Polona Kogovšek

Single-step RT real-time PCR for sensitive detection and discrimination of Potato virus Y isolates

Potato virus Y (PVY) has a worldwide distribution and infects several economically important crops from the Solanaceae family. The emergence and spread of the PVYNTN strain, which is the causative agent of potato tuber necrotic ringspot disease (PTNRD), has lead to large economic losses and highlighted the need for accurate discrimination of the different PVY strains. Detection and differentiation of PVY isolates is mainly based on a combination of ELISA, RT-PCR and bioassays; however, PVYNTN isolates are particularly difficult to differentiate from standard PVYN without the use of time-consuming bioassays. A strong correlation has been identified previously between the ability to induce PTNRD and the presence of a recombination point in the virus coat protein. An RT real-time PCR assay has been developed to enable detection of isolates with the recombination point, therefore, enabling rapid differentiation between potentially tuber necrotic PVYNTN isolates and standard PVYN isolates. The assay is also able to detect the presence of PVYO isolates. To aid with routine testing, immuno-capture and post-ELISA virus release were introduced; when coupled with RT real-time PCR the sensitivity of the assays were up to seven orders of magnitude higher than ELISA. The assay was shown to be a suitable method for rapid large-scale diagnostic testing of PVY in different types of plant material including tubers, and specific screening for potentially tuber necrotic recombinant isolates.

PVYNTN elicits a diverse gene expression response in different potato genotypes in the first 12 h after inoculation

Host gene expression changes in the early response to potato virus Y(NTN) interaction were compared in two differently sensitive potato cultivars: the resistant cultivar Santé and the sensitive cultivar Igor. Hybridization of potato TIGR cDNA microarrays allowed us to monitor the expression of approximately 10,000 genes simultaneously at 0.5 and 12 h post-inoculation (hpi). Microarray data, analysed by statistics and data mining, were complemented by subtraction library construction and sequence analysis to validate the findings. The expression profiles of the two cultivars were similar and faint at 0.5 hpi, but they differed substantially at 12 hpi. Although, at 0.5 hpi, cv. Santé responded by the differential expression of a greater number of genes, at 12 hpi the number was higher in cv. Igor. The majority of genes in this cultivar were down-regulated at 12 hpi, indicating a host gene shut-off. Suites of genes that exhibited altered transcript abundance in response to the virus were identified, and included genes involved in the processes of photosynthesis, perception, signalling and defence responses. The expression of the considerable number of genes associated with photosynthesis was surprisingly up-regulated as early as 0.5 hpi and down-regulated at 12 hpi in both cultivars. The expression of genes involved in perception and signalling was increased in the sensitive cultivar at 12 hpi. By contrast, a simultaneous strong defence response at the transcriptional level was evident in the resistant cultivar, as shown by the up-regulation of genes involved in brassinosteroid, polyamine and secondary metabolite biosynthesis, and of genes coding for pathogenesis-related proteins.

Dynamics of Responses in Compatible Potato - Potato virus Y Interaction Are Modulated by Salicylic Acid

To investigate the dynamics of the potato – Potato virus Y (PVY) compatible interaction in relation to salicylic acid - controlled pathways we performed experiments using non-transgenic potato cv. Désirée, transgenic NahG-Désirée, cv. Igor and PVYNTN, the most aggressive strain of PVY. The importance of salicylic acid in viral multiplication and symptom development was confirmed by pronounced symptom development in NahG-Désirée, depleted in salicylic acid, and reversion of the effect after spraying with 2,6-dichloroisonicotinic acid (a salicylic acid - analogue). We have employed quantitative PCR for monitoring virus multiplication, as well as plant responses through expression of selected marker genes of photosynthetic activity, carbohydrate metabolism and the defence response. Viral multiplication was the slowest in inoculated potato of cv. Désirée, the only asymptomatic genotype in the study. The intensity of defence-related gene expression was much stronger in both sensitive genotypes (NahG-Désirée and cv. Igor) at the site of inoculation than in asymptomatic plants (cv. Désirée). Photosynthesis and carbohydrate metabolism gene expression differed between the symptomatic and asymptomatic phenotypes. The differential gene expression pattern of the two sensitive genotypes indicates that the outcome of the interaction does not rely simply on one regulatory component, but similar phenotypical features can result from distinct responses at the molecular level.

LAMP assay and rapid sample preparation method for on‐site detection of flavescence dorée phytoplasma in grapevine

In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.

Distribution of Potato virus Y in potato plant organs, tissues, and cells.

The distribution of Potato virus Y (PVY) in the systemically infected potato (Solanum tuberosum) plants of the highly susceptible cultivar Igor was investigated. Virus presence and accumulation was analyzed in different plant organs and tissues using real-time polymerase chain reaction and transmission electron microscopy (TEM) negative staining methods. To get a complete insight into the location of viral RNA within the tissue, in situ hybridization was developed and optimized for the detection of PVY RNA at the cellular level. PVY was shown to accumulate in all studied leaf and stem tissues, in shoot tips, roots, and tubers; however, the level of virus accumulation was specific for each organ or tissue. The highest amounts of viral RNA and viral particles were found in symptomatic leaves and stem. By observing cell ultrastructure with TEM, viral cytoplasmic inclusion bodies were localized in close vicinity to the epidermis and in trichomes. Our results show that viral RNA, viral particles, and cytoplasmic inclusion bodies colocalize within the same type of cells or in close vicinity.

Primary Metabolism, Phenylpropanoids and Antioxidant Pathways Are Regulated in Potato as a Response to Potato virus Y Infection

Potato production is one of the most important agricultural sectors, and it is challenged by various detrimental factors, including virus infections. To control losses in potato production, knowledge about the virus—plant interactions is crucial. Here, we investigated the molecular processes in potato plants as a result of Potato virus Y (PVY) infection, the most economically important potato viral pathogen. We performed an integrative study that links changes in the metabolome and gene expression in potato leaves inoculated with the mild PVYN and aggressive PVYNTN isolates, for different times through disease development. At the beginning of infection (1 day post-inoculation), virus-infected plants showed an initial decrease in the concentrations of metabolites connected to sugar and amino-acid metabolism, the TCA cycle, the GABA shunt, ROS scavangers, and phenylpropanoids, relative to the control plants. A pronounced increase in those metabolites was detected at the start of the strong viral multiplication in infected leaves. The alterations in these metabolic pathways were also seen at the gene expression level, as analysed by quantitative PCR. In addition, the systemic response in the metabolome to PVY infection was analysed. Systemic leaves showed a less-pronounced response with fewer metabolites altered, while phenylpropanoid-associated metabolites were strongly accumulated. There was a more rapid onset of accumulation of ROS scavengers in leaves inoculated with PVYN than those inoculated with PVYNTN. This appears to be related to the lower damage observed for leaves of potato infected with the milder PVYN strain, and at least partially explains the differences between the phenotypes observed.

Potato virus Y infection hinders potato defence response and renders plants more vulnerable to Colorado potato beetle attack

In the field, plants are challenged by more than one biotic stressor at the same time. In this study, the molecular interactions between potato (Solanum tuberosum L.), Colorado potato beetle (Leptinotarsa decemlineata Say; CPB) and Potato virus YNTN (PVYNTN) were investigated through analyses of gene expression in the potato leaves and the gut of the CPB larvae, and of the release of potato volatile compounds. CPB larval growth was enhanced when reared on secondary PVYNTN‐infected plants, which was linked to decreased accumulation of transcripts associated with the antinutritional properties of potato. In PVYNTN‐infected plants, ethylene signalling pathway induction and induction of auxin response transcription factors were attenuated, while no differences were observed in jasmonic acid (JA) signalling pathway. Similarly to rearing on virus‐infected plants, CPB larvae gained more weight when reared on plants silenced in JA receptor gene (coi1). Although herbivore‐induced defence mechanism is regulated predominantly by JA, response in coi1‐silenced plants only partially corresponded to the one observed in PVYNTN‐infected plants, confirming the role of other plant hormones in modulating this response. The release of β‐barbatene and benzyl alcohol was different in healthy and PVYNTN‐infected plants before CPB larvae infestation, implicating the importance of PVYNTN infection in plant communication with its environment. This was reflected in gene expression profiles of neighbouring plants showing different degree of defence response. This study thus contributes to our understanding of plant responses in agro‐ecosystems.

β-1,3-glucanase class III promotes spread of PVY(NTN) and improves in planta protein production.

Glucanases are enzymes regulating the size exclusion limit and permeability of plasmodesmata and play a role in biotic stress. In plant genomes, they are encoded as relatively large gene families divided into four classes. Most studies of plant virus interactions have focused on glucanases from classes I and II. In our study, we have evaluated the role of the β-1,3-glucanase class III (Glu-III) gene in the potato–potato virus YNTN (PVYNTN) interaction and implemented the findings to plant biotechnology application. Potato cultivars Désirée and Santé, which are tolerant and extremely resistant to PVYNTN, respectively, were stably transformed with Agrobacterium tumefaciens harbouring constructs for Glu-III overexpression. Localization of Glu-III protein in patches within the cell wall was determined by tagging the Glu-III protein with green fluorescent protein. Transgenic and non-transgenic plants were challenged with PVYNTN and its multiplication and spreading was followed. Differences in viral spread were observed between transgenic lines overexpressing Glu-III and non-transgenic lines, with stronger and faster viral spread in transgenic Désirée, and some multiplication in transgenic Santé. In addition, the ability of Glu-III to improve in planta protein production after agroinfiltration was tested. The results have shown that Glu-III overexpression enables faster spreading of vectors between cells and better protein production, which could be beneficial in improving in planta protein production system using viral vectors.

Physiology of the Potato–Potato Virus Y Interaction

Many morphological, physiological, and phytochemical changes in plants are caused by biotic stress. Response of plants to various pathogenic microorganisms and pests is described at different levels: changed morphology of the plant and development of symptoms like lesions and necrosis; alterations in hormone concentrations; signaling molecules; secondary metabolism; pathogenesis-related proteins; and mRNA or gene expression. Due to its small size and therefore early description of the genome, Arabidopsis thaliana is a model plant in which metabolic processes are well described and constitutes a basis for research on other plants (Dardick et al. 2000; Pieterse et al. 2009; Simon et al. 2010; Wan et al. 2002). Nevertheless, plants from other families possess specific metabolic pathways not observed in this model plant (e.g., tuberization is specific for potato), which gives urgency to research on a wider variety of plant families. There are only limited reports on the responses of potato plants, especially to virus infection. The availability of the potato genome sequence opens up the possibility of advancing the understanding of the physiology of potato response to virus infection.

Assessment of SNaPshot and single step RT-qPCR methods for discriminating Potato virus Y (PVY) subgroups.

Potato virus Y (PVY) is the most important virus infecting potato (Solanum tuberosum), causing potato tuber necrotic ringspot disease (PTNRD), with a great impact on seed potato production. Numerous PVY strain groups with different pathogenicity and economical impact are distributed worldwide. Tools for accurate and reliable detection and discrimination of PVY strain groups are therefore essential for successful disease management. Two state of the art characterization tools based on detecting molecular markers - RT-qPCR (Kogovsek et al., 2008) and SNaPshot (Rolland et al., 2008) - were assessed for their ability to assign PVY accurately to the correct group. The results were validated by bioassay, ELISA and in silico sequence analysis. The spectrum of PVY strain groups distinguished by SNaPshot is broader than that by RT-qPCR. However, the latter was more reliable in discriminating the PVY(NTN) group members, known for their ability to induce PTNRD on selected potato cultivars. The difference in discrimination precision was due to different molecular markers being targeted by RT-qPCR and SNaPshot. Both tools use genotypic markers for detecting PVY(NTN) strain groups. Future development, however, should be focused on identifying the genomic determinants of the tuber necrosis property. Until then, the RT-qPCR and SNaPshot methods remain the most powerful diagnostic tools for detecting the PVY subgroup isolates found in Europe.