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.

Methodologies for Discovery and Quantitative Profiling of sRNAs in Potato.

Small RNAs (sRNAs) are short noncoding RNAs involved in the regulation of a wide range of biological processes in plants. Advances in high-throughput sequencing and development of new computational tools had facilitated the discovery of different classes of sRNAs, their quantification, and elucidation of their functional role in gene expression regulation by target transcript predictions. The workflow presented here allows identification of different sRNA species: known and novel potato miRNAs, and their sequence variants (isomiRs), as well as identification of phased small interfering RNAs (phasiRNAs). Moreover, it includes steps for differential expression analysis to search for regulated sRNAs across different tested biological conditions. In addition, it describes two different methods for predicting sRNA targets, in silico prediction, and degradome sequencing data analysis. All steps of the workflow are written in a clear and user-friendly way; thus they can be followed also by the users with minimal bioinformatics knowledge. We also included several in-house scripts together with valuable notes to facilitate data (pre)processing steps and to reduce the analysis time.

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.

Cultivar-specific transcriptome and pan-transcriptome reconstruction of tetraploid potato.

Although the reference genome of Solanum tuberosum Group Phureja double-monoploid (DM) clone is available, knowledge on the genetic diversity of the highly heterozygous tetraploid Group Tuberosum, representing most cultivated varieties, remains largely unexplored. This lack of knowledge hinders further progress in potato research. In conducted investigation, we first merged and manually curated the two existing partially-overlapping DM genome-based gene models, creating a union of genes in Phureja scaffold. Next, we compiled available and newly generated RNA-Seq datasets (cca. 1.5 billion reads) for three tetraploid potato genotypes (cultivar Désirée, cultivar Rywal, and breeding clone PW363) with diverse breeding pedigrees. Short-read transcriptomes were assembled using several de novo assemblers under different settings to test for optimal outcome. For cultivar Rywal, PacBio Iso-Seq full-length transcriptome sequencing was also performed. EvidentialGene redundancy-reducing pipeline complemented with in-house developed scripts was employed to produce accurate and complete cultivar-specific transcriptomes, as well as to attain the pan-transcriptome. The generated transcriptomes and pan-transcriptome represent a valuable resource for potato gene variability exploration, high-throughput omics analyses, and breeding programmes.

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.

Multiomics analysis of tolerant interaction of potato with potato virus Y.

Potato virus Y (PVY) is the most economically important viral pathogen of potato worldwide. Different potato cultivars react to the pathogen differently, resulting in resistant, tolerant or disease outcome of the interaction. Here we focus on tolerant interaction between potato cv. Désirée and PVYNTN. To capture the response in its full complexity, we analyzed the dynamic changes on multiple molecular levels, including transcriptomics, sRNAomics, degradomics, proteomics and hormonomics. The analysis was complemented by the measurements of viral accumulation, photosynthetic activity and phenotypisation of the symptoms. Besides cv. Désirée we also studied its transgenic counterpart depleted for the accumulation of salicylic acid (NahG-Désirée). This multiomics analysis provides better insights into the mechanisms leading to tolerant response of potato to viral infection and can be used as a base in further studies of plant immunity regulation.

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.

A model species for agricultural pest genomics: the genome of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases.

Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.

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.

Bimodal dynamics of primary metabolism-related responses in tolerant potato-Potato virus Y interaction.

BACKGROUND: Potato virus Y (PVY) is a major pathogen that causes substantial economic losses in worldwide potato production. Different potato cultivars differ in resistance to PVY, from severe susceptibility, through tolerance, to complete resistance. The aim of this study was to better define the mechanisms underlying tolerant responses of potato to infection by the particularly aggressive PVY(NTN) strain. We focused on the dynamics of the primary metabolism-related processes during PVY(NTN) infection. RESULTS: A comprehensive analysis of the dynamic changes in primary metabolism was performed, which included whole transcriptome analysis, nontargeted proteomics, and photosynthetic activity measurements in potato cv. Désirée and its transgenic counterpart depleted for accumulation of salicylic acid (NahG-Désirée). Faster multiplication of virus occurred in the NahG-Désirée, with these plants developing strong disease symptoms. We show that while the dynamics of responses at the transcriptional level are extensive and bimodal, this is only partially translated to the protein level, and to the final functional outcome. Photosynthesis-related genes are transiently induced before viral multiplication is detected and it is down-regulated later on. This is reflected as a deficiency of the photosynthetic apparatus at the onset of viral multiplication only. Interestingly, specific and constant up-regulation of some RuBisCO transcripts was detected in Désirée plants, which might be important, as these proteins have been shown to interact with viral proteins. In SA-deficient and more sensitive NahG-Désirée plants, consistent down-regulation of photosynthesis-related genes was detected. A constant reduction in the photochemical efficiency from the onset of viral multiplication was identified; in nontransgenic plants this decrease was only transient. The transient reduction in net photosynthetic rate occurred in both genotypes with the same timing, and coincided with changes in stomatal conductivity. CONCLUSIONS: Down-regulation of photosynthesis-related gene expression and decreased photosynthetic activity is in line with other studies that have reported the effects of biotic stress on photosynthesis. Here, we additionally detected induction of light-reaction components in the early stages of PVY(NTN) infection of tolerant interaction. As some of these components have already been shown to interact with viral proteins, their overproduction might contribute to the absence of symptoms in cv. Désirée.

Clitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases.

Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a major potato pest that adapts readily to insecticides. Several types of protease inhibitors have previously been investigated as potential control agents, but with limited success. Recently, cysteine protease inhibitors from parasol mushroom, the macrocypins, were reported to inhibit growth of CPB larvae. To further investigate the insecticidal potential and mode of action of cysteine protease inhibitors of fungal origin, clitocypin, a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis), was evaluated for its lethal effects on CPB larvae. Clitocypin isolated from fruiting bodies and recombinant clitocypin produced in Escherichia coli slowed growth and reduced survival of CPB larvae in a concentration dependent manner. Clitocypin was also expressed by transgenic potato, but only at low levels. Nevertheless, it reduced larval weight gain and delayed development. We have additionally shown that younger larvae are more susceptible to the action of clitocypin. The inhibition of digestive cysteine proteases, intestains, by clitocypin was shown to be the underlying mode of action. Protease inhibitors from mushrooms are confirmed as promising candidates for biopesticides.

Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot.

Here we report on the first assessment of droplet digital PCR (ddPCR) for detection and absolute quantification of two quarantine plant pathogenic bacteria that infect many species of the Rosaceae and Solanaceae families: Erwinia amylovora and Ralstonia solanacearum. An open-source R script was written for the ddPCR data analysis. Analysis of a set of samples with known health status aided the assessment and selection of different threshold settings (QuantaSoft analysis, definetherain pipeline and manual threshold), which led to optimal diagnostic specificity. The interpretation of the E. amylovora ddPCR was straightforward, and the analysis approach had little influence on the final results and the concentrations determined. The sensitivity and linear range were similar to those for real-time PCR (qPCR), for the analysis of both bacterial suspensions and plant material, making ddPCR a viable choice when both detection and quantification are desired. With the R. solanacearum ddPCR, the use of a high global threshold was necessary to exclude false-positive reactions that are sometimes observed in healthy plant material. ddPCR significantly improved the analytical sensitivity over that of qPCR, and improved the detection of low concentrations of R. solanacearum in potato tuber samples. Accurate and rapid absolute quantification of both of these bacteria in pure culture was achieved by direct ddPCR. Our data confirm the suitability of these ddPCR assays for routine detection and quantification of plant pathogens and for preparation of defined in-house reference materials with known target concentrations.

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.

Inhibition of the growth of colorado potato beetle larvae by macrocypins, protease inhibitors from the parasol mushroom.

Proteins from higher fungi have attracted interest because of their exceptional characteristics. Macrocypins, cysteine protease inhibitors from the parasol mushroom Macrolepiota procera , were evaluated for their adverse effects and their mode of action on the major potato pest Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). They were shown to reduce larval growth when expressed in potato or when their recombinant analogues were added to the diet. Macrocypins target a specific set of digestive cysteine proteases, intestains. Additionally, protein-protein interaction analysis revealed potential targets among other digestive enzymes and proteins related to development and primary metabolism. No effect of dietary macrocypins on gene expression of known adaptation-related digestive enzymes was observed in CPB guts. Macrocypins are the first fungal protease inhibitors to be reported as having a negative effect on growth and development of CPB larvae and could also be evaluated as control agents for other pests.

A complex of genes involved in adaptation of Leptinotarsa decemlineata larvae to induced potato defense.

The Colorado potato beetle (Leptinotarsa decemlineata) is the most important pest of potato in many areas of the world. One of the main reasons for its success lies in the ability of its larvae to counteract plant defense compounds. Larvae adapt to protease inhibitors (PIs) produced in potato leaves through substitution of inhibitor-sensitive digestive cysteine proteases with inhibitor-insensitive cysteine proteases. To get a broader insight into the basis of larval adaptation to plant defenses, we created a "suppression subtractive hybridisation" library using cDNA from the gut of L. decemlineata larvae fed methyl jasmonate-induced or uninduced potato leaves. Four hundred clones, randomly selected from the library, were screened for their relevance to adaptation with DNA microarray hybridizations. Selected enzyme systems of beetle digestion were further inspected for changes in gene expression using quantitative PCR and enzyme activity measurements. We identified two new groups of digestive cysteine proteases, intestains D and intestains E. Intestains D represent a group of structurally distinct digestive cysteine proteases, of which the tested members are strongly upregulated in response to induced plant defenses. Moreover, we found that other digestive enzymes also participate in adaptation, namely, cellulases, serine proteases, and an endopolygalacturonase. In addition, juvenile hormone binding protein-like (JHBP-like) genes were upregulated. All studied genes were expressed specifically in larval guts. In contrast to earlier studies that reported experiments based on PI-enriched artificial diets, our results increase understanding of insect adaptation under natural conditions.

The identification and interpretation of differences in the transcriptomes of organically and conventionally grown potato tubers.

In the European integrated research project SAFEFOODS, one of the aims was to further establish the potential of transcriptomics for the assessment of differences between plant varieties grown under different environmental conditions. Making use of the knowledge of cellular processes and interactions is one of the ways to obtain a better understanding of the differences found with transcriptomics. For the present study the potato genotype Santé was grown under both organic and conventional fertilizer, and each combined with either organic or conventional crop protection, giving four different treatments. Samples were derived from the European project QualityLowInputFood (QLIF). Microarray data were analyzed using different statistical tools (multivariate, principal components analysis (PCA); univariate, analysis of variance (ANOVA)) and with pathway analysis (hypergeometric distribution (HGD) and gene set enrichment analysis (GSEA)). Several biological processes were implicated as a result of the different treatments of the plants. Most obvious were the lipoxygenase pathway, with higher expression in organic fertilizer and lower expression in organic crop protection; the starch synthase pathway, with higher expression in both organic crop protection and fertilizer; and the biotic stress pathway, with higher expression in organic fertilizer. This study confirmed that gene expression profiling in combination with pathway analysis can identify and characterize differences between plants grown under different environmental conditions.

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.