subjected to various biotic and abiotic stresses. Both root-knot nematodes (biotic stress) and water deficiency (abiotic stress) lead to similar drought symptoms in the plant canopy. In this work, hyperspectral imaging was used for early detection of nematode infestation and water deficiency (drought) stress in tomato plants. Hyperspectral data in the range from 400 to 2500 nm of plants subjected to different watering regimes and nematode infestation levels were analysed by partial least squares – discriminant analysis (PLS-DA) and partial least squares – support vector machine (PLS-SVM) classification. PLS-SVM classification achieved up to 100% accuracy differentiating between well-watered and water-deficient plants, and between 90 and 100% when identifying nematode-infested plants. Grouping the data according to the time of imaging increased the accuracy of classification. Shortwave infrared spectral regions associated with the OH and CH stretches were most relevant for the identification of nematode infested plants and severity of infestation. This study demonstrates the capability of hyperspectral imaging to identify and discriminate between biotic and abiotic plant stresses.
Ključne besede: nematode, Hyperspectral imaging, Drought stress, Root-knot nematode, Tomato
Objavljeno v DiRROS: 20.07.2018; Ogledov: 4056; Prenosov: 2499
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