Assessing lethal and sublethal effects of pesticides on honey bees in a multifactorial context.

The registration of novel pesticides that are subsequently banned because of their unexpected negative effects on non-target species can have a huge environmental impact. Therefore, the pre-emptive evaluation of the potential effects of new compounds is essential. To this aim both lethal and sublethal effects should be assessed in a realistic scenario including the other stressors that can interact with pesticides. However, laboratory studies addressing such interactive effects are rare, while standardized laboratory-based protocols focus on lethal effects and not on sub-lethal effects. We propose to assess both lethal and sublethal effects in a multifactorial context including the other stressors affecting the non-target species. We tested this approach by studying the impact on honey bees of the insecticide sulfoxaflor in combination with a common parasite, a sub-optimal temperature and food deprivation. We studied the survival and the transcriptome of honey bees, to assess both the lethal and the potential sublethal effects of the insecticide, respectively. With this method we show that a field realistic concentration of sulfoxaflor in food does not affect the survival of honey bees; however, the significant impact on some key genes indicates that sublethal effects are possible in a realistically complex scenario. Moreover, our results demonstrate the feasibility and reliability of a novel approach to hazard assessment considering the interactive effects of pesticides. We anticipate our approach to be a starting point for a paradigm shift in toxicology: from an unifactorial, mortality-centered assessment to a multifactorial, comprehensive approach. This is something of the utmost importance to preserve pollination, thus contributing to the sustainability of our food production system.

An integrated approach to explore the microbial biodiversity of natural milk cultures for cheesemaking.

The use of natural milk culture (NMC) represents a key factor in Protected Designation of Origin (PDO) Montasio cheese, contributing to its distinctive sensory profile. The complex microbial ecosystem of NMC is the result of heat treatment and incubation conditions, which can vary considerably among different production plants. In this study, the microbiota of NMC collected from 10 PDO Montasio cheese dairies was investigated by employing colony counts and metagenomic analysis. Furthermore, residual sugars, organic acids, and volatile profiles were quantitatively investigated. Results showed that Streptococcus thermophilus was the dominant species in all NMC, and a subdominant population made of other streptococci and Ligilactobacillus salivarius was also present. The incubation temperature appeared to be the main driver of biodiversity in NMC. Metagenomics allowed us to evidence the presence of minor species involving safety (e.g., Staphylococcus aureus) as well as possible functional aspects (Next Generation Probiotics). Statistical analysis based on residual sugars, organic acids, and volatiles' content allowed to correlate the presence of specific microbial groups with metabolites of great technological and sensory relevance, which can contribute to giving value to the artisanal production procedures of NMC and clarify their role in the creation of the characteristics of PDO Montasio cheese.

Nitrogen nutrition and xylem sap composition in Zea mays: effect of urea, ammonium and nitrate on ionomic and metabolic profiles.

In plants the communication between organs is mainly carried out via the xylem and phloem. The concentration and the molecular species of some phytohormones, assimilates and inorganic ions that are translocated in the xylem vessel play a key role in the systemic nutritional signaling in plants. In this work the composition of the xylem sap of maize was investigated at the metabolic and ionomic level depending on the N form available in the nutrient solution. Plants were grown up to 7 days in hydroponic system under N-free nutrient solution or nutrient solution containing N in form of nitrate, urea, ammonium or a combination of urea and ammonium. For the first time this work provides evidence that the ureic nutrition reduced the water translocation in maize plants more than mineral N forms. This result correlates with those obtained from the analyses of photosynthetic parameters (stomatal conductance and transpiration rate) suggesting a parsimonious use of water by maize plants under urea nutrition. A peculiar composition in amino acids and phytohormones (i.e. S, Gln, Pro, ABA) of the xylem sap under urea nutrition could explain differences in xylem sap exudation in comparison to plants treated with mineral N forms. The knowledge improvement of urea nutrition will allow to further perform good agronomic strategies to improve the resilience of maize crop to water stress.

Age-related response to mite parasitization and viral infection in the honey bee suggests a trade-off between growth and immunity.

Host age at parasites' exposure is often neglected in studies on host-parasite interactions despite the important implications for epidemiology. Here we compared the impact of the parasitic mite Varroa destructor, and the associated pathogenic virus DWV on different life stages of their host, the western honey bee Apis mellifera. The pre-imaginal stages of the honey bee proved to be more susceptible to mite parasitization and viral infection than adults. The higher viral load in mite-infested bees and DWV genotype do not appear to be the drivers of the observed difference which, instead, seems to be related to the immune-competence of the host. These results support the existence of a trade-off between immunity and growth, making the pupa, which is involved in the highly energy-demanding process of metamorphosis, more susceptible to parasites and pathogens. This may have important implications for the evolution of the parasite's virulence and in turn for honey bee health. Our results highlight the important role of host's age and life stage at exposure in epidemiological modelling. Furthermore, our study could unravel new aspects of the complex honey bee-Varroa relationship to be addressed for a sustainable management of this parasite.

Epidemiology of a major honey bee pathogen, deformed wing virus: potential worldwide replacement of genotype A by genotype B.

The western honey bee (Apis mellifera) is of major economic and ecological importance, with elevated rates of colony losses in temperate regions over the last two decades thought to be largely caused by the exotic ectoparasitic mite Varroa destructor and deformed wing virus (DWV), which the mite transmits. DWV currently exists as two main genotypes: the formerly widespread DWV-A and the more recently described and rapidly expanding DWV-B. It is an excellent system to understand viral evolution and the replacement of one viral variant by another. Here we synthesise published results on the distribution and prevalence of DWV-A and -B over the period 2008-2021 and present novel data for Germany, Italy and the UK to suggest that (i) DWV-B has rapidly expanded worldwide since its first description in 2004 and (ii) that it is potentially replacing DWV-A. Both genotypes are also found in wild bee species. Based on a simple mathematical model, we suggest that interference between viral genotypes when co-infecting the same host is key to understanding their epidemiology. We finally discuss the consequences of genotype replacement for beekeeping and for wild pollinator species.

Infection by phloem-limited phytoplasma affects mineral nutrient homeostasis in tomato leaf tissues.

Phytoplasmas are sieve-elements restricted wall-less, pleomorphic pathogenic microorganisms causing devastating damage to over 700 plant species worldwide. The invasion of sieve elements by phytoplasmas has several consequences on nutrient transport and metabolism, anyway studies about changes of the mineral-nutrient profile following phytoplasma infections are scarce and offer contrasting results. Here, we examined changes in macro- and micronutrient concentration in tomato plant upon 'Candidatus Phytoplasma solani' infection. To investigate possible effects of 'Ca. P. solani' infection on mineral element allocation, the mineral elements were separately analysed in leaf midrib, leaf lamina and root. Moreover, we focused our analysis on the transcriptional regulation of genes encoding trans-membrane transporters of mineral nutrients. To this aim, a manually curated inventory of differentially expressed genes encoding transporters in tomato leaf midribs was mined from the transcriptional profile of healthy and infected tomato leaf midribs. Results highlighted changes in ion homeostasis in the host plant, and significant modulations at transcriptional level of genes encoding ion transporters and channels.

Systemic effects of Tuber melanosporum inoculation in two Corylus avellana genotypes.

Roots of the European hazelnut (Corylus avellana L.), i.e., one of the most economically important nut species, form symbiosis with ectomycorrhizal (ECM) fungi, including truffles. Although physical interactions only occur in roots, the presence of mycorrhizal fungi can lead to metabolic changes at a systemic level, i.e., in leaves. However, how root colonization by ECM fungi modifies these processes in the host plant has so far not been widely studied. This work aimed to investigate the response in two C. avellana genotypes, focusing on leaves from plants inoculated with the black truffle Tuber melanosporum Vittad. Transcriptomic profiles of leaves of colonized plants were compared with those of non-colonized plants, as well as sugar and polyphenolic content. Results suggested that T. melanosporum has the potential to support plants in stressed conditions, leading to the systemic regulation of several genes involved in signaling and defense responses. Although further confirmation is needed, our results open new perspectives for future research aimed to highlight novel aspects in ECM symbiosis.

ddRAD-seq reveals the genetic structure and detects signals of selection in Italian brown trout.

BACKGROUND: Brown trout is one of the most widespread fresh-water fish species in Europe. The evolutionary history of and phylogenetic relationships between brown trout populations are complex, and this is especially true for Italian populations, which are heavily influenced in different ways by stocking practices. The characterization of the genetic structure of Italian brown trout populations may give information on the risk of losing endemic Italian populations due to lack of genetic diversity or to admixture with stocking populations. The identification of signatures of selection, and the information deriving from dense genotyping data will help genotype-informed breeding programs. We used a ddRAD-seq approach to obtain more than 100,000 single nucleotide polymorphisms (SNPs), and to characterize the population structure and signatures of selection in 90 brown trout samples. RESULTS: Italian brown trout populations are genetically differentiated, although the stocking practices have introduced strong admixture in endemic Italian trout, especially with the Atlantic lineage. Most of the analysed populations showed high levels of kinship and inbreeding. We detected putative signatures of selection using different approaches, and investigated if the regions were enriched for functional categories. Several regions putatively under selection and characterized by a reduction in heterozygosity across all the studied populations are enriched for genes involved in the response to viral infections. CONCLUSIONS: Our results, which show evidence of admixture with the Atlantic lineage (commonly used for stocking), confirm the need for controlling stocking practices, in order to avoid the erosion of the endemic gene pool; given the apparently high levels of kinship and inbreeding in local populations, our results also show the need to take action for increasing gene diversity. In addition, we used the genetically-distinct lineages to detect signatures of selection and we identified putative signatures of selection in several regions associated with resistance to infectious diseases. These constitute candidate regions for the study of resistance to infections in wild and farmed trout.

Evaluation of sensitivity and specificity in RNA-Seq-based detection of grapevine viral pathogens.

Virus detection is a crucial step for the implementation of clean stock programs that preserve healthy crop species. Viral infections in grapevine, a vegetatively propagated perennial crop, cannot be eradicated from the vineyards by the application of agrochemicals and must be curtailed at the stage of nursery production during the propagation of planting material. Viral detection is routinely performed using enzyme-linked immunosorbent assays (ELISA) or Reverse Transcription-quantitative Polymerase Chain Reactions (RT-qPCR). High throughput sequencing (HTS) approaches have the potential to detect all viral pathogens in a plant specimen. However, to date, no published HTS-based study has used threshold selection based on ROC curves for discriminating positive from negative samples. To fill this gap, we assessed the specificity and sensitivity of different sequencing and bioinformatics approaches for nine common viruses, which were tested in the same specimens using ELISA and/or RT-qPCR. The normalized detection thresholds giving the best results were 19.28 Fragments Per Kilobase of transcript per Million mapped reads (FPKM) for alignment-based total RNA-Seq approaches, 386 Reads Per Million mapped reads (RPM) for metagenomics-based total RNA-Seq, 1572 FPKM for alignment-based small RNA-Seq analysis and 0.97 % of contigs for de novo analysis of small RNA-Seq data. Validation of the proposed thresholds using independent specimens collected over time from the same stocks and other specimens collected from nearby stocks that had derived from the same propagating material showed that HTS approaches are accurate, with RNA-Seq approaches showing better performance than small RNA-Seq.

The genomes of 204 Vitis vinifera accessions reveal the origin of European wine grapes.

In order to elucidate the still controversial processes that originated European wine grapes from its wild progenitor, here we analyse 204 genomes of Vitis vinifera and show that all analyses support a single domestication event that occurred in Western Asia and was followed by numerous and pervasive introgressions from European wild populations. This admixture generated the so-called international wine grapes that have diffused from Alpine countries worldwide. Across Europe, marked differences in genomic diversity are observed in local varieties that are traditionally cultivated in different wine producing countries, with Italy and France showing the largest diversity. Three genomic regions of reduced genetic diversity are observed, presumably as a consequence of artificial selection. In the lowest diversity region, two candidate genes that gained berry-specific expression in domesticated varieties may contribute to the change in berry size and morphology that makes the fruit attractive for human consumption and adapted for winemaking.

Power calculator for detecting allelic imbalance using hierarchical Bayesian model.

OBJECTIVE: Allelic imbalance (AI) is the differential expression of the two alleles in a diploid. AI can vary between tissues, treatments, and environments. Methods for testing AI exist, but methods are needed to estimate type I error and power for detecting AI and difference of AI between conditions. As the costs of the technology plummet, what is more important: reads or replicates? RESULTS: We find that a minimum of 2400, 480, and 240 allele specific reads divided equally among 12, 5, and 3 replicates is needed to detect a 10, 20, and 30%, respectively, deviation from allelic balance in a condition with power > 80%. A minimum of 960 and 240 allele specific reads divided equally among 8 replicates is needed to detect a 20 or 30% difference in AI between conditions with comparable power. Higher numbers of replicates increase power more than adding coverage without affecting type I error. We provide a Python package that enables simulation of AI scenarios and enables individuals to estimate type I error and power in detecting AI and differences in AI between conditions.

Open chromatin in grapevine marks candidate CREs and with other chromatin features correlates with gene expression.

Vitis vinifera is an economically important crop and a useful model in which to study chromatin dynamics. In contrast to the small and relatively simple genome of Arabidopsis thaliana, grapevine contains a complex genome of 487 Mb that exhibits extensive colonization by transposable elements. We used Hi-C, ChIP-seq and ATAC-seq to measure how chromatin features correlate to the expression of 31 845 grapevine genes. ATAC-seq revealed the presence of more than 16 000 open chromatin regions, of which we characterize nearly 5000 as possible distal enhancer candidates that occur in intergenic space > 2 kb from the nearest transcription start site (TSS). A motif search identified more than 480 transcription factor (TF) binding sites in these regions, with those for TCP family proteins in greatest abundance. These open chromatin regions are typically within 15 kb from their nearest promoter, and a gene ontology analysis indicated that their nearest genes are significantly enriched for TF activity. The presence of a candidate cis-regulatory element (cCRE) > 2 kb upstream of the TSS, location in the active nuclear compartment as determined by Hi-C, and the enrichment of H3K4me3, H3K4me1 and H3K27ac at the gene are correlated with gene expression. Taken together, these results suggest that regions of intergenic open chromatin identified by ATAC-seq can be considered potential candidates for cis-regulatory regions in V. vinifera. Our findings enhance the characterization of a valuable agricultural crop, and help to clarify the understanding of unique plant biology.

Transcriptomic and metabolomic profiles of Zea mays fed with urea and ammonium.

The simultaneous presence of different N-forms in the rhizosphere leads to beneficial effects on nitrogen (N) nutrition in plants. Although widely used as fertilizers, the occurrence of cross connection between urea and ammonium nutrition has been scarcely studied in plants. Maize fed with a mixture of urea and ammonium displayed a better N-uptake efficiency than ammonium- or urea-fed plants (Buoso et al., Plant Physiol Biochem, 2021a; 162: 613-623). Through multiomic approaches, we provide the molecular characterization of maize response to urea and ammonium nutrition. Several transporters and enzymes involved in N-nutrition were upregulated by all three N-treatments (urea, ammonium, or urea and ammonium). Already after 1 day of treatment, the availability of different N-forms induced specific transcriptomic and metabolomic responses. The combination of urea and ammonium induced a prompt assimilation of N, characterized by high levels of some amino acids in shoots. Moreover, ZmAMT1.1a, ZmGLN1;2, ZmGLN1;5, ZmGOT1, and ZmGOT3, as well transcripts involved in glycolysis-TCA cycle were induced in roots by urea and ammonium mixture. Depending on N-form, even changes in the composition of phytohormones were observed in maize. This study paves the way to formulate guidelines for the optimization of N fertilization to improve N-use efficiency in maize and therefore limit N-losses in the environment.

A forward genetics approach integrating genome-wide association study and expression quantitative trait locus mapping to dissect leaf development in maize (Zea mays).

The characterization of the genetic basis of maize (Zea mays) leaf development may support breeding efforts to obtain plants with higher vigor and productivity. In this study, a mapping panel of 197 biparental and multiparental maize recombinant inbred lines (RILs) was analyzed for multiple leaf traits at the seedling stage. RNA sequencing was used to estimate the transcription levels of 29 573 gene models in RILs and to derive 373 769 single nucleotide polymorphisms (SNPs), and a forward genetics approach combining these data was used to pinpoint candidate genes involved in leaf development. First, leaf traits were correlated with gene expression levels to identify transcript-trait correlations. Then, leaf traits were associated with SNPs in a genome-wide association (GWA) study. An expression quantitative trait locus mapping approach was followed to associate SNPs with gene expression levels, prioritizing candidate genes identified based on transcript-trait correlations and GWAs. Finally, a network analysis was conducted to cluster all transcripts in 38 co-expression modules. By integrating forward genetics approaches, we identified 25 candidate genes highly enriched for specific functional categories, providing evidence supporting the role of vacuolar proton pumps, cell wall effectors, and vesicular traffic controllers in leaf growth. These results tackle the complexity of leaf trait determination and may support precision breeding in maize.

Testcrosses are an efficient strategy for identifying cis-regulatory variation: Bayesian analysis of allele-specific expression (BayesASE).

Allelic imbalance (AI) occurs when alleles in a diploid individual are differentially expressed and indicates cis acting regulatory variation. What is the distribution of allelic effects in a natural population? Are all alleles the same? Are all alleles distinct? The approach described applies to any technology generating allele-specific sequence counts, for example for chromatin accessibility and can be applied generally including to comparisons between tissues or environments for the same genotype. Tests of allelic effect are generally performed by crossing individuals and comparing expression between alleles directly in the F1. However, a crossing scheme that compares alleles pairwise is a prohibitive cost for more than a handful of alleles as the number of crosses is at least (n2-n)/2 where n is the number of alleles. We show here that a testcross design followed by a hypothesis test of AI between testcrosses can be used to infer differences between nontester alleles, allowing n alleles to be compared with n crosses. Using a mouse data set where both testcrosses and direct comparisons have been performed, we show that the predicted differences between nontester alleles are validated at levels of over 90% when a parent-of-origin effect is present and of 60%-80% overall. Power considerations for a testcross, are similar to those in a reciprocal cross. In all applications, the testing for AI involves several complex bioinformatics steps. BayesASE is a complete bioinformatics pipeline that incorporates state-of-the-art error reduction techniques and a flexible Bayesian approach to estimating AI and formally comparing levels of AI between conditions. The modular structure of BayesASE has been packaged in Galaxy, made available in Nextflow and as a collection of scripts for the SLURM workload manager on github (https://github.com/McIntyre-Lab/BayesASE).

A Transcriptomic Approach Provides Insights on the Mycorrhizal Symbiosis of the Mediterranean Orchid Limodorum abortivum in Nature.

The study of orchid mycorrhizal interactions is particularly complex because of the peculiar life cycle of these plants and their diverse trophic strategies. Here, transcriptomics has been applied to investigate gene expression in the mycorrhizal roots of Limodorum abortivum, a terrestrial mixotrophic orchid that associates with ectomycorrhizal fungi in the genus Russula. Our results provide new insights into the mechanisms underlying plant-fungus interactions in adult orchids in nature and in particular into the plant responses to the mycorrhizal symbiont(s) in the roots of mixotrophic orchids. Our results indicate that amino acids may represent the main nitrogen source in mycorrhizal roots of L. abortivum, as already suggested for orchid protocorms and other orchid species. The upregulation, in mycorrhizal L. abortivum roots, of some symbiotic molecular marker genes identified in mycorrhizal roots from other orchids as well as in arbuscular mycorrhiza, may mirror a common core of plant genes involved in endomycorrhizal symbioses. Further efforts will be required to understand whether the specificities of orchid mycorrhiza depend on fine-tuned regulation of these common components, or whether specific additional genes are involved.

Transcriptome Analysis of Amyloodinium ocellatum Tomonts Revealed Basic Information on the Major Potential Virulence Factors.

The ectoparasite protozoan Amyloodinium ocellatum (AO) is the etiological agent of amyloodiniosis in European seabass (Dicentrarchus labrax) (ESB). There is a lack of information about basic molecular data on AO biology and its interaction with the host. Therefore, de novo transcriptome sequencing of AO tomonts was performed. AO trophonts were detached from infested ESB gills, and quickly becoming early tomonts were purified by Percoll® density gradient. Tomont total RNA was processed and quality was assessed immediately. cDNA libraries were constructed using TruSeq® Stranded mRNA kit and sequenced using Illumina sequencer. CLC assembly was used to generate the Transcriptome assembly of AO tomonts. Out of 48,188 contigs, 56.12% belong to dinophyceae wherein Symbiodinium microadriaticum had 94.61% similarity among dinophyceae. Functional annotations of contigs indicated that 12,677 had associated GO term, 9005 with KEGG term. The contigs belonging to dinophyceae resulted in the detection of several peptidases. A BLAST search for known virulent factors from the virulence database resulted in hits to Rab proteins, AP120, Ribosomal phosphoprotein, Heat-shock protein70, Casein kinases, Plasmepsin IV, and Brucipain. Hsp70 and casein kinase II alpha were characterized in-silico. Altogether, these results provide a reference database in understanding AO molecular biology, aiding to the development of novel diagnostics and future vaccines.

On the origin of European sheep as revealed by the diversity of the Balkan breeds and by optimizing population-genetic analysis tools.

BACKGROUND: In the Neolithic, domestic sheep migrated into Europe and subsequently spread in westerly and northwesterly directions. Reconstruction of these migrations and subsequent genetic events requires a more detailed characterization of the current phylogeographic differentiation. RESULTS: We collected 50 K single nucleotide polymorphism (SNP) profiles of Balkan sheep that are currently found near the major Neolithic point of entry into Europe, and combined these data with published genotypes from southwest-Asian, Mediterranean, central-European and north-European sheep and from Asian and European mouflons. We detected clines, ancestral components and admixture by using variants of common analysis tools: geography-informative supervised principal component analysis (PCA), breed-specific admixture analysis, across-breed [Formula: see text] profiles and phylogenetic analysis of regional pools of breeds. The regional Balkan sheep populations exhibit considerable genetic overlap, but are clearly distinct from the breeds in surrounding regions. The Asian mouflon did not influence the differentiation of the European domestic sheep and is only distantly related to present-day sheep, including those from Iran where the mouflons were sampled. We demonstrate the occurrence, from southeast to northwest Europe, of a continuously increasing ancestral component of up to 20% contributed by the European mouflon, which is assumed to descend from the original Neolithic domesticates. The overall patterns indicate that the Balkan region and Italy served as post-domestication migration hubs, from which wool sheep reached Spain and north Italy with subsequent migrations northwards. The documented dispersal of Tarentine wool sheep during the Roman period may have been part of this process. Our results also reproduce the documented 18th century admixture of Spanish Merino sheep into several central-European breeds. CONCLUSIONS: Our results contribute to a better understanding of the events that have created the present diversity pattern, which is relevant for the management of the genetic resources represented by the European sheep population.

A draft genome of sweet cherry (Prunus avium L.) reveals genome-wide and local effects of domestication.

Sweet cherry (Prunus avium L.) trees are both economically important fruit crops but also important components of natural forest ecosystems in Europe, Asia and Africa. Wild and domesticated trees currently coexist in the same geographic areas with important questions arising on their historical relationships. Little is known about the effects of the domestication process on the evolution of the sweet cherry genome. We assembled and annotated the genome of the cultivated variety "Big Star*" and assessed the genetic diversity among 97 sweet cherry accessions representing three different stages in the domestication and breeding process (wild trees, landraces and modern varieties). The genetic diversity analysis revealed significant genome-wide losses of variation among the three stages and supports a clear distinction between wild and domesticated trees, with only limited gene flow being detected between wild trees and domesticated landraces. We identified 11 domestication sweeps and five breeding sweeps covering, respectively, 11.0 and 2.4 Mb of the P. avium genome. A considerable fraction of the domestication sweeps overlaps with those detected in the related species, Prunus persica (peach), indicating that artificial selection during domestication may have acted independently on the same regions and genes in the two species. We detected 104 candidate genes in sweep regions involved in different processes, such as the determination of fruit texture, the regulation of flowering and fruit ripening and the resistance to pathogens. The signatures of selection identified will enable future evolutionary studies and provide a valuable resource for genetic improvement and conservation programs in sweet cherry.

'Candidatus Phytoplasma solani' interferes with the distribution and uptake of iron in tomato.

BACKGROUND: 'Candidatus Phytoplasma solani' is endemic in Europe and infects a wide range of weeds and cultivated plants. Phytoplasmas are prokaryotic plant pathogens that colonize the sieve elements of their host plant, causing severe alterations in phloem function and impairment of assimilate translocation. Typical symptoms of infected plants include yellowing of leaves or shoots, leaf curling, and general stunting, but the molecular mechanisms underlying most of the reported changes remain largely enigmatic. To infer a possible involvement of Fe in the host-phytoplasma interaction, we investigated the effects of 'Candidatus Phytoplasma solani' infection on tomato plants (Solanum lycopersicum cv. Micro-Tom) grown under different Fe regimes. RESULTS: Both phytoplasma infection and Fe starvation led to the development of chlorotic leaves and altered thylakoid organization. In infected plants, Fe accumulated in phloem tissue, altering the local distribution of Fe. In infected plants, Fe starvation had additive effects on chlorophyll content and leaf chlorosis, suggesting that the two conditions affected the phenotypic readout via separate routes. To gain insights into the transcriptional response to phytoplasma infection, or Fe deficiency, transcriptome profiling was performed on midrib-enriched leaves. RNA-seq analysis revealed that both stress conditions altered the expression of a large (> 800) subset of common genes involved in photosynthetic light reactions, porphyrin / chlorophyll metabolism, and in flowering control. In Fe-deficient plants, phytoplasma infection perturbed the Fe deficiency response in roots, possibly by interference with the synthesis or transport of a promotive signal transmitted from the leaves to the roots. CONCLUSIONS: 'Candidatus Phytoplasma solani' infection changes the Fe distribution in tomato leaves, affects the photosynthetic machinery and perturbs the orchestration of root-mediated transport processes by compromising shoot-to-root communication.