Plant Signals Anticipate the Induction of the Type III Secretion System in Pseudomonas syringae pv. actinidiae, Facilitating Efficient Temperature-Dependent Effector Translocation.

Disease resistance in plants depends on a molecular dialogue with microbes that involves many known chemical effectors, but the time course of the interaction and the influence of the environment are largely unknown. The outcome of host-pathogen interactions is thought to reflect the offensive and defensive capabilities of both players. When plants interact with Pseudomonas syringae, several well-characterized virulence factors contribute to early bacterial pathogenicity, including the type III secretion system (T3SS), which must be activated by signals from the plant and environment to allow the secretion of virulence effectors. The manner in which these signals regulate T3SS activity is still unclear. Here, we strengthen the paradigm of the plant-pathogen molecular dialogue by addressing overlooked details concerning the timing of interactions, specifically the role of plant signals and temperature on the regulation of bacterial virulence during the first few hours of the interaction. Whole-genome expression profiling after 1 h revealed that the perception of plant signals from kiwifruit or tomato extracts anticipated T3SS expression in P. syringae pv. actinidiae compared to apoplast-like conditions, facilitating more efficient effector transport in planta, as revealed by the induction of a temperature-dependent hypersensitive response in the nonhost plant Arabidopsis thaliana Columbia-0 (Col-0). Our results show that in the arms race between plants and bacteria, the temperature-dependent timing of bacterial virulence versus the induction of plant defenses is probably one of the fundamental parameters governing the outcome of the interaction. IMPORTANCE Plant diseases-their occurrence and severity-result from the impact of three factors: the host, the pathogen, and the environmental conditions, interconnected in the disease triangle. Time was further included as a fourth factor accounting for plant disease, leading to a more realistic three-dimensional disease pyramid to represent the evolution of disease over time. However, this representation still considers time only as a parameter determining when and to what extent a disease will occur, at a scale from days to months. Here, we show that time is a factor regulating the arms race between plants and pathogens, at a scale from minutes to hours, and strictly depends on environmental factors. Thus, besides the arms possessed by pathogens and plants per se, the opportunity and the timing of arms mobilization make the difference in determining the outcome of an interaction and thus the occurrence of plant disease.

Host-specific signal perception by PsaR2 LuxR solo induces Pseudomonas syringae pv. actinidiae virulence traits.

Plant-associated bacteria, including pathogens, recognise host-derived signals to activate specific responses. The genome of Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of bacterial canker of kiwifruit, encodes for three putative LuxR-like receptors. Proteins of this family are usually involved in the quorum sensing system, through the perception of autoinducers (AHLs) produced by a cognate LuxI. However, Psa does not produce AHLs according to the lack of LuxI-encoding gene. It has been proposed that the so-called LuxR solos may be involved in the perception of environmental stimuli. We thus hypothesised that Psa LuxR-like receptors could be involved in host-derived signal sensing. Psa virulence traits, i.e., biofilm formation, motility and endophytic colonisation, were stimulated by growing the pathogen in host plant extracts, but not in non-host plant extracts or rich medium. Moreover, the phenotypic analyses of Psa mutant strains lacking the LuxR solo-encoding genes, demonstrated that PsaR2 plays a major role in host recognition and induction of virulence responses. The heterologous expression of PsaR2, followed by affinity chromatography and fraction activity assessment, confirmed the specific recognition of plant-derived components by this sensor. Overall, these data provide a deeper understanding of the regulation of Psa virulence through interkingdom communication, which represents a interesting target for the development of tolerant/resistant genotypes or innovative control strategies.

The Grapevine E3 Ubiquitin Ligase VriATL156 Confers Resistance against the Downy Mildew Pathogen Plasmopara viticola.

Downy mildew, caused by Plasmopara viticola, is one of the most severe diseases of grapevine (Vitis vinifera L.). Genetic resistance is an effective and sustainable control strategy, but major resistance genes (encoding receptors for specific pathogen effectors) introgressed from wild Vitis species, although effective, may be non-durable because the pathogen can evolve to avoid specific recognition. Previous transcriptomic studies in the resistant species Vitis riparia highlighted the activation of signal transduction components during infection. The transfer of such components to V. vinifera might confer less specific and therefore more durable resistance. Here, we describe the generation of transgenic V. vinifera lines constitutively expressing the V. riparia E3 ubiquitin ligase gene VriATL156. Phenotypic and molecular analysis revealed that the transgenic plants were less susceptible to P. viticola than vector-only controls, confirming the role of this E3 ubiquitin ligase in the innate immune response. Two independent transgenic lines were selected for detailed analysis of the resistance phenotype by RNA-Seq and microscopy, revealing the profound reprogramming of transcription to achieve resistance that operates from the earliest stages of pathogen infection. The introduction of VriATL156 into elite grapevine cultivars could therefore provide an effective and sustainable control measure against downy mildew.

Transcriptional Profiling of Three Pseudomonas syringae pv. actinidiae Biovars Reveals Different Responses to Apoplast-Like Conditions Related to Strain Virulence on the Host.

Pseudomonas syringae pv. actinidiae is a phytopathogen that causes devastating bacterial canker in kiwifruit. Among five biovars defined by genetic, biochemical, and virulence traits, P. syringae pv. actinidiae biovar 3 (Psa3) is the most aggressive and is responsible for the most recent reported outbreaks; however, the molecular basis of its heightened virulence is unclear. Therefore, we designed the first P. syringae multistrain whole-genome microarray, encompassing biovars Psa1, Psa2, and Psa3 and the well-established model P. syringae pv. tomato, and analyzed early bacterial responses to an apoplast-like minimal medium. Transcriptomic profiling revealed i) the strong activation in Psa3 of all hypersensitive reaction and pathogenicity (hrp) and hrp conserved (hrc) cluster genes, encoding components of the type III secretion system required for bacterial pathogenicity and involved in responses to environmental signals; ii) potential repression of the hrp/hrc cluster in Psa2; and iii) activation of flagellum-dependent cell motility and chemotaxis genes in Psa1. The detailed investigation of three gene families encoding upstream regulatory proteins (histidine kinases, their cognate response regulators, and proteins with diguanylate cyclase or phosphodiesterase domains) indicated that cyclic di-GMP may be a key regulator of virulence in P. syringae pv. actinidiae biovars. The gene expression data were supported by the quantification of biofilm formation. Our findings suggest that diverse early responses to the host apoplast, even among bacteria belonging to the same pathovar, can lead to different virulence strategies and may explain the differing outcomes of infections. Based on our detailed structural analysis of hrp operons, we also propose a revision of hrp cluster organization and operon regulation in P. syringae.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Activity and Mechanism of Action of the Bioceramic Silicon Nitride as an Environmentally Friendly Alternative for the Control of the Grapevine Downy Mildew Pathogen Plasmopara viticola.

Downy mildew of grapevine, caused by Plasmopara viticola (Berk. and Curt.) Berl. and de Toni, is one of the most devastating diseases of grapevine, severely affecting grape and wine production and quality worldwide. Infections are usually controlled by the intensive application of synthetic fungicides or by copper-based products in organic farming, rising problems for soil contamination and adverse impacts on environment and human health. While strict regulations attempt to minimize their harmful consequences, the situation calls for the development of alternative fungicidal strategies. This study presents the unprecedented case of a bioceramic, silicon nitride, with antimicrobial properties against P. viticola, but without adverse effects on human cells and environment, opening the way to the possible extension of silicon nitride applications in agriculture. Raman spectroscopic assessments of treated sporangia in conjunction with microscopic observations mechanistically showed that the nitrogen-chemistry of the bioceramic surface affects pathogen's biochemical components and cell viability, thus presenting a high potential for host protection from P. viticola infections.

N-Acyl Homoserine Lactones and Lux Solos Regulate Social Behaviour and Virulence of Pseudomonas syringae pv. actinidiae.

The phyllosphere is a complex environment where microbes communicate through signalling molecules in a system, generally known as quorum sensing (QS). One of the most common QS systems in Gram-negative proteobacteria is based on the production of N-acyl homoserine lactones (AHLs) by a LuxI synthase and their perception by a LuxR sensor. Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of the bacterial canker of kiwifruit, colonises plant phyllosphere before penetrating via wounds and natural openings. Since Psa genome encodes three LuxR solos without a cognate LuxI, this bacterium may perceive diffusible signals, but it cannot produce AHLs, displaying a non-canonical QS system. The elucidation of the mechanisms underlying the perception of environmental cues in the phyllosphere by this pathogen and their influence on the onset of pathogenesis are of crucial importance for a long-lasting and sustainable management of the bacterial canker of kiwifruit. Here, we report the ability of Psa to sense its own population density and the presence of surrounding bacteria. Moreover, we show that Psa can perceive AHLs, indicating that AHL-producing neighbouring bacteria may regulate Psa virulence in the host. Our results suggest that the ecological environment is important in determining Psa fitness and pathogenic potential. This opens new perspectives in the use of more advanced biochemical and microbiological tools for the control of bacterial canker of kiwifruit.

Inhibition of Virulence-Related Traits in Pseudomonas syringae pv. actinidiae by Gunpowder Green Tea Extracts.

Green tea is a widely-consumed healthy drink produced from the leaves of Camellia sinensis. It is renowned for its antioxidant and anticarcinogenic properties, but also displays significant antimicrobial activity against numerous human pathogens. Here we analyzed the antimicrobial activity of Gunpowder green tea against Pseudomonas syringae pv. actinidiae (Psa), the agent that causes kiwifruit bacterial canker. At the phenotypic level, tea extracts strongly inhibited Psa growth and swimming motility, suggesting it could reduce Psa epiphytic survival during plant colonization. The loss of bacterial virulence-related traits following treatment with tea extracts was also investigated by large-scale transcriptome analysis, which confirmed the in vitro phenotypes and revealed the induction of adaptive responses in the treated bacteria allowing them to cope with iron deficiency and oxidative stress. Such molecular changes may account for the ability of Gunpowder green tea to protect kiwifruit against Psa infection.

Plant and fungus transcriptomic data from grapevine berries undergoing artificially-induced noble rot caused by Botrytis cinerea.

Noble rot is a latent infection of grape berries caused by the necrotrophic fungus Botrytis cinerea, which develops under specific climatic conditions. The infected berries undergo biochemical and metabolic changes, associated with a rapid withering, which altogether offer interesting organoleptic features to sweet white wines. In this paper, we provide RNAseq datasets (raw and normalized counts as well as differentially expressed genes lists) of the transcriptome profiles of both grapevine berries (Vitis vinifera cv. Garganega) and B. cinerea during the establishment of noble rot, artificially induced in controlled conditions. The sequencing data are available in the NCBI GEO database under accession number GSE116741. These data were exploited in a comprehensive meta-analysis of gene expression during noble rot infection, gray mold and post-harvest withering. This highlighted an important common transcriptional reprogramming in different botrytized grape berry varieties and led to the identification of key genes specifically modulated during noble rot infection, which are described in the article entitled "Specific molecular interactions between Vitis vinifera and Botrytis cinerea are required for noble rot development in grape berries" Lovato et al., 2019.

Co-expression network analysis and cis-regulatory element enrichment determine putative functions and regulatory mechanisms of grapevine ATL E3 ubiquitin ligases.

Arabidopsis thaliana Toxicos en Levadura (ATL) proteins are a subclass of the RING-H2 zinc finger binding E3 ubiquitin ligases. The grapevine (Vitis vinifera) ATL family was recently characterized, revealing 96 members that are likely to be involved in several physiological processes through protein ubiquitination. However, the final targets and biological functions of most ATL E3 ligases are still unknown. We analyzed the co-expression networks among grapevine ATL genes across a set of transcriptomic data related to defense and abiotic stress, combined with a condition-independent dataset. This revealed strong correlations between ATL proteins and diverse signal transduction components and transcriptional regulators, in particular those involved in immunity. An enrichment analysis of cis-regulatory elements in ATL gene promoters and related co-expressed genes highlighted the importance of hormones in the regulation of ATL gene expression. Our work identified several ATL proteins as candidates for further studies aiming to decipher specific grapevine resistance mechanisms activated in response to pathogens.

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine.

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the prediction of family functions based on several features, such as the presence of sequence motifs or of particular sites for post-translational modification and the expression profile of family members in different conditions. This work describes a detailed protocol for gene family characterization. Here, the procedure is applied to the characterization of the Arabidopsis Tóxicos in Levadura (ATL) E3 ubiquitin ligase family in grapevine. The methods include the genome-wide identification of family members, the characterization of gene localization, structure, and duplication, the analysis of conserved protein motifs, the prediction of protein localization and phosphorylation sites as well as gene expression profiling across the family in different datasets. Such procedure, which could be extended to further analyses depending on experimental purposes, could be applied to any gene family in any plant species for which genomic data are available, and it provides valuable information to identify interesting candidates for functional studies, giving insights into the molecular mechanisms of plant adaptation to their environment.

The Induction of Noble Rot (Botrytis cinerea) Infection during Postharvest Withering Changes the Metabolome of Grapevine Berries (Vitis vinifera L., cv. Garganega).

The natural or induced development of noble rot caused by the fungus Botrytis cinerea during the late stages of grapevine (Vitis vinifera L.) berry ripening is used in some traditional viticulture areas to produce high-quality wines such as Sauternes and Tokaji. In this research, we wanted to verify if by changing the environmental conditions during post-harvest withering we could induce the noble rot development on harvested berries in order to positively change the wine produced from withered Garganega berries. Therefore, we exposed the berries to postharvest withering under normal or artificially humid conditions, the latter to induce noble rot. The presence of noble rot symptoms was associated with the development of B. cinerea in the berries maintained under humid conditions. The composition of infected and non-infected berries was investigated by untargeted metabolomics using liquid chromatography/mass spectrometry. We also explored the effects of the two withering methods on the abundance of volatile organic compounds in wine by yeast-inoculated micro-fermentation followed by targeted gas chromatography/mass spectrometry. These experiments revealed significant metabolic differences between berries withered under normal and humid conditions, indicating that noble rot affects berry metabolism and composition. As well as well-known botrytization markers, we detected two novel lipids that have not been observed before in berries infected with noble rot. Unraveling the specific metabolic profile of berries infected with noble rot may help to determine the compounds responsible for the organoleptic quality traits of botrytized Garganega wines.

Application of Chemical Genomics to Plant-Bacteria Communication: A High-Throughput System to Identify Novel Molecules Modulating the Induction of Bacterial Virulence Genes by Plant Signals.

The life cycle of bacterial phytopathogens consists of a benign epiphytic phase, during which the bacteria grow in the soil or on the plant surface, and a virulent endophytic phase involving the penetration of host defenses and the colonization of plant tissues. Innovative strategies are urgently required to integrate copper treatments that control the epiphytic phase with complementary tools that control the virulent endophytic phase, thus reducing the quantity of chemicals applied to economically and ecologically acceptable levels. Such strategies include targeted treatments that weaken bacterial pathogens, particularly those inhibiting early infection steps rather than tackling established infections. This chapter describes a reporter gene-based chemical genomic high-throughput screen for the induction of bacterial virulence by plant molecules. Specifically, we describe a chemical genomic screening method to identify agonist and antagonist molecules for the induction of targeted bacterial virulence genes by plant extracts, focusing on the experimental controls required to avoid false positives and thus ensuring the results are reliable and reproducible.

Genome-wide characterisation and expression profile of the grapevine ATL ubiquitin ligase family reveal biotic and abiotic stress-responsive and development-related members.

The Arabidopsis Tóxicos en Levadura (ATL) protein family is a class of E3 ubiquitin ligases with a characteristic RING-H2 Zn-finger structure that mediates diverse physiological processes and stress responses in plants. We carried out a genome-wide survey of grapevine (Vitis vinifera L.) ATL genes and retrieved 96 sequences containing the canonical ATL RING-H2 domain. We analysed their genomic organisation, gene structure and evolution, protein domains and phylogenetic relationships. Clustering revealed several clades, as already reported in Arabidopsis thaliana and rice (Oryza sativa), with an expanded subgroup of grapevine-specific genes. Most of the grapevine ATL genes lacked introns and were scattered among the 19 chromosomes, with a high level of duplication retention. Expression profiling revealed that some ATL genes are expressed specifically during early or late development and may participate in the juvenile to mature plant transition, whereas others may play a role in pathogen and/or abiotic stress responses, making them key candidates for further functional analysis. Our data offer the first genome-wide overview and annotation of the grapevine ATL family, and provide a basis for investigating the roles of specific family members in grapevine physiology and stress responses, as well as potential biotechnological applications.

Grapevine Downy Mildew Plasmopara viticola Infection Elicits the Expression of Allergenic Pathogenesis-Related Proteins.

BACKGROUND: Downy mildews are a group of microorganisms belonging to the Chromista kingdom that can infect specific plants. When growing on plant tissues these microbes can elicit the expression of pathogenesis-related proteins (PRs), a group of stress-induced proteins frequently described as allergens in many plant species. Our aim was to verify by a proteomic approach whether the allergic reactions experienced by a farmer working in a vineyard infected by Plasmopara viticola (Pv), the etiological agent of downy mildew, are elicited by PRs expressed by the grapevine upon infection or by allergens present in Pv. METHODS: A skin prick test and prick-to-prick test with infected field grapevine leaves and control leaves were carried out. Field leaves and ad hoc Pv-inoculated leaves were compared by SDS-PAGE and IgE-immunoblotting with extracts from control leaves and Pv sporangia. IgE-binding proteins were further separated by two-dimensional electrophoresis and the positive spots analyzed by nanoHPLC-Chip and tandem mass spectrometry (MS/MS) for identification. RESULTS: Only infected leaves showed IgE-binding protein bands at 42 and 36 kDa. This agreed with the positive skin prick test experienced by the patient only with the infected leaves extract. Two-dimensional electrophoresis followed by MS/MS analysis led to the identification of PR-2 (ß-1,3-glucanase) and harpin-binding protein 1 as putative allergens, the latter having never been reported before. CONCLUSION: The results indicate that Pv infection might represent a new source of plant allergens.

Construction of a synthetic infectious cDNA clone of Grapevine Algerian latent virus (GALV-Nf) and its biological activity in Nicotiana benthamiana and grapevine plants.

BACKGROUND: Grapevine Algerian latent virus (GALV) is a tombusvirus first isolated in 1989 from an Algerian grapevine (Vitis spp.) plant and more recently from water samples and commercial nipplefruit and statice plants. No further reports of natural GALV infections in grapevine have been published in the last two decades, and artificial inoculations of grapevine plants have not been reported. We developed and tested a synthetic GALV construct for the inoculation of Nicotiana benthamiana plants and different grapevine genotypes to investigate the ability of this virus to infect and spread systemically in different hosts. METHODS: We carried out a phylogenetic analysis of all known GALV sequences and an epidemiological survey of grapevine samples to detect the virus. A GALV-Nf clone under the control of the T7 promoter was chemically synthesized based on the full-length sequence of the nipplefruit isolate GALV-Nf, the only available sequence at the time the project was conceived, and the infectious transcripts were tested in N. benthamiana plants. A GALV-Nf-based binary vector was then developed for the agroinoculation of N. benthamiana and grapevine plants. Infections were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in both species. RESULTS: Sequence analysis showed that the GALV coat protein is highly conserved among diverse isolates. The first epidemiological survey of cDNAs collected from 152 grapevine plants with virus-like symptoms did not reveal the presence of GALV in any of the samples. The agroinoculation of N. benthamiana and grapevine plants with the GALV-Nf binary vector promoted efficient infections, as revealed by serological and molecular analysis. The GALV-Nf infection of grapevine plants was characterized in more detail by inoculating different cultivars, revealing distinct patterns of symptom development. Ultrastructural changes induced by GALV-Nf in N. benthamiana were similar to those induced by tombusviruses in other hosts, but the cytopathological alterations in grapevine plants were less severe. CONCLUSIONS: This is the first report describing the development of a synthetic GALV-Nf cDNA clone, its artificial transmission to grapevine plants and the resulting symptoms and cytopathological alterations.

Genome-wide identification and analysis of mitogen activated protein kinase kinase kinase gene family in grapevine (Vitis vinifera).

BACKGROUND: Mitogen-activated protein kinase kinase kinases (MAPKKKs; MAP3Ks) are important components of MAPK cascades, which are highly conserved signal transduction pathways in animals, yeast and plants, play important roles in plant growth and development. MAPKKKs have been investigated on their evolution and expression patterns in limited plants including Arabidopsis, rice and maize. RESULTS: In this study, we performed a genome-wide survey and identified 45 MAPKKK genes in the grapevine genome. Chromosome location, phylogeny, gene structure and conserved protein motifs of MAPKKK family in grapevine have been analyzed to support the prediction of these genes. In the phylogenetic analysis, MAPKKK genes of grapevine have been classified into three subgroups as described for Arabidopsis, named MEKK, ZIK and RAF, also confirmed in grapevine by the analysis of conserved motifs and exon-intron organizations. By analyzing expression profiles of MAPKKK genes in grapevine microarray databases, we highlighted the modulation of different MAPKKKs in different organs and distinct developmental stages. Furthermore, we experimentally investigated the expression profiles of 45 grape MAPKKK genes in response to biotic (powdery mildew) and abiotic stress (drought), as well as to hormone (salicylic acid, ethylene) and hydrogen peroxide treatments, and identified several candidate MAPKKK genes that might play an important role in biotic and abiotic responses in grapevine, for further functional characterization. CONCLUSIONS: This is the first comprehensive experimental survey of the grapevine MAPKKK gene family, which provides insights into their potential roles in regulating responses to biotic and abiotic stresses, and the evolutionary expansion of MAPKKKs is associated with the diverse requirement in transducing external and internal signals into intracellular actions in MAPK cascade in grapevine.

Investigating topic models' capabilities in expression microarray data classification.

In recent years a particular class of probabilistic graphical models-called topic models-has proven to represent an useful and interpretable tool for understanding and mining microarray data. In this context, such models have been almost only applied in the clustering scenario, whereas the classification task has been disregarded by researchers. In this paper, we thoroughly investigate the use of topic models for classification of microarray data, starting from ideas proposed in other fields (e.g., computer vision). A classification scheme is proposed, based on highly interpretable features extracted from topic models, resulting in a hybrid generative-discriminative approach; an extensive experimental evaluation, involving 10 different literature benchmarks, confirms the suitability of the topic models for classifying expression microarray data.

Proteomic analysis of the compatible interaction between Vitis vinifera and Plasmopara viticola.

We analyzed the proteome of grapevine (Vitis vinifera) leaves 24, 48 and 96 h post infection (hpi) with the downy mildew pathogen Plasmopara viticola. Total proteins were separated on 2-DE gels. By MS analysis, we identified 82 unique grapevine proteins differentially expressed after infection. Upregulated proteins were often included in the functional categories of general metabolism and stress response, while proteins related to photosynthesis and energy production were mostly downregulated. As expected, the activation of a defense reaction was observed more often at the late time point, consistent with the establishment of a compatible interaction. Most proteins involved in resistance were isoforms of different PR-10 pathogenesis-related proteins. Although >50 differentially expressed protein isoforms were observed at 24 and 96 hpi, only 18 were detected at 48 hpi and no defense-related proteins were among this group. This profile suggests a transient breakdown in defense responses accompanying the onset of disease, further supported by gene expression analyses and by a western blot analysis of a PR-10 protein. Our data reveal the complex modulation of plant metabolism and defense responses during compatible interactions, and provide insight into the underlying molecular processes which may eventually yield novel strategies for pathogen control in the field.

Distinct colonization patterns and cDNA-AFLP transcriptome profiles in compatible and incompatible interactions between melon and different races of Fusarium oxysporum f. sp. melonis.

BACKGROUND: Fusarium oxysporum f. sp. melonis Snyd. & Hans. (FOM) causes Fusarium wilt, the most important infectious disease of melon (Cucumis melo L.). The four known races of this pathogen can be distinguished only by infection on appropriate cultivars. No molecular tools are available that can discriminate among the races, and the molecular basis of compatibility and disease progression are poorly understood. Resistance to races 1 and 2 is controlled by a single dominant gene, whereas only partial polygenic resistance to race 1,2 has been described. We carried out a large-scale cDNA-AFLP analysis to identify host genes potentially related to resistance and susceptibility as well as fungal genes associated with the infection process. At the same time, a systematic reisolation procedure on infected stems allowed us to monitor fungal colonization in compatible and incompatible host-pathogen combinations. RESULTS: Melon plants (cv. Charentais Fom-2), which are susceptible to race 1,2 and resistant to race 1, were artificially infected with a race 1 strain of FOM or one of two race 1,2 w strains. Host colonization of stems was assessed at 1, 2, 4, 8, 14, 16, 18 and 21 days post inoculation (dpi), and the fungus was reisolated from infected plants. Markedly different colonization patterns were observed in compatible and incompatible host-pathogen combinations. Five time points from the symptomless early stage (2 dpi) to obvious wilting symptoms (21 dpi) were considered for cDNA-AFLP analysis. After successful sequencing of 627 transcript-derived fragments (TDFs) differentially expressed in infected plants, homology searching retrieved 305 melon transcripts, 195 FOM transcripts expressed in planta and 127 orphan TDFs. RNA samples from FOM colonies of the three strains grown in vitro were also included in the analysis to facilitate the detection of in planta-specific transcripts and to identify TDFs differentially expressed among races/strains. CONCLUSION: Our data suggest that resistance against FOM in melon involves only limited transcriptional changes, and that wilting symptoms could derive, at least partially, from an active plant response.We discuss the pathogen-derived transcripts expressed in planta during the infection process and potentially related to virulence functions, as well as transcripts that are differentially expressed between the two FOM races grown in vitro. These transcripts provide candidate sequences that can be further tested for their ability to distinguish between races.Sequence data from this article have been deposited in GenBank, Accession Numbers: HO867279-HO867981.

General and species-specific transcriptional responses to downy mildew infection in a susceptible (Vitis vinifera) and a resistant (V. riparia) grapevine species.

BACKGROUND: Downy mildew is a destructive grapevine disease caused by Plasmopara viticola (Berk. and Curt.) Berl. and de Toni, which can only be controlled by intensive fungicide treatments. Natural sources of resistance from wild grapevine (Vitis) species are used in conventional breeding approaches, but the signals and effectors involved in resistance in this important crop species are not well understood. RESULTS: Early transcriptional changes associated with P. viticola infection in susceptible V. vinifera and resistant V. riparia plants were analyzed using the Combimatrix microarray platform. Transcript levels were measured 12 and 24 h post-inoculation, reflecting the time points immediately preceding the onset of resistance in V. riparia, as determined by microscopic analysis. Our data indicate that resistance in V. riparia is induced after infection, and is not based on differences in basal gene expression between the two species. The strong and rapid transcriptional reprogramming involves the induction of pathogenesis-related proteins and enzymes required for the synthesis of phenylpropanoid-derived compounds, many of which are also induced, albeit to a lesser extent, in V. vinifera. More interestingly, resistance in V. riparia also involves the specific modulation of numerous transcripts encoding components of signal transduction cascades, hypersensitive reaction markers and genes involved in jasmonate biosynthesis. The limited transcriptional modulation in V. vinifera represents a weak attempted defense response rather than the activation of compatibility-specific pathways. CONCLUSIONS: Several candidate resistance genes were identified that could be exploited in future biotechnological approaches to increase disease resistance in susceptible grapevine species. Measurements of jasmonic acid and methyl jasmonate in infected leaves suggest that this hormone may also be involved in V. riparia resistance to P. viticola.