Chloroplast redox state changes mark cell-to-cell signaling in the hypersensitive response.

Hypersensitive response (HR)-conferred resistance is associated with induction of programmed cell death and pathogen spread restriction in its proximity. The exact role of chloroplastic reactive oxygen species and its link with salicylic acid (SA) signaling in HR remain unexplained. To unravel this, we performed a detailed spatiotemporal analysis of chloroplast redox response in palisade mesophyll and upper epidermis to potato virus Y (PVY) infection in a resistant potato genotype and its transgenic counterpart with impaired SA accumulation and compromised resistance. Besides the cells close to the cell death zone, we detected individual cells with oxidized chloroplasts further from the cell death zone. These are rare in SA-deficient plants, suggesting their role in signaling for resistance. We confirmed that chloroplast redox changes play important roles in signaling for resistance, as blocking chloroplast redox changes affected spatial responses at the transcriptional level. Through spatiotemporal study of stromule induction after PVY infection, we show that stromules are induced by cell death and also as a response to PVY multiplication at the front of infection. Overall induction of stromules is attenuated in SA-deficient plants.

Analysis of Virus Spread Around the Cell Death Zone at Spatiotemporal Resolution Using Confocal Microscopy.

The role of programmed cell death (PCD) in hypersensitive response (HR)-conferred resistance depends on the type of host-pathogen interaction and therefore has to be studied for each individual pathosystem. Here we present and explain the protocol for studying the role of PCD in HR-conferred resistance in potato plants in the interaction with the viral pathogen. As an experimental system, we use genotype Rywal, where the virus spread is restricted and HR PCD develops 3 days post potato virus Y (PVY) inoculation. As a control of virus multiplication and spread, we include its transgenic counterpart impaired in salicylic acid (SA) accumulation (NahG-Rywal), in which the HR-PCD occurs but the spread of the virus is not restricted. To follow the occurrence of virus-infected cells and/or virus multiplication outside the cell death zone, we use GFP-tagged PVY (PVY-N605(123)-GFP) which can be monitored by confocal microscopy. Any other plant-pathogen system which results in PCD development could be studied using a modified version of this protocol.

RNA Sequencing Analyses for Deciphering Potato Molecular Responses.

Understanding the molecular mechanisms of potato development and responses to environmental stressors is of utmost importance for achieving stable crop yields. RNA sequencing (RNA-Seq) provides an insight into responses of all of the organism genes to the environmental and developmental cues and thus provides insights into underlying modes of action. In this chapter, we guide a researcher through some of the most important steps in the analysis of transcriptomics data. The initial topic of experimental design is followed by a more wet-lab-oriented section on RNA-Seq sample preparation. Next, we present intermediate steps of data retrieval, quality control, mapping, and differential expression of the dataset and a section on how to expose your data to the public (i.e., public repositories) and make it findable, accessible, interoperable, and reusable (FAIR). In the last four sections, we describe specific tools or Web applications, which ease the exploration of generated results in the context of their gene function and network-based visualizations, specifically GoMapMan, GSEA, DiNAR, and Biomine Explorer. All sections are accompanied by potato dataset examples and include general hints and tricks, as well as potato specificities that one should be aware of.

Precision transcriptomics of viral foci reveals the spatial regulation of immune-signaling genes and identifies RBOHD as an important player in the incompatible interaction between potato virus Y and potato.

Whereas the activation of resistance (R) proteins has been intensively studied, the downstream signaling mechanisms leading to the restriction of the pathogen remain mostly unknown. We studied the immunity network response conditioned by the potato Ny-1 gene against potato virus Y. We analyzed the processes in the cell death zone and surrounding tissue on the biochemical and gene expression levels in order to reveal the spatiotemporal regulation of the immune response. We show that the transcriptional response in the cell death zone and surrounding tissue is dependent on salicylic acid (SA). For some genes the spatiotemporal regulation is completely lost in the SA-deficient line, whereas other genes show a different response, indicating multiple connections between hormonal signaling modules. The induction of NADPH oxidase RBOHD expression occurs specifically on the lesion border during the resistance response. In plants with silenced RBOHD, the functionality of the resistance response is perturbed and the spread of the virus is not arrested at the site of infection. RBOHD is required for the spatial accumulation of SA, and conversely RBOHD is under the transcriptional regulation of SA. Using spatially resolved RNA-seq, we also identified spatial regulation of an UDP-glucosyltransferase, another component in feedback activation of SA biosynthesis, thus deciphering a novel aspect of resistance signaling.

Plant Molecular Responses to Potato Virus Y: A Continuum of Outcomes from Sensitivity and Tolerance to Resistance.

Potato virus Y (PVY) is the most economically important virus affecting potato production. PVY manipulates the plant cell machinery in order to successfully complete the infecting cycle. On the other side, the plant activates a sophisticated multilayer immune defense response to combat viral infection. The balance between these mechanisms, depending on the plant genotype and environment, results in a specific outcome that can be resistance, sensitivity, or tolerance. In this review, we summarize and compare the current knowledge on molecular events, leading to different phenotypic outcomes in response to PVY and try to link them with the known molecular mechanisms.

Network Modeling Unravels Mechanisms of Crosstalk between Ethylene and Salicylate Signaling in Potato.

To develop novel crop breeding strategies, it is crucial to understand the mechanisms underlying the interaction between plants and their pathogens. Network modeling represents a powerful tool that can unravel properties of complex biological systems. In this study, we aimed to use network modeling to better understand immune signaling in potato (Solanum tuberosum). For this, we first built on a reliable Arabidopsis (Arabidopsis thaliana) immune signaling model, extending it with the information from diverse publicly available resources. Next, we translated the resulting prior knowledge network (20,012 nodes and 70,091 connections) to potato and superimposed it with an ensemble network inferred from time-resolved transcriptomics data for potato. We used different network modeling approaches to generate specific hypotheses of potato immune signaling mechanisms. An interesting finding was the identification of a string of molecular events illuminating the ethylene pathway modulation of the salicylic acid pathway through Nonexpressor of PR Genes1 gene expression. Functional validations confirmed this modulation, thus supporting the potential of our integrative network modeling approach for unraveling molecular mechanisms in complex systems. In addition, this approach can ultimately result in improved breeding strategies for potato and other sensitive crops.

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 Y(NTN) (PVY(NTN) ) 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 PVY(NTN) -infected plants, which was linked to decreased accumulation of transcripts associated with the antinutritional properties of potato. In PVY(NTN) -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 PVY(NTN) -infected plants, confirming the role of other plant hormones in modulating this response. The release of ß-barbatene and benzyl alcohol was different in healthy and PVY(NTN) -infected plants before CPB larvae infestation, implicating the importance of PVY(NTN) 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.

Involvement of potato (Solanum tuberosum L.) MKK6 in response to potato virus Y.

Mitogen-activated protein kinase (MAPK) cascades have crucial roles in the regulation of plant development and in plant responses to stress. Plant recognition of pathogen-associated molecular patterns or pathogen-derived effector proteins has been shown to trigger activation of several MAPKs. This then controls defence responses, including synthesis and/or signalling of defence hormones and activation of defence related genes. The MAPK cascade genes are highly complex and interconnected, and thus the precise signalling mechanisms in specific plant-pathogen interactions are still not known. Here we investigated the MAPK signalling network involved in immune responses of potato (Solanum tuberosum L.) to Potato virus Y, an important potato pathogen worldwide. Sequence analysis was performed to identify the complete MAPK kinase (MKK) family in potato, and to identify those regulated in the hypersensitive resistance response to Potato virus Y infection. Arabidopsis has 10 MKK family members, of which we identified five in potato and tomato (Solanum lycopersicum L.), and eight in Nicotiana benthamiana. Among these, StMKK6 is the most strongly regulated gene in response to Potato virus Y. The salicylic acid treatment revealed that StMKK6 is regulated by the hormone that is in agreement with the salicylic acid-regulated domains found in the StMKK6 promoter. The involvement of StMKK6 in potato defence response was confirmed by localisation studies, where StMKK6 accumulated strongly only in Potato-virus-Y-infected plants, and predominantly in the cell nucleus. Using a yeast two-hybrid method, we identified three StMKK6 targets downstream in the MAPK cascade: StMAPK4_2, StMAPK6 and StMAPK13. These data together provide further insight into the StMKK6 signalling module and its involvement in plant defence.

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 PVY(NTN), 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.

PVY(NTN) 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.

Finding differentially expressed genes in two-channel DNA microarray datasets: how to increase reliability of data preprocessing.

Due to the great variety of preprocessing tools in two-channel expression microarray data analysis it is difficult to choose the most appropriate one for a given experimental setup. In our study, two independent two-channel inhouse microarray experiments as well as a publicly available dataset were used to investigate the influence of the selection of preprocessing methods (background correction, normalization, and duplicate spots correlation calculation) on the discovery of differentially expressed genes. Here we are showing that both the list of differentially expressed genes and the expression values of selected genes depend significantly on the preprocessing approach applied. The choice of normalization method to be used had the highest impact on the results. We propose a simple but efficient approach to increase the reliability of obtained results, where two normalization methods which are theoretically distinct from one another are used on the same dataset. Then the intersection of results, that is, the lists of differentially expressed genes, is used in order to get a more accurate estimation of the genes that were de facto differentially expressed.

Jasmonic acid stimulates taxane production in cell suspension culture of yew (Taxus x media).

Cell lines of Taxus x media Rehd. were established via embryo and callus culture and grown on B5 medium containing 10 microM 2,4-D and 1 microM kinetin. Jasmonic acid (JA) was used to elicit taxane synthesis in a selected yew cell line. 100 microM JA was added 7 days after subculture. JA strongly increased taxane content in the cells and in the medium. At the time of the highest amount in elicited cultures, the enhancement of paclitaxel production was 19-fold and 4-fold in the cells and medium, respectively, compared to controls (non-elicited cultures). The time course of taxane accumulation in the cells and in the medium and the release of taxanes into the medium were not changed after JA elicitation.